Fusion protein of TACI / BCMA chimera and use thereof

Abstract

Provided are a novel TACI / BCMA chimera and a fusion protein comprising same, and a nucleic acid encoding the chimera or fusion protein, a vector comprising the nucleic acid, and a host cell comprising the nucleic acid or vector. Further provided are a treatment method and the use of the fusion protein in the treatment of immune-related diseases.

Description

TACI / BCMA chimeric fusion protein and its use

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on Chinese patent application number 202410396507.7, filed on April 2, 2024, Chinese patent application number 202411500688.X, filed on October 25, 2024, and Chinese patent application number 202510355955.7, filed on March 25, 2025, and claims priority to the three Chinese patent applications. The entire contents of the three Chinese patent applications are hereby introduced into this application as a reference.

[0003] The present invention relates to a novel TACI / BCMA chimera and fusion proteins comprising the same. The present invention also relates to nucleic acids encoding the chimera or fusion protein, vectors comprising the nucleic acids, and host cells comprising the nucleic acids or vectors. The present invention also relates to therapeutic methods and uses of the fusion proteins for treating immune-related diseases. Background Art

[0004] The cytokine tumor necrosis factor (TNF) was first described in 1968 as a cytotoxic factor that induces tumor necrosis. This protein is produced as a 233-amino acid type II transmembrane protein that is released as a soluble homotrimer into the extracellular space through proteolytic cleavage by the metalloproteinase TNF-α converting enzyme (TACE / ADAM17). TNF-α (TNFSF1B) and TNF-β (TNFSF1A) bind to two specific receptors, TNFR1 and TNFR2.

[0005] The TNF superfamily (TNFSF) now includes 19 molecules, while the TNF receptor superfamily (TNFRSF) includes 29 different protein members. In addition to one-to-one selectivity between specific ligands and receptors, cross-reactivity has also been reported, suggesting differential signaling and multiple putative cellular and molecular actions.

[0006] The TNF receptor superfamily (TNFRSF) refers to a group of cell surface cytokine receptors, all of which are type I (N-terminal extracellular) transmembrane glycoproteins containing one to six cysteine-rich domains (CRDs) in their extracellular domains. Molecules are classified as members of the superfamily based on shared structural features, including one or more cysteine-rich domains (CRDs) present in their N-terminal extracellular regions, which typically play a role in the binding of proteins to their cognate binding partners or ligands. TNFRSF proteins may have only one or several CRDs (e.g., CRD1, CRD2, etc.). Typically, the ECD or extracellular domain of TNFRSF members contains 1 to 6 CRD pseudorepeats. For example, BAFF receptor and BCMA each contain one CRD, while TACI contains two CRDs (CRD1 and CRD2). TNFRSF members are typically trimerized or multimerized complexes that are stabilized by disulfide bonds within their cysteines. Binding of TNFRSF proteins to their ligands promotes a variety of biological activities in cells, such as induction of apoptotic cell death or cell survival and proliferation.

[0007] BAFF (B cell activating factor) and APRIL (A proliferation-inducing ligand) are B cell activating and regulatory factors belonging to the TNF family. They promote B cell development and proliferation, stimulate the secretion of various immunoglobulins, and increase their serum expression, playing a crucial role in regulating the immune response. They bind to the cell membrane receptors TACI (Transmembrane activator and CAML-interactor) and BCMA (B cell maturation antigen). BAFF also binds to another receptor, BAFFR (B cell-activating factor receptor). Through signaling from these receptors, BAFF and APRIL regulate lymphocyte activation, development, and proliferation. Overexpression of BAFF and APRIL is a contributing factor to various autoimmune diseases. Studies have shown that serum concentrations of BAFF and APRIL are significantly elevated in patients with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. Therefore, targeting BAFF / APRIL has become an effective therapeutic approach for autoimmune diseases.

[0008] The transmembrane activator and CAML-interacting protein (TACI), also known as tumor necrosis factor receptor superfamily member 13B (TNFRSF13B), was initially discovered due to its ability to interact with calcium regulator and cyclophilin ligand (CAML). TACI was later found to play a crucial role in humoral immunity by interacting with two members of the TNF family, BAFF and APRIL.

[0009] Prior art has discovered fusion proteins containing TACI for the treatment of immune diseases. For example, Rongchang Biopharma developed an optimized TACI-Fc fusion protein, Taitasip (CN101323643B), which utilizes the full-length extracellular domain of TACI. However, the resulting drug product exhibits poor molecular stability, requiring only lyophilized powders. Furthermore, the clinical dosing cycle is two injections per week, making it impossible to compensate for the increased dosing frequency by increasing the dosage.

[0010] Therefore, there is still a need in the art to develop TACI fusion proteins with good drugability and stable molecules. Summary of the Invention

[0011] The present invention relates to a TACI / BCMA chimera and a fusion protein comprising the chimera, such as a fusion protein with an Fc region.

[0012] The Fc region fusion protein of the TACI / BCMA chimera of the present invention has better stability, such as thermal stability, compared with known TACI-Fc fusion proteins; it also has a longer half-life and / or a lower clearance rate.

[0013] In some embodiments, the present invention relates to a TACI / BCMA chimera, comprising a chimeric protein in which the N-terminus and / or C-terminus of a functional fragment of the TACI extracellular domain ECD is replaced with the N-terminus and / or C-terminus of BCMA.

[0014] In a specific embodiment, the TACI / BCMA chimera comprises the following structure:

[0015] BCMAN terminal amino acid-TACI portion, wherein the TACI portion is the extracellular domain ECD of TACI lacking the N-terminus or a functional fragment thereof, or

[0016] BCMAN terminal amino acid-TACI part-BCMAC terminal amino acid, wherein the TACI part is the extracellular domain ECD of TACI lacking the N-terminus and C-terminus or a functional fragment thereof.

[0017] In a specific embodiment, the extracellular region ECD functional fragment of TACI in the TACI / BCMA chimera contains CRD2 of TACI and does not contain CRD1 or any fragment of CRD1. In one embodiment, the CRD2 is the amino acid sequence of TACI corresponding to positions 71-104 shown in SEQ ID NO:1, and CRD1 is the amino acid sequence of TACI corresponding to positions 34-66 shown in SEQ ID NO:1. In one embodiment, the extracellular region ECD functional fragment of TACI further contains part of the stem region of TACI and / or any amino acid sequence between positions 68-70 of TACI corresponding to SEQ ID NO:1. In one embodiment, the extracellular region ECD functional fragment of TACI is or contains the following fragment of TACI corresponding to the amino acid sequence shown in SEQ ID NO:1: amino acid residues 68-110. In one embodiment, the extracellular region ECD functional fragment of TACI contains

[0018] (i) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 31, 32 or 33;

[0019] (ii) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 33 and has an amino acid sequence of Y102D;

[0020] (iii) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 32 and has K77E, F78Y and Y102D;

[0021] (iv) the amino acid sequence shown in SEQ ID NO: 31, 32 or 33; or

[0022] (v) consisting of the sequence of any one of (i) to (iv).

[0023] In a specific embodiment, the N-terminus of the extracellular domain ECD functional fragment of TACI refers to the N-terminal amino acid of TACI before amino acid Y79 corresponding to SEQ ID NO:1.

[0024] In a specific embodiment, the C-terminus of the extracellular ECD functional fragment of TACI refers to the C-terminal amino acid after amino acid position 99, amino acid position 100, amino acid position 101, amino acid position 102, amino acid position 103, amino acid position 104, amino acid position 105, amino acid position 106, amino acid position 107, amino acid position 108, amino acid position 109 or amino acid position 110 of TACI corresponding to SEQ ID NO: 1, preferably the C-terminal amino acid after amino acid position 99 or 105.

[0025] In a specific embodiment, the N-terminal amino acid of BCMA is selected from the amino acid sequence of BCMA corresponding to positions 1-13, 2-13, 3-13, 4-13, 5-13, 6-13, or 7-13 of SEQ ID NO: 2. In a specific embodiment, the N-terminal amino acid sequence of BCMA comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 40-46.

[0026] In a specific embodiment, the C-terminal amino acid sequence of BCMA is the amino acid sequence of BCMA corresponding to positions 37-47, 37-46, 37-45, 37-44 of SEQ ID NO: 2, or the amino acid sequence corresponding to positions 43-44 or 43-46 of SEQ ID NO: 2.

[0027] In a specific embodiment, the C-terminal amino acid of BCMA comprises a mutation, such as a substitution, that improves binding affinity, increases stability and / or improves druggability, such as a mutation at position 39 and / or position 42, such as a substitution, such as N42A or N42Q or R39D or N42A-R39D.

[0028] In a specific embodiment, the C-terminal amino acid sequence of BCMA comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 47-55.

[0029] In a specific embodiment, the TACI ECD functional fragment comprises an amino acid site mutation that reduces the risk of aggregation, such as a mutation such as a substitution at a druggability risk site. For example, the druggability risk site is selected from amino acids 69, 72, 73, 74, 77, 78, 85, 102 or 103 corresponding to SEQ ID NO: 1. Preferably, the amino acid at the druggability risk site is mutated to A or D, for example, the mutation is Y102D.

[0030] In a specific embodiment, the TACI portion comprises the amino acid sequence set forth in any one of SEQ ID NOs: 31-38, or comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical thereto.

[0031] In a specific embodiment, the TACI / BCMA chimera comprises the amino acid sequence set forth in any one of SEQ ID NOs: 59-80, or comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical thereto.

[0032] In one aspect, the present invention relates to a fusion protein comprising the TACI / BCMA chimera described herein and an Fc region.

[0033] In a specific embodiment, the Fc region is human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc.

[0034] In a specific embodiment, the Fc region comprises one or more of the following mutations:

[0035] (i) Lysine K deleted at the C-terminus (K447del);

[0036] (ii) mutations that reduce effector function mediated by the Fc region;

[0037] (iii) mutations that reduce binding to Fcγ receptors, such as L234A / L235A mutations or L234A / L235E or L234A / L235E / G237A mutations;

[0038] (iv) mutations that enhance the binding of the Fc fragment to FcRn, such as M252Y / S254T / T256E and / or M482L / N434S.

[0039] In a specific embodiment, the Fc region comprises

[0040] (i) the amino acid sequence of SEQ ID NO: 81 or 82, or an amino acid sequence that is at least 90% identical thereto, such as 95%, 96%, 97%, 99% or more identical thereto;

[0041] (ii) the amino acid sequence shown in SEQ ID NO: 83 or 84, or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity thereto, optionally lacking the C-terminal lysine;

[0042] (iii) an amino acid sequence as set forth in any one of SEQ ID NOs: 85-88 and 100-103, or an amino acid sequence that is at least 90% identical thereto, such as 95%, 96%, 97%, 98%, 99% or more identical thereto;

[0043] (iv) an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 85 or 87 and comprises the mutations L234A / L235E / G237A;

[0044] (v) an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 86 or 88 and comprises the mutations L234A / L235E / G237A and a C-terminal deleted lysine;

[0045] (vi) an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 100 or 102 and comprises the mutations L234A / L235E / G237A and M252Y / S254T / T256E;

[0046] (vii) an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 101 or 103 and comprises the mutations L234A / L235E / G237A, M252Y / S254T / T256E, and a C-terminal deleted lysine; or

[0047] (viii) consisting of the amino acid sequence of any one of (i) to (vii) above.

[0048] In a specific embodiment, the TACI / BCMA chimera is fused to Fc directly or through a linker. Preferably, the C-terminus of the chimera is fused to the N-terminus of Fc directly or through a linker. In a specific embodiment, the linker is selected from (GSGGGGS) n , (GS) n 、(GSGGS) n、(GGGGS) n or (GGGS) n , wherein n is an integer of at least 1, such as 1, 2, 3, 4 or 5; for example (GSGGGGS) n , wherein n=1-3, for example, the linker is the amino acid sequence shown in SEQ ID NO:39.

[0049] In a specific embodiment, the fusion protein

[0050] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 5-28; or

[0051] (ii) comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 5-28

[0052] In one aspect, the present invention relates to a fusion protein dimer comprising a first monomer and a second monomer, wherein the first monomer and the second monomer respectively comprise or consist of the fusion protein chain described herein, preferably the first monomer and the second monomer are identical.

[0053] In one aspect, the present invention relates to a polynucleotide encoding the TACI / BCMA chimera or fusion protein or fusion protein dimer of the present invention.

[0054] In one aspect, the present invention relates to an expression vector comprising the polynucleotide of the present invention, for example, the expression vector is a pCDNA expression vector, such as a pCDNA3.1 expression vector.

[0055] In one aspect, the present invention relates to a host cell comprising the polynucleotide or expression vector of the present invention.

[0056] In one aspect, the present invention relates to a method for preparing a TACI / BCMA chimera or a fusion protein or fusion protein dimer thereof, wherein the method comprises culturing the host cells of the present invention under conditions suitable for the expression of the TACI / BCMA chimera or its fusion protein or fusion protein dimer, and optionally recovering the TACI / BCMA chimera or its fusion protein or fusion protein dimer from the host cells (or host cell culture medium).

[0057] In one aspect, the present invention relates to a pharmaceutical composition comprising the TACI / BCMA chimera or fusion protein or fusion protein dimer of the present invention, and optionally a pharmaceutically acceptable excipient.

[0058] In one aspect, the present invention relates to a pharmaceutical combination or combination product comprising a TACI / BCMA chimera or fusion protein or fusion protein dimer of the present invention and one or more other therapeutic agents (e.g., cytokines, hormones, cytotoxic agents or inhibitors (e.g., cytostatic agents that affect T cell and / or B cell proliferation), antibodies or small molecule drugs or immunomodulators (e.g., immunosuppressants)).

[0059] In one aspect, the present invention relates to a method for preventing or treating a subject's disease, such as a B cell or autoantibody-related disease or immune system disease (e.g., an autoimmune disease) or inflammation, the method comprising administering to the subject a TACI / BCMA chimera or fusion protein or fusion protein dimer or pharmaceutical composition or combination product of the present invention. In a specific embodiment, the B cell or autoantibody-related disease is a B cell or autoantibody-mediated disease, such as an autoimmune disease mediated by a B cell or autoantibody. In a specific embodiment, the B cell or autoantibody-related disease or immune system disease or inflammation is a disease (e.g., an autoimmune disease) in which B cells abnormally proliferate or are abnormally activated in an individual compared to a sample of a healthy individual. In a specific embodiment, the disease is lupus, such as systemic lupus erythematosus, chronic kidney disease such as rheumatoid arthritis, IgA nephropathy (IgAN) or membranous nephropathy, Sjögren's syndrome, myasthenia gravis, idiopathic thrombocytopenic purpura (ITP), warm antibody autoimmune hemolytic anemia (wAIHA), multiple sclerosis (MS), coronary heart disease (CAD) or thyroid eye disease. In a specific embodiment, the administration further comprises the combined administration of one or more other therapeutic agents (e.g., cytokines, hormones, cytotoxic agents or inhibitors (e.g., cytostatic agents that affect T cell and / or B cell proliferation), antibodies or small molecule drugs or immunomodulators (e.g., immunosuppressants)). BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 shows the structural analysis of the complexes of TACI and BCMA with APRIL.

[0061] Figure 2 shows the structural alignment and analysis of TACI and BCMA.

[0062] FIG3 shows the amino acid sequence alignment of TACI and BCMA.

[0063] FIG4 shows the ability of TACI / BCMA chimeric fusion protein to inhibit B cell proliferation.

[0064] FIG5 shows that TACI / BCMA chimeric fusion protein inhibits the proliferation of mouse spleen cells and spleen B cells induced by KLH immunization.

[0065] FIG6 shows that TACI / BCMA chimeric fusion protein inhibits the levels of IgA, IgM and IgG in the serum of mice immunized with KLH.

[0066] FIG7 shows the pharmacokinetics of TACI / BCMA chimeric fusion protein in mice.

[0067] Figure 8 shows the toxicokinetics of TACI / BCMA chimeric fusion protein in cynomolgus monkeys.

[0068] Figure 9 shows the pharmacokinetics of TACI / BCMA chimeric fusion protein and its YTE molecule in cynomolgus monkeys.

[0069] FIG10 shows the changes in immunoglobulin levels of TACI / BCMA chimeric fusion protein and its YTE molecule in cynomolgus monkey serum.

[0070] Detailed Description of the Invention

[0071] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, protocol and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention, which will only be limited by the appended claims.

[0072] I. Definition

[0073] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0074] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0075] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5%, 4%, 3%, 2% or 1% less than the specified numerical value and an upper limit that is 5%, 4%, 3%, 2% or 1% greater than the specified numerical value.

[0076] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0077] As used herein, the terms "comprising" or "including" mean including the recited elements, integers, or steps, but not excluding any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the recited elements, integers, or steps is also encompassed. For example, when reference is made to "comprising" or "including" a chimera, it is intended to encompass a composition having only that chimera.

[0078] Herein, the parent TACI refers to a template for introducing mutations of the present invention, which can be wild-type TACI, such as naturally occurring TACI protein, such as natural TACI derived from humans, mice, rats, non-human primates, including unprocessed (e.g., signal peptide not removed) forms and processed (e.g., N-terminal methionine removed) forms; or, for example, naturally occurring TACI allelic variants and splice variants, isoforms, homologs, and species homologs; or, for example, a TACI variant, for example, the variant can have at least 95%, 96%, 97%, 98% or 99% or higher identity with natural TACI or have no more than 1-10 or 1-5 amino acid mutations (e.g., conservative substitutions), and preferably has substantially the same BAFF binding affinity and / or APRIL binding affinity as the natural TACI protein. In one embodiment, the parent TACI refers to a functional fragment of TACI, such as a fragment comprising the extracellular domain (ECD) of TACI or the CRD2 domain of TACI, such as the extracellular domain (ECD) of TACI, or the CRD2 domain of TACI, or other functional fragments of TACI.

[0079] In the present invention, when referring to amino acid positions in TACI protein or TACI sequence segments, reference is made to the wild-type human TACI protein (also referred to as TACI WT ) is determined by comparing the amino acid sequence of SEQ ID NO: 1. The corresponding amino acid positions on other TACI proteins or polypeptides (including full-length sequences or truncated fragments) can be identified by amino acid sequence alignment with SEQ ID NO: 1. Therefore, in the present invention, unless otherwise indicated, the amino acid positions of TACI proteins or polypeptides are the amino acid positions numbered according to SEQ ID NO: 1. For example, when referring to "Y102", it refers to the tyrosine residue Y at position 102 of SEQ ID NO: 1, or the amino acid residue at the corresponding position on other TACI polypeptide sequences after alignment.

[0080] In the present invention, when referring to amino acid positions in BCMA protein or BCMA sequence segments, reference is made to the wild-type human BCMA protein (also referred to as BCMA WT) is determined by comparing the amino acid sequence of SEQ ID NO: 2. The corresponding amino acid positions on other BCMA proteins or polypeptides (including full-length sequences or truncated fragments) can be identified by amino acid sequence alignment with SEQ ID NO: 2. Therefore, in the present invention, unless otherwise indicated, the amino acid positions of BCMA proteins or polypeptides are the amino acid positions numbered according to SEQ ID NO: 2. For example, when referring to "Q3", it refers to the tyrosine residue Q at position 3 of SEQ ID NO: 2, or the amino acid residue at the corresponding position on other TACI polypeptide sequences after alignment.

[0081] In this article, when referring to mutant proteins, single amino acid substitutions are described as follows: [original amino acid residue / position / substituted amino acid residue]. For example, the substitution of tyrosine at position 102 with aspartic acid can be represented as Y102D. When a given position (e.g., position Y102) can have multiple optional amino acid substitutions (e.g., D, E), the amino acid substitution can be represented as: Y102D / E. Accordingly, the single amino acid substitutions can be connected by a plus sign "+" or "-" to represent a combination mutation at multiple given positions. For example, a combination mutation at positions K77E, F78Y, and Y102D can be represented as: K77E-F78Y-Y102D, or K77E+F78Y+Y102D.

[0082] As used herein, "N-terminal amino acid" or "N-terminus" are used interchangeably and refer to one or more amino acid segments starting from the N-terminus of BCMA or TACI or a functional fragment thereof.

[0083] As used herein, "C-terminal amino acid" or "C-terminus" are used interchangeably and refer to one or more amino acid segments ending at the C-terminus of BCMA or TACI or a functional fragment thereof.

[0084] As used herein, the term "chimera" refers to a fusion protein formed by genetically engineering two or more fragments of different proteins. Such chimeras typically retain the original functions of each fragment and may exhibit new biological properties or enhanced stability.

[0085] As used herein, the term "drugability risk site" refers to a site on a pharmaceutically acceptable protein, such as the chimera or fusion protein of the present invention, that affects its drugability (e.g., stability, etc.), including post-translational modification sites such as isomerization (D), deamidation (N), glycosylation (N*S / T), free cysteine ​​(C), oxidation (M / W); and a large area of ​​hydrophobic amino acids or charged amino acid residues enriched in the exposed surface of the protein (patch).

[0086] In this article, "percentage of sequence identity" can be determined by comparing two optimally aligned sequences within a comparison window. Preferably, sequence identity is determined over the full length of a reference sequence (e.g., SEQ ID NO: 1). Methods for comparing sequences are well known in the art. Suitable algorithms for determining percentages of sequence identity include, for example, BLAST and BLAST 2.0 algorithms (see Altschul et al., Nuc. Acids Res. 25: 3389-402, 1977 and Altschul et al. J. Mol. Biol. 215: 403-10, 1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. For the purposes of this application, percentage identity is determined using the Basic Local Alignment Search Tool available from https: / / blast.ncbi.nlm.nih.gov / Blast.cgi using default parameters.

[0087] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the biological function of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Typical conservative amino acid substitutions refer to substitutions of one amino acid with another amino acid having similar chemical properties (e.g., charge or hydrophobicity). Conservative substitution tables for functionally similar amino acids are well known in the art. In some embodiments, the following are exemplary conservative substitutions:

[0088] For example, the parent TACI protein may have conservative amino acid substitutions relative to one of SEQ ID NO: 1, or only conservative amino acid substitutions, and in a preferred embodiment, the conservative substitutions do not exceed 10 amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues. For another example, the mutant TACI protein of the present invention may have conservative amino acid substitutions relative to the TACI mutant protein sequences specifically given herein, or only conservative amino acid substitutions, and in a preferred embodiment, the conservative substitutions do not exceed 10 amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues.

[0089] "Affinity" or "binding affinity" can be used to reflect the intrinsic binding ability of an interaction between members of a binding pair. The affinity of a molecule X for its binding partner Y can be expressed by the equilibrium dissociation constant (K D ) indicates that the equilibrium dissociation constant is the dissociation rate constant and the association rate constant (k dis and k on Binding affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio affinity determination technology described herein.

[0090] The terms "extracellular domain", "extracellular domain" or "ECD" used interchangeably herein refer to a region of a membrane protein (such as a transmembrane protein) that is located outside the vacuole membrane (e.g., the space outside the cell) when the full-length form of the membrane protein is expressed from a cell. For the purposes of this article, it will be understood that reference to the ECD refers to the sequences and domains that make up this region, and does not require that the protein containing the ECD be a membrane protein or that the domain be present outside the cell. For example, a soluble immunomodulatory protein can contain the ECD sequence of a membrane protein fused to another portion (such as a multimerization domain, such as an Fc region). The extracellular domain typically interacts with a specific ligand or a specific cell surface receptor, for example, by specifically binding to a binding domain of a ligand or cell surface receptor. Examples of binding domains include cysteine-rich domains (CRDs). The extracellular domains of members of the TNFR superfamily contain TD domains (e.g., CRD domains). Therefore, reference to the ECD herein includes the full-length sequence of the ECD of a membrane protein, as well as specific binding fragments thereof containing a CRD or a portion thereof that bind to a ligand.

[0091] In this article, antibody Fc fragment refers to the C-terminal region of the immunoglobulin heavy chain containing the constant region of at least a portion, and can include native sequence Fc fragments and variant Fc fragments.Native sequence Fc fragments cover naturally occurring various immunoglobulin Fc sequences, such as various Ig subtypes and the Fc districts of their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes:from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.) In some embodiments, the Fc districts are from the Fc districts of IgG, such as the Fc districts of human IgG. In some embodiments, the Fc districts are from the Fc districts of IgG1, IgG2, IgG3 or IgG4, such as the Fc districts of human IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc districts are the Fc districts of human IgG1, IgG2, IgG3 or IgG4. In one embodiment, the human IgG heavy chain Fc fragment extends from Cys226 of the heavy chain or from Pro230 to the carboxyl terminus. In another embodiment, the C-terminal lysine (Lys447) of the Fc- fragment may or may not be present. In other embodiments, the Fc fragment is a variant Fc fragment comprising a mutation, for example, comprising an L234A-L235A mutation. Unless otherwise noted herein, the numbering of the amino acid residues in the Fc fragment is according to the EU numbering system, also referred to as the EU index, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242. In some embodiments, the antibody Fc fragment can have an IgG1 hinge sequence or a partial IgG1 hinge sequence at the N-terminus, for example, according to EU numbering, a sequence from E216 to T225 or a sequence from D221 to T225. The hinge sequence may contain a mutation, such as C220S.

[0092] As used herein, the term "linker" refers to any molecule that enables direct connection of the different parts of the fusion protein. Examples of linkers for establishing covalent connections between different parts of the fusion protein include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes or copolymers of polyethylene glycol and polypropylene glycol. The term "peptide linker" according to the present invention refers to an amino acid sequence, wherein the sequence connects the amino acid sequence of the first part of the fusion protein to the second part of the fusion protein. For example, a peptide linker can connect the TACI / BCMA portion of the fusion protein to the Fc domain or a fragment thereof. For example, a peptide linker can also connect an antibody to TACI / BCMA, such as connecting the C-terminus of the antibody heavy chain to TACI / BCMA. Preferably, the peptide linker has a length that is sufficient to connect the two entities in a manner that allows them to maintain their conformation relative to each other so as not to interfere with the desired activity. The peptide linker may or may not primarily include the following amino acid residues: Gly, Ser, Ala or Thr. Useful linkers include glycine-serine polymers, including, for example, (GSGGGGS)n(SEQ ID NO:89), (GS)n(SEQ ID NO:90), (GSGGS)n(SEQ ID NO:91), (GGGGS)n(SEQ ID NO:92), or (GGGS)n(SEQ ID NO:93), wherein n is an integer of at least 1 (and, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10). Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. In some embodiments, the linker of the present invention is (GSGGGGS)n(SEQ ID NO:89). Preferably, the linker of the present invention is SEQ ID NO:39.

[0093] As used herein, the term "fusion" refers to a fusion formed by linking two or more initially separate proteins / genes / compounds. If the entity constituting the fusion is a protein, it is referred to as a fusion protein. Fusion proteins are encompassed within the scope of the fusions of this application. For example, TACI linked to an Fc dimer can constitute a TACI-Fc fusion protein. The connection between the two entity molecules constituting the fusion can be achieved with or without a linker.

[0094] As used herein, the terms "first" and "second" are used with respect to an Fc domain (Fc region) or monomer, etc., to facilitate distinction when there is more than one of each type of module. Unless explicitly stated otherwise, the use of these terms is not intended to confer a particular order or orientation on the fusion proteins.

[0095] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating immune diseases or inflammation, such as immune diseases or inflammation, including but not limited to cytokines, hormones, cytotoxic agents or inhibitors (e.g., cytostatic agents that affect T cell and / or B cell proliferation), antibodies or small molecule drugs or immunomodulators (e.g., immunosuppressants).

[0096] The term "effective amount" refers to an amount or dosage of an antibody, fragment, composition, or combination of the invention that, after administration to a patient in a single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. "Effective amount" may encompass a "therapeutically effective amount" or a "prophylactically effective amount."

[0097] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter by at least about 40%, even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%, relative to an untreated subject. A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic outcome at the desired dosage and for the desired period of time. Typically, because prophylactic doses are used in subjects prior to or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0098] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity as the initially transformed cell are included herein.

[0099] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change in a detectable substrate compound or composition in the case of an enzymatic label. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of the probe or antibody by reacting with another reagent of the direct label.

[0100] The term "biological half-life" refers to the length of time it takes for a substance (e.g., an immunomodulatory protein) to lose half of its pharmacological or physiological activity or concentration. Biological half-life may be affected by elimination, excretion, degradation (e.g., enzymatic degradation / digestion) of the substance, or absorption and concentration in certain organs or tissues of the body. In some embodiments, biological half-life can be assessed by determining the time it takes for the plasma concentration of the substance to reach half of its steady-state level ("plasma half-life").

[0101] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0102] The term "immune system disease or disorder" refers to any disease or disorder associated with a malfunction of the immune system, including but not limited to autoimmune diseases or inflammatory diseases such as systemic lupus erythematosus and rheumatoid arthritis.

[0103] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0104] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0105] The term "drug combination" or "combination product" refers to a non-fixed combination or a fixed combination, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) the chimera or fusion protein of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without specific time restrictions, or sequentially at the same or different time intervals, as separate entities, wherein such administration provides prophylactically or therapeutically effective levels of two or more active agents in the patient's body. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0106] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0107] As used herein, "treat," ...

[0108] As used herein, "prevention" includes the inhibition of the development or progression of a disease or condition, or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0109] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0110] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from a fresh, frozen and / or preserved organ; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. Tissue samples may contain compounds that are not naturally contaminated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0111] II. TACI / BCMA Chimera and Fusion Proteins Thereof

[0112] In some embodiments, the present invention provides a TACI mutant protein having more uniform charge on the protein surface, better stability, and better drugability.

[0113] In some embodiments, the TACI mutant protein of the present invention reduces the area of ​​surface charge groups and hydrophobic groups after mutation, thereby reducing the aggregation risk of TACI.

[0114] In some embodiments, the TACI mutant protein of the present invention is a TACI mutant extracellular domain or a functional fragment thereof, wherein a portion of the domain is replaced by a corresponding domain of BCMA. Therefore, the TACI mutant protein comprising such a mutation is also referred to as a TACI / BCMA chimera in the present invention.

[0115] In some embodiments, the TACI / BCMA chimera of the present invention comprises the extracellular domain of TACI or a functional fragment thereof, wherein the N-terminal amino acid and / or C-terminal amino acid of the extracellular domain or the functional fragment thereof is replaced by the N-terminal amino acid and / or C-terminal amino acid of the extracellular domain of BCMA.

[0116] In some embodiments, the TACI / BCMA chimera of the present invention comprises a mutated extracellular domain of TACI or a functional fragment thereof, wherein the N-terminal amino acid of the extracellular domain or the functional fragment thereof is replaced by the N-terminal amino acid of the extracellular domain of BCMA.

[0117] In some embodiments, the TACI / BCMA chimera of the present invention comprises a mutated extracellular domain of TACI or a functional fragment thereof, wherein the N-terminal amino acid and C-terminal amino acid of the extracellular domain or the functional fragment thereof are replaced by the N-terminal amino acid and C-terminal amino acid of the extracellular domain of BCMA, respectively.

[0118] In some embodiments, the TACI / BCMA chimeras of the present invention further comprise substitutions at druggability risk sites. In some embodiments, the druggability risk sites are selected from positions 69, 72, 73, 74, 77, 78, 85, 102, and / or 103 of TACI. In some embodiments, the amino acid at the druggability risk site is mutated to A or D, such as D. In some embodiments, the substitution at the druggability risk site is Y102D.

[0119] In some embodiments, the present invention also provides a fusion protein comprising the TACI / BCMA chimera of the present invention. In a preferred embodiment, the TACI / BCMA chimera of the present invention is fused to another polypeptide that can confer improved pharmacokinetic properties, such as albumin, preferably an antibody Fc fragment.

[0120] In some embodiments, the present invention also provides a fusion protein dimer formed by dimerization of two fusion protein chains of the present invention.

[0121] The following describes in detail the components of the TACI / BCMA chimera or fusion protein thereof of the present invention. Those skilled in the art will appreciate that, unless the context clearly indicates otherwise, any combination of the technical features of these components is contemplated by the present invention. Furthermore, those skilled in the art will appreciate that, unless the context clearly indicates otherwise, the TACI / BCMA chimera or fusion protein thereof of the present invention may include any such combination of features.

[0122] II-1TACI extracellular domain or its functional fragment

[0123] TACI is a member of the tumor necrosis factor receptor family, characterized by an extracellular domain (ECD) containing a cysteine-rich pseudo-repeat domain (CRD). TACI is a membrane-bound receptor with an extracellular domain containing two cysteine-rich pseudo-repeat sequences (CRD1 and CRD2), a transmembrane domain, and a cytoplasmic domain that interacts with CAML (calcium regulator and cyclophilin ligand), an integral membrane protein located in intracellular vesicles that is a co-inducer of NF-AT activation when overexpressed in Jurkat cells. TACI is associated with subsets of B cells and T cells. The TACI receptor binds to two members of the tumor necrosis factor (TNF) ligand family. One ligand is named BAFF (B cell activating factor of the TNF family). The other ligand has been named APRIL. Both ligands are also bound by the B cell maturation receptor (BCMA). Binding of the TACI receptor to its ligands BAFF or APRIL stimulates B cell responses, including T cell-independent B cell antibody responses, isotype switching, and B cell homeostasis. Binding of BAFF or APRIL stimulates B cell responses, including T cell-independent B cell antibody responses, isotype switching, and B cell homeostasis.

[0124] In some embodiments, TACI is human TACI. In some embodiments, human TACI comprises the amino acid sequence set forth in SEQ ID NO: 1, or comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the full-length sequence of human TACI comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the human TACI protein is a type III membrane protein and lacks a signal peptide; upon expression in eukaryotic cells, the N-terminal methionine is removed. In some embodiments, the mature TACI protein does not contain the N-terminal methionine as set forth in SEQ ID NO: 1.

[0125] The structure and characterization of wild-type human TACI can be found in Uniprot o14836 (https: / / www.uniprot.org / uniprotkb / O14836 / entry). Human TACI typically consists of three domains: an extracellular domain, corresponding to amino acids 1-165; a transmembrane region, corresponding to amino acids 166-186; and a cytoplasmic domain, corresponding to amino acids 187-293.

[0126] The extracellular domain of TACI (e.g., amino acid residues 1-165 of SEQ ID NO: 1; the ECD shown in SEQ ID NO: 29) contains two cysteine-rich domains (CRDs), each of which exhibits affinity binding to BAFF and APRIL. The first cysteine-rich domain (CRD1) corresponds to or contains amino acid residues 34-66 of the sequence shown in SEQ ID NO: 1. The second cysteine-rich domain (CRD2) corresponds to or contains amino acids 71-104 of the sequence shown in SEQ ID NO: 1. TACI also contains a stem region of approximately 60 amino acids following the second cysteine ​​repeat sequence in the extracellular domain, corresponding to or containing amino acid residues 105-165 of the sequence shown in SEQ ID NO: 1.

[0127] In some embodiments, the extracellular domain of TACI comprises the amino acid sequence of positions 1-165 of SEQ ID NO: 1, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or consists of said amino acid sequence. In some embodiments, the extracellular domain of TACI comprises the amino acid sequence of SEQ ID NO: 29, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or consists of said amino acid sequence. In some embodiments, CRD2 comprises the amino acid sequence of SEQ ID NO: 58, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or consists of said amino acid sequence.

[0128] In some embodiments, a functional fragment of the TACI ECD comprises an amino acid sequence that binds to APRIL, BAFF, or an APRIL / BAFF heterotrimer.

[0129] In some embodiments, a functional fragment of a TACI ECD lacks a portion of the N-terminal residues of the ECD corresponding to a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, or 67 consecutive amino acids from the N-terminus of the ECD sequence shown in SEQ ID NO:1.

[0130] In some embodiments, a functional fragment of a TACI ECD lacks a portion of the stem region of the ECD, for example, lacking 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 consecutive amino acids from the C-terminus.

[0131] In some embodiments, the functional fragment of TACI ECD comprises CRD2, or comprises only complete CRD2. In some embodiments, the functional fragment of TACI ECD consists of CRD2.

[0132] In some embodiments, the functional fragment of TACI ECD comprises CRD2 and a portion of the stem region. In some embodiments, the functional fragment of TACI ECD further comprises a sequence between CRD1 and CRD2, such as any amino acid sequence corresponding to positions 68-70 of SEQ ID NO: 1. In some embodiments, the functional fragment of TACI ECD does not comprise CRD1 or any fragment thereof.

[0133] In some embodiments, the functional fragment of TACI ECD contains a portion of the stem region, for example, a fragment of TACI corresponding to the amino acid sequence of SEQ ID NO: 1 comprising amino acid residue 105, amino acid residues 105 to 106, amino acid residues 105 to 107, amino acid residues 105 to 108, amino acid residues 105 to 109, amino acid residues 105 to 110, amino acid residues 105 to 111, amino acid residues 105 to 112, amino acid residues 105 to 113, amino acid residues 105 to 114, amino acid residues 105 to 115, amino acid residues 105 to 116, 116, amino acid residues 105 to 117, amino acid residues 105 to 118, amino acid residues 105 to 119, amino acid residues 105 to 120, amino acid residues 105 to 121, amino acid residues 105 to 122, amino acid residues 105 to 123, amino acid residues 105 to 124, amino acid residues 105 to 125, amino acid residues 105 to 126, amino acid residues 105 to 127, amino acid residues 105 to 128, amino acid residues 1 05 to 129, amino acid residues 105 to 130, amino acid residues 105 to 131, amino acid residues 105 to 132, amino acid residues 105 to 133, amino acid residues 105 to 134, amino acid residues 105 to 135, amino acid residues 105 to 136, amino acid residues 105 to 137, amino acid residues 105 to 138, amino acid residues 105 to 139, amino acid residues 105 to 140, amino acid residues 105 to 141, amino acid residues residues 105 to 142, amino acid residues 105 to 143, amino acid residues 105 to 144, amino acid residues 105 to 145, amino acid residues 105 to 146, amino acid residues 105 to 147, amino acid residues 105 to 148, amino acid residues 105 to 149, amino acid residues 105 to 150, amino acid residues 105 to 151, amino acid residues 105 to 152, amino acid residues 105 to 153 and amino acid residues 105 to 154.

[0134] In some embodiments, a functional fragment of the TACI ECD comprises the following fragment of TACI corresponding to the amino acid sequence set forth in SEQ ID NO: 1: amino acid residues 67 to 118, amino acid residues 67 to 117, amino acid residues 67 to 116, amino acid residues 67 to 115, amino acid residues 67 to 114, amino acid residues 67 to 113, amino acid residues 67 to 112, amino acid residues 67 to 111, amino acid residues 67 to 110, amino acid residues 67 to 109, amino acid residues 67 to 108, amino acid residues 67 to 107, amino acid residues 67 to 106, amino acid residues 67 to 105, or amino acid residues 67 to 104; to 116, amino acid residues 68 to 115, amino acid residues 68 to 114, amino acid residues 68 to 113, amino acid residues 68 to 112, amino acid residues 68 to 111, amino acid residues 68 to 110, amino acid residues 68 to 109, amino acid residues 68 to 108, amino acid residues 68 to 107, amino acid residues 68 to 106, amino acid residues 68 to 105 or amino acid residues 68 to 104; amino acid residues 69 to 118, amino acid residues 69 to 117, amino acid residues 69 to 116, amino acid residues 69 to 115, amino acid residues 69 to 114, amino acid residues 69 to 113, amino acid residues 69 to 1 12. amino acid residues 69 to 111, amino acid residues 69 to 110, amino acid residues 69 to 109, amino acid residues 69 to 108, amino acid residues 69 to 107, amino acid residues 69 to 106, amino acid residues 69 to 105, or amino acid residues 69 to 104; amino acid residues 70 to 118, amino acid residues 70 to 117, amino acid residues 70 to 116, amino acid residues 70 to 115, amino acid residues 70 to 114, amino acid residues 70 to 113, amino acid residues 70 to 112, amino acid residues 70 to 111, amino acid residues 70 to 110, amino acid residues 70 to 109, or amino acid residues 70 to 108 , amino acid residues 70 to 107, amino acid residues 70 to 106, amino acid residues 70 to 105 or amino acid residues 70 to 104; amino acid residues 71 to 118, amino acid residues 71 to 117, amino acid residues 71 to 116, amino acid residues 71 to 115, amino acid residues 71 to 114, amino acid residues 71 to 113, amino acid residues 71 to 112, amino acid residues 71 to 111, amino acid residues 71 to 110, amino acid residues 71 to 109, amino acid residues 71 to 108, amino acid residues 71 to 107, amino acid residues 71 to 106, amino acid residues 71 to 105 or amino acid residues 71 to 104.In some embodiments, a functional fragment of TACI ECD comprises amino acids 13-118 of ECD, or comprises amino acids 68-110, or consists of said contiguous amino acid sequence.

[0135] In some embodiments, the functional fragment of TACI ECD comprises the amino acid sequence shown in SEQ ID NO:30, 31 or 58, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of the amino acid sequence.

[0136] In some embodiments, the functional fragment of TACI ECD consists of the amino acid sequence shown in SEQ ID NO:31.

[0137] In some embodiments of the present invention, TACI ECD or a functional fragment thereof also encompasses ECD variants or functional fragments thereof having mutations. In some embodiments, the functional fragment of TACI ECD comprises K77E, F78Y and / or Y102D.

[0138] In some embodiments, the functional fragment of TACI ECD comprises Y102D. In some embodiments, the functional fragment of TACI ECD comprises the amino acid sequence set forth in SEQ ID NO: 33, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or consists of said amino acid sequence. In some embodiments, the functional fragment of TACI ECD comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto and having Y102D. In some embodiments, the functional fragment of TACI ECD consists of the amino acid sequence set forth in SEQ ID NO: 33.

[0139] In some embodiments, the functional fragment of TACI ECD comprises K77E, F78Y, and Y102D. In some embodiments, the functional fragment of TACI ECD comprises the amino acid sequence set forth in SEQ ID NO: 32, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or consists of said amino acid sequence. In some embodiments, the functional fragment of TACI ECD comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto and having K77E, F78Y, and Y102D. In some embodiments, the functional fragment of TACI ECD consists of the amino acid sequence set forth in SEQ ID NO: 32.

[0140] II-2TACI / BCMA chimera

[0141] The present invention discovered that the protein structure of BCMA (6-42, PDB ID: 1XU2), a protein in the TACI family, is highly homologous to that of TACI and shares essentially the same ligand-binding sites. Furthermore, the N-termini and C-termini of both proteins are not involved in ligand binding (Figure 1). More importantly, analysis using Discovery Studio software revealed that BCMA exhibits a balanced surface charge distribution and exhibits a significantly lower aggregation risk than TACI.

[0142] Therefore, the present invention designs a series of chimeric molecules of BCMA and TACI extracellular domains. On the basis of retaining the TACI and ligand binding sites, the N-terminal amino acid or C-terminal amino acid of TACI is replaced with the corresponding sequence of BCMA, or both the N-terminal amino acid and the C-terminal amino acid of TACI are replaced with the corresponding sequence of BCMA, and amino acids are optionally replaced at some druggability risk sites, thereby reducing the area of ​​charge groups and hydrophobic groups on the surface of the TACI protein and reducing the aggregation risk of TACI.

[0143] BCMA is a member of the tumor necrosis factor receptor family, characterized by an extracellular domain (ECD) containing a cysteine-rich pseudo-repeat domain (CRD). BCMA is a membrane-bound receptor with an extracellular domain containing a single CRD, a transmembrane domain, and a cytoplasmic domain containing a TRAF binding site for binding to TRAF signaling molecules. BCMA binds to its cognate ligands APRIL and BAFF, but with weaker affinity for BAFF. BCMA binding to BAFF has been reported to be two to three orders of magnitude weaker than the binding between BAFF and its other cognate receptors, BAFF-R and TACI.

[0144] In some embodiments, BCMA is human BCMA. In some embodiments, human BCMA comprises the amino acid sequence set forth in SEQ ID NO: 2, or comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the full-length human BCMA sequence comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the BCMA protein is a type II membrane protein and lacks a signal peptide; upon expression in eukaryotic cells, the N-terminal methionine is removed. In some embodiments, the mature BCMA protein does not contain the N-terminal methionine as set forth in SEQ ID NO: 2.

[0145] The structure and characterization of wild-type human BCMA can be found in Uitprot Q02223 (https: / / www.uniprot.org / uniprotkb / Q02223 / entry). Human BCMA generally contains three domains, namely the extracellular domain, corresponding to amino acids 1-54; the transmembrane region, corresponding to amino acids 55-77; and the cytoplasmic domain, corresponding to amino acids 78-184. The extracellular domain of BCMA contains a cysteine-rich domain (CRD), which exhibits affinity for binding to APRIL and, to a lesser extent, to BAFF. In some embodiments, the CRD contains amino acid residues 7-41 of the sequence shown in SEQ ID NO: 2.

[0146] In some embodiments, the extracellular domain of BCMA comprises amino acids 1-54 of SEQ ID NO: 2, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or consists of said amino acid sequence. In some embodiments, the extracellular domain of BCMA comprises amino acids 1-54 of SEQ ID NO: 2, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or consists of said amino acid sequence.

[0147] In some embodiments, the N-terminus and / or C-terminus of TACI ECD or a functional fragment thereof (eg, an ECD containing only CRD2, such as TACI (68-110)) is replaced with the corresponding sequence of BCMA.

[0148] In some embodiments, the N-terminus of TACI ECD or a functional fragment thereof refers to the N-terminal amino acid of TACI preceding amino acid Y79 of SEQ ID NO: 1.

[0149] In some embodiments, the C-terminus of TACI ECD or a functional fragment thereof refers to the C-terminal amino acid of TACI corresponding to amino acid position 99, amino acid position 100, amino acid position 101, amino acid position 102, amino acid position 103, amino acid position 104, amino acid position 105, amino acid position 106, amino acid position 107, amino acid position 108, amino acid position 109 or amino acid position 110 of SEQ ID NO:1.

[0150] In some embodiments, the C-terminus of TACI ECD or a functional fragment thereof refers to the C-terminal amino acid following amino acid position 99 of TACI corresponding to SEQ ID NO: 1. In some embodiments, the C-terminus of TACI ECD or a functional fragment thereof refers to the C-terminal amino acid following amino acid position 105 of TACI corresponding to SEQ ID NO: 1.

[0151] In some embodiments, the corresponding N-terminal amino acid sequence of BCMA that can be used to replace the N-terminus of TACI ECD or a functional fragment thereof is selected from the amino acid sequence of BCMA corresponding to positions 1-13, 2-13, 3-13, 4-13, 5-13, 6-13, or 7-13 of SEQ ID NO: 2.

[0152] In some embodiments, the corresponding N-terminal amino acid sequence of BCMA used for replacement comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 40-46.

[0153] In some embodiments, the corresponding C-terminal amino acid sequence of BCMA that can be used to replace the C-terminus of TACI ECD or a functional fragment thereof is selected from the BCMA amino acid sequence corresponding to positions 37-47, 37-46, 37-45, 37-44 of SEQ ID NO: 2, or the amino acids corresponding to positions 43-44 or 43-46 of SEQ ID NO: 2.

[0154] In some embodiments, the corresponding N-terminal amino acid or C-terminal amino acid sequence of BCMA used for replacement may contain mutations, such as substitutions, for improving binding affinity, increasing stability, and / or improving drugability. In some embodiments, the C-terminal amino acid sequence of BCMA used for replacement contains mutations at position 39 and / or position 42.

[0155] In some embodiments, the C-terminal amino acid sequence of the BCMA used for replacement comprises N42A. In some embodiments, the C-terminal amino acid sequence of the BCMA used for replacement comprises N42Q. In some embodiments, the C-terminal amino acid sequence of the BCMA used for replacement comprises R39D. In some embodiments, the C-terminal amino acid sequence of the BCMA used for replacement comprises N42A-R39D.

[0156] In some embodiments, the corresponding C-terminal amino acid sequence of BCMA used for replacement comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 47-55.

[0157] In some embodiments, the number of amino acids in the N-terminal or C-terminal amino acid sequence of BCMA to be replaced is equal to the number of amino acids in the N-terminal or C-terminal amino acid sequence of the replaced ECD of TACI or a functional fragment thereof, or the difference is within 1-3 amino acids.

[0158] Thus, in some embodiments, the TACI / BCMA chimera of the present invention comprises the following structure:

[0159] BCMAN terminal amino acid-TACI portion, wherein the TACI portion is an ECD or a functional fragment thereof lacking the N-terminus, or

[0160] BCMAN terminal amino acid-TACI portion-BCMAC terminal amino acid, wherein the TACI portion is the ECD or a functional fragment thereof lacking the N-terminus and C-terminus.

[0161] Therefore, as used herein, the term "BCMAN terminal amino acid-TACI moiety" refers to a polypeptide obtained by replacing the N-terminal amino acid of TACI with the N-terminal amino acid of BCMA. The term "BCMAN terminal amino acid-TACI moiety-BCMAC terminal amino acid" refers to a polypeptide obtained by replacing the N-terminal amino acid of TACI with the N-terminal amino acid of BCMA and the C-terminal amino acid of TACI with the C-terminal amino acid of BCMA.

[0162] In some embodiments, the TACI portion of a TACI / BCMA chimera suitable for use in the present invention may comprise a mutation, such as a substitution.

[0163] In some embodiments, the TACI portion of the TACI / BCMA chimera suitable for use in the present invention further comprises a mutation at a druggability risk site, such as an amino acid site with aggregation risk, such as replacing an amino acid at one or more druggability risk sites in the TACI portion. In some embodiments, if the TACI portion comprises a druggability risk site, it is replaced with an amino acid that reduces that risk. In some embodiments, if the TACI portion comprises an amino acid with aggregation risk, it is replaced with an amino acid that reduces that aggregation risk. In some embodiments, the druggability risk site is selected from amino acids 69, 72, 73, 74, 77, 78, 85, 102, or 103 corresponding to SEQ ID NO: 1. For example, the amino acid at the druggability risk site is mutated to A or D.

[0164] In some embodiments, the TACI moiety comprises a mutation, eg, a substitution, at position Y102, eg, Y102D.

[0165] In some embodiments, the TACI portion comprises the amino acid sequence set forth in any one of SEQ ID NOs: 31-38, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical thereto, or comprises an amino acid sequence that has 1, 2, 3, 4 or 5 amino acid changes (e.g., substitutions, deletions or additions, such as conservative substitutions; or, for example, substitutions at druggability risk sites) thereto, or consists of the amino acid sequence.

[0166] In some embodiments, the TACI portion consists of the amino acid sequence set forth in any one of SEQ ID NOs: 31-38.

[0167] In some embodiments, the BCMA portion of the TACI / BCMA chimera suitable for use in the present invention (e.g., at the BCMAN end or the BCMAC end, preferably the BCMAC end) further comprises a mutation for improving binding affinity, increasing stability and / or improving drugability, such as a substitution, for example, at a drugability risk site such as a mutation that results in an N-glycosylation site, for example, replacing one or more amino acids at a drugability risk site of the BCMA portion. In some embodiments, if the BCMA portion comprises a drugability risk site, it is replaced with an amino acid that reduces the risk. In some embodiments, if the BCMA portion comprises an amino acid with glycosylation (e.g., N-glycosylation), it is replaced with another amino acid. In some embodiments, the drugability risk site is the 39th or 42nd amino acid corresponding to SEQ ID NO: 2. For example, the amino acid at the drugability risk site is mutated to D, A, or Q.

[0168] In some embodiments, the BCMAN terminus comprises the amino acid sequence shown in any one of SEQ ID NOs: 40-46, or comprises an amino acid sequence having at least 80% or 90% identity thereto, or comprises an amino acid sequence having 1, 2, or 3 amino acid changes (e.g., substitutions, deletions, or additions, such as conservative substitutions or substitutions at druggability risk sites) thereto, or consists of the amino acid sequence.

[0169] In some embodiments, the BCMAN terminus consists of the amino acid sequence set forth in any one of SEQ ID NOs: 40-46.

[0170] In some embodiments, the BCMAC terminus comprises a mutation at position R39, such as a substitution, for example, R39D. In some embodiments, the BCMAC terminus comprises a mutation at position N42, such as a substitution, for example, N42A or N42Q. In some embodiments, the BCMAC terminus comprises a mutation at position R39 and a mutation at position N42, for example, R39D-N42A.

[0171] In some embodiments, the BCMAC terminus comprises the amino acid sequence set forth in any one of SEQ ID NOs: 47-55, or comprises an amino acid sequence having at least 80% or 90% identity thereto, or comprises an amino acid sequence having 1 or 2 amino acid changes (e.g., substitutions, deletions, or additions, such as conservative substitutions or substitutions at druggability risk sites) thereto, or consists of said amino acid sequence.

[0172] In some embodiments, the BCMAC terminus consists of the amino acid sequence set forth in any one of SEQ ID NOs: 47-55.

[0173] The TACI / BCMA chimera of the present invention comprises the amino acid sequence shown in any one of SEQ ID NOs: 59-80, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identity thereto, or consists of the amino acid sequence.

[0174] II-3. TACI / BCMA chimeric fusion protein

[0175] In one aspect, the present invention also provides a fusion protein comprising a TACI / BCMA chimera of the present invention. In a preferred embodiment, the TACI / BCMA chimera of the present invention is fused to another polypeptide that can confer improved pharmacokinetic properties, such as albumin, more preferably an antibody Fc fragment. In some embodiments, FcRn-mediated recycling in the body can extend the half-life of the TACI / BCMA chimera-Fc fusion protein.

[0176] In one embodiment, the present invention provides a TACI / BCMA chimera fusion protein comprising the TACI / BCMA chimera of the present invention fused to an Fc region. Preferably, the Fc region is fused to the C-terminus of the TACI / BCMA chimera of the present invention, for example, the N-terminus of the Fc region is fused to the C-terminus of the chimera.

[0177] In some embodiments, the Fc region is a human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In some embodiments, the Fc region comprises a complete hinge region at its N-terminus, i.e., starting from position E216 of the human IgG1 heavy chain constant region to the C-terminus. In some embodiments, the Fc region comprises a cysteine ​​mutated to a serine at position 220 of the N-terminal hinge region corresponding to IgG1 (i.e., comprising a 220S mutation).

[0178] The Fc region may also lack part of the hinge region, for example, starting from position D221 to the C-terminus corresponding to the human IgG1 heavy chain constant region.

[0179] In one embodiment, the Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 81 or 82, or an amino acid sequence that is at least 90% identical thereto, such as 95%, 96%, 97%, 98%, 99% or more identical thereto.

[0180] In one embodiment, the Fc region of the fusion protein suitable for use in the present invention lacks lysine K at the C-terminus (K447del).

[0181] In one embodiment, the Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO: 83 or 84, or an amino acid sequence that is at least 90% identical thereto, e.g., 95%, 96%, 97%, 98%, 99% or more identical thereto (optionally also lacking the C-terminal lysine).

[0182] The Fc region of the binding molecules of the invention, such as antibodies, can also be mutated to obtain desired properties. Mutations in the Fc region are known in the art.

[0183] In one embodiment, the Fc region is modified with respect to the properties of the effector functions of the Fc region (e.g., complement activation function of the Fc region). In one embodiment, the effector functions have been reduced or eliminated relative to the wild-type Fc region. In one embodiment, the effector functions are reduced or eliminated by a method selected from the following: using an Fc isotype that naturally has reduced or eliminated effector functions, and Fc region modifications. In a preferred embodiment, the Fc region has reduced effector functions mediated by the Fc region, such as reduced or eliminated ADCC or ADCP or CDC effector functions, for example, comprising mutations that achieve the above functions.

[0184] In one embodiment, modifications that alter binding affinity for one or more Fc receptors may also be included in the Fc region. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region comprises mutations that reduce binding to Fcγ receptors. In another preferred embodiment, the Fc region may have mutations that result in increased serum half-life, such as mutations that improve binding of the Fc fragment to FcRn.

[0185] For example, in some embodiments, the Fc region used in the present invention has a mutation that reduces binding to Fcγ receptors. The Fc fragment used in the present invention has a mutation that reduces binding to Fcγ receptors, such as L234A / L235A mutation, L234A / L235E mutation, G237A mutation, or L234A / L235E / G237A. For example, in some embodiments, the Fc region used in the present invention has a mutation that improves binding of the Fc fragment to FcRn, such as a YTE mutation (M252Y / S254T / T256E) or a LS mutation (M428L / N434S) that enhances binding of the Fc fragment to FcRn. For example, exemplary mutations are described in WO2002060919A2, which is incorporated herein by reference in its entirety.

[0186] In some embodiments, the Fc region comprises both the mutation that reduces binding to Fcγ receptors and the mutation that improves binding of the Fc fragment to FcRn.

[0187] In some embodiments, the Fc region comprises L234A / L235E / G237A and M252Y / S254T / T256E, and optionally a C-terminal deleted lysine.

[0188] In some embodiments, the Fc region comprises L234A / L235E / G237A and M428L / N434S, and optionally a C-terminal deleted lysine.

[0189] In one embodiment, the Fc region comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs: 85-88 and 100-103, or an amino acid sequence that is at least 90% identical thereto, such as 95%, 96%, 97%, 98%, 99% or more identical thereto.

[0190] In one embodiment, the Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 85 or 87 and comprises the mutations L234A / L235E / G237A.

[0191] In one embodiment, the Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 86 or 88 and comprises the mutations L234A / L235E / G237A and a C-terminal deleted lysine.

[0192] In one embodiment, the Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 100 or 102 and comprises the mutations L234A / L235E / G237A and M252Y / S254T / T256E.

[0193] In one embodiment, the Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 101 or 103 and comprises the mutations L234A / L235E / G237A, M252Y / S254T / T256E, and a C-terminal deleted lysine.

[0194] In some embodiments, the TACI / BCMA chimera is fused to the Fc region directly or through a linker, for example, the C-terminus of the chimera is fused to the N-terminus of the Fc region directly or through a linker. As will be appreciated by those skilled in the art, the linker suitable for connecting the TACI / BCMA and Fc regions in the fusion proteins and dimer molecules of the present invention can be any linker known in the art. In some embodiments, the linker can comprise an IgG1 hinge, or can comprise a linker sequence selected from the following: (GSGGGGS) n (SEQ ID NO:94), (GS) n (SEQ ID NO:95), (GSGGS) n(SEQ ID NO:96), (GGGGS) n (SEQ ID NO:97) or (GGGS) n (SEQ ID NO:98), wherein n is an integer of at least 1, such as 1, 2, 3, 4 or 5.

[0195] In one embodiment, the linker is (GSGGGGS) n (SEQ ID NO: 99), wherein n = 1 to 3. In one embodiment, the linker comprises or is the amino acid sequence shown in SEQ ID NO: 39.

[0196] In yet another aspect, the present invention also provides a dimeric molecule comprising a TACI / BCMA chimera of the present invention fused to an Fc region.

[0197] In some embodiments, the present invention provides a TACI / BCMA chimera-Fc dimer protein, wherein the first monomer and the second monomer, respectively, comprise or consist of the following from N-terminus to C-terminus: i) a TACI / BCMA chimera; ii) a linker (optionally present or absent); and iii) an Fc region. In some embodiments, the dimer protein is a homodimer. In some embodiments, the monomers have the same amino acid sequence. In some embodiments, the TACI / BCMA chimera-Fc protein dimer of the present invention comprises two identical monomers.

[0198] In some embodiments, the TACI / BCMA chimera-Fc fusion protein of the present invention or its monomer

[0199] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 5-28; or

[0200] (ii) comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 5-28; or

[0201] (iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes compared to the amino acid sequence of any one of SEQ ID NOs: 5-28.

[0202] In some embodiments, the TACI / BCMA chimera-Fc protein of the present invention or the protein dimer monomer of the present invention consists of the amino acid sequence of any one of SEQ ID NOs: 5-28.

[0203] III. Polynucleotides, Vectors, and Hosts

[0204] The present invention provides nucleic acids encoding any of the above-described TACI / BCMA chimeras, or any monomer or domain thereof in a fusion protein or fusion protein dimer. Polynucleotide sequences encoding the muteins of the present invention can be generated by de novo solid-phase DNA synthesis or by PCR mutagenesis of existing sequences encoding wild-type TACI using methods well known in the art. Furthermore, the polynucleotides and nucleic acids of the present invention may include a segment encoding a secretory signal peptide, which can be operably linked to a segment encoding a mutein of the present invention, thereby directing secretory expression of the mutein of the present invention.

[0205] The present invention also provides vectors comprising the nucleic acid of the present invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In a preferred embodiment, the expression vector of the present invention is a pCDNA expression vector, such as a pCDNA3.1 expression vector.

[0206] The present invention also provides a host cell comprising the nucleic acid or the vector. Host cells suitable for replicating and supporting the expression of TACI / BCMA chimera or its fusion protein or fusion protein dimer are well known in the art.

[0207] Such cells can be transfected or transduced with specific expression vectors, and large quantities of vector-containing cells can be grown for inoculating large-scale fermenters, thereby obtaining sufficient amounts of TACI / BCMA chimera or its fusion protein or fusion protein dimer for clinical use.

[0208] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293 cells).

[0209] Examples of useful mammalian host cell lines include monkey kidney CV1 line (COS-7) transformed by SV40, human embryonic kidney line (293 or 293T cells or HEK293 cells), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT060562), TRI cells, MRC5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr-CHO cells, and myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0.

[0210] In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell such as a Chinese hamster ovary (CHO) cell, HEK293 cell, human embryonic kidney (HEK) cell or a lymphocyte (eg, Y0, NS0, Sp20 cell).

[0211] IV. Preparation Method

[0212] In another aspect, the present invention provides a method for preparing the TACI / BCMA chimera or its fusion protein or fusion protein dimer of the present invention, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the TACI / BCMA chimera or its fusion protein or fusion protein dimer, as provided above, under conditions suitable for the expression of the TACI / BCMA chimera or its fusion protein or fusion protein dimer, and optionally recovering the TACI / BCMA chimera or its fusion protein or fusion protein dimer from the host cell (or host cell culture medium).

[0213] In one embodiment, a vector containing a nucleic acid encoding a TACI / BCMA chimera or its fusion protein or fusion protein dimer is transferred into cells for expression, and then the cells (or cell culture supernatant) are collected, the TACI / BCMA chimera or its fusion protein or fusion protein dimer is extracted, and purified to obtain the TACI / BCMA chimera or its fusion protein or fusion protein dimer.

[0214] In a specific embodiment, the purification method is affinity purification. In another specific embodiment, the purification method is ion exchange purification. In some embodiments, the purification is performed by filtration using a gel filtration chromatography column.

[0215] V. Properties of the TACI / BCMA Chimera or Its Fusion Protein or Fusion Protein Dimer of the Present Invention

[0216] The TACI / BCMA chimera or fusion protein or fusion protein dimer provided herein can be identified, screened, or characterized for its physical / chemical properties and / or biological activities by various assays known in the art.

[0217] The protein surface charge of the TACI / BCMA chimera obtained in the present invention is more uniform, and therefore has better stability and better drugability.

[0218] The TACI / BCMA chimeras and fusion proteins obtained in the present invention exhibit improved stability (e.g., thermal stability) and pharmacokinetic data, as well as a longer half-life, compared to TACI proteins and fusion proteins known in the prior art. Under accelerated conditions, the TACI / BCMA chimera fusion proteins obtained in the present invention exhibit improved stability compared to TACI fusion proteins or variants known in the prior art, with some molecules exhibiting significantly better thermal stability than ALPN303.

[0219] In some embodiments, the TACI / BCMA chimeric fusion protein obtained by the present invention has one or more properties selected from the following:

[0220] a) High expression in cells and easy to purify;

[0221] b) Specific binding to BAFF and APRIL, as determined by biointerferometry (ForteBio) assays, with a K of 1. D less than or equal to about 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM or 0.4 nM, or greater than or equal to about 0.1 nM or 0.2 nM, or any value between said values; and with a K of APRIL D Less than or equal to about 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, or 0.4 nM, or greater than or equal to about 0.1 nM or 0.2 nM, or between any of the recited values;

[0222] c) effectively inhibiting the binding of BAFF to IM-9 cells, for example, with an IC50 of less than or equal to about 5 nM, 4 nM, 3 nM, 2 nM, or 1.5 nM when assayed by FACS; effectively inhibiting the binding of APRIL to IM-9 cells, for example, with an IC50 of less than or equal to about 5 nM, 4 nM, 3 nM, 2 nM 1.5 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, or 0.2 nM when assayed by FACS;

[0223] d) effectively inhibiting BAFF / APRIL-induced surface expression of (human) TACI, for example, with an IC50 of less than or equal to about 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM or 5.5 nM as determined by a TACI / NF-κB Reporter Jurkat signal assay;

[0224] e) effectively inhibits BAFF / APRIL-induced surface expression of (human) BCMA, for example, with an IC50 of less than or equal to about 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 Nm, 5.5 nM, 5 nM, 4.5 nM or 4 nM as determined by a BCMA / NF-κB Reporter Jurkat signal assay;

[0225] f) effectively inhibiting BAFF / APRIL-induced splenocyte proliferation, e.g., with an IC50 of less than or equal to about 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, or 0.6 nM;

[0226] g) Effectively inhibits BAFF / APRIL-induced B cell proliferation;

[0227] h) has better thermal stability;

[0228] i) Better drugability;

[0229] j) have better accelerated stability than, for example, known molecules RC-18 and / or ALPN-303;

[0230] k) effectively inhibiting BAFF-induced proliferation of splenocytes and / or B cells and / or different B cell subsets in the spleen;

[0231] l) effectively inhibiting the proliferation of splenocytes and / or B cells and / or different B cell subsets in the spleen, for example in a KLH immunization model, for example better than the known molecule RC-18;

[0232] m) can effectively reduce the levels of IgA, IgM and / or IgG in serum induced by BAFF;

[0233] n) more effectively inhibit the proliferation of MZ cells, GC B cells, and plasma cells, for example, better than the known molecule ALPN-303;

[0234] o) have better pharmacokinetics, for example, better than known molecules RC-18 and / or ALPN-303;

[0235] p) has a longer half-life (e.g., plasma half-life), e.g., a half-life (e.g., plasma half-life) greater than or equal to about 45 hours, e.g., greater than or equal to about 46, 47, 48, 49, or 50 hours; and / or

[0236] q) has a lower clearance, such as a clearance of less than about 1.8, 1.7, 1.6, or 1.5 mL / kg / h.

[0237] VI. Pharmaceutical Compositions and Pharmaceutical Formulations

[0238] The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising a TACI / BCMA chimera or a fusion protein or fusion protein dimer thereof, or compositions comprising a polynucleotide encoding a TACI / BCMA chimera or a fusion protein or fusion protein dimer thereof. These compositions may also optionally contain suitable pharmaceutical excipients, such as pharmaceutical carriers and excipients known in the art, including buffers.

[0239] Pharmaceutical compositions comprising the TACI / BCMA chimera or its fusion protein or fusion protein dimer of the present invention can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, encapsulating, or lyophilizing processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants that facilitate processing of the protein into pharmaceutically acceptable formulations. The appropriate formulation depends on the chosen route of administration.

[0240] The TACI / BCMA chimera or its fusion protein or fusion protein dimer can be formulated into a composition in free acid or base, neutral, or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include acid addition salts, such as those formed with free amino groups of the proteinaceous composition, or with inorganic acids (such as hydrochloric acid or phosphoric acid) or with organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid.

[0241] VII. Combination

[0242] In one aspect, the present invention also provides a pharmaceutical combination or combination product comprising the TACI / BCMA chimera of the present invention or its fusion protein or fusion protein dimer, and one or more other therapeutic agents (e.g., cytokines, hormones, cytotoxic agents or inhibitors (e.g., cytostatic agents that affect T cell and / or B cell proliferation), antibodies or small molecule drugs, or immunomodulators (e.g., immunosuppressants)). The pharmaceutical combination or combination product of the present invention can be used in the treatment methods of the present invention.

[0243] In some embodiments, the pharmaceutical combination or combination product is used to prevent or treat a B cell-related disease or immune system disease or inflammation.

[0244] In some embodiments, the pharmaceutical combination or combination product is used to prepare a medicament for preventing or treating a B cell-related disease or an immune system disease or inflammation.

[0245] VIII. Methods of Treatment and Use

[0246] In one aspect, the present invention relates to a method for preventing or treating a subject's disease, such as a B cell or autoantibody-related disease or an immune system disease (e.g., an autoimmune disease) or inflammation, comprising administering to the subject an effective amount of any TACI / BCMA chimera or its fusion protein or fusion protein dimer or pharmaceutical composition or drug combination described herein. In some embodiments, the disease is a disease associated with abnormal expression (increased concentration) or activity of BAFF and / or APRIL or BAFF / APRIL heterotrimers, for example, compared to a sample (e.g., blood or serum) of a healthy individual.

[0247] In some embodiments, the B cell or autoantibody-related disease or immune system disease is a B cell or autoantibody-mediated disease, such as a B cell or autoantibody-mediated autoimmune disease. In some embodiments, the B cell-mediated disease is a disease associated with B cell proliferation or abnormal activation, such as an autoimmune disease of B cell proliferation or abnormal activation. In some embodiments, the autoantibody-mediated disease refers to the production of unwanted autoantibodies due to abnormal proliferation or activation of B cells, or the production of abnormal autoantibodies.

[0248] In some embodiments, the disease associated with B cell expansion is a disease in which B cells proliferate abnormally (e.g., an autoimmune disease), for example, compared to a sample (e.g., blood or serum) of a healthy individual. In some embodiments, the disease mediated by B cells or autoantibodies is caused by excessive production of autoantibodies against self-antigens or abnormal production of autoantibodies due to abnormal activation of B cells.

[0249] In one aspect, the present invention relates to a method of inhibiting B cell proliferation in an individual, comprising administering to the subject an effective amount of any TACI / BCMA chimera described herein, or a fusion protein or fusion protein dimer thereof.

[0250] In some embodiments, the treatment of the disease will benefit from inhibition of BAFF and / or APRIL or BAFF / APRIL heterotrimer-related signaling pathways. In some embodiments, the treatment of the disease will benefit from inhibition of B cell proliferation or lymphocyte proliferation or inhibition of autoantibodies.

[0251] In some embodiments, the autoimmune disease includes but is not limited to lupus, such as systemic lupus erythematosus, rheumatoid arthritis, chronic kidney disease such as IgA nephropathy (IgAN) or membranous nephropathy, Sjögren's syndrome, myasthenia gravis, idiopathic thrombocytopenic purpura (ITP), warm antibody autoimmune hemolytic anemia (wAIHA), multiple sclerosis (MS), coronary heart disease (CAD), or thyroid eye disease.

[0252] The TACI / BCMA chimera or its fusion protein or fusion protein dimer of the present invention (and pharmaceutical compositions comprising the same, optionally with additional therapeutic agents) can be administered by any suitable method, including parenteral administration, intrapulmonary administration and intranasal administration, and, if required for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Depending on whether the medication is short-term or long-term to a certain extent, it can be administered by any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various medication schedules are contemplated herein, including, but not limited to, single administration or multiple administration at multiple time points, bolus administration, and pulse infusion.

[0253] For the prevention or treatment of disease, the appropriate dosage of the TACI / BCMA chimera or its fusion protein or fusion protein dimer of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the administration is for preventive or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient as a single treatment or over a series of treatments.

[0254] On the other hand, the present invention also provides the use of the TACI / BCMA chimera or its fusion protein or fusion protein dimer or pharmaceutical composition or drug combination of the present invention in the preparation of a drug for the aforementioned method (e.g., for treating the B cell or autoantibody-related diseases or immune system diseases (e.g., autoimmune diseases) or inflammation).

[0255] On the other hand, the present invention also provides the TACI / BCMA chimera of the present invention or its fusion protein or fusion protein dimer or pharmaceutical composition or pharmaceutical combination, which is used for therapy, such as for the aforementioned method (e.g., for treating the B cell or autoantibody-related diseases or immune system diseases (e.g., autoimmune diseases) or inflammation) or use.

[0256] In another aspect, the present invention also provides the use of the TACI / BCMA chimera or its fusion protein or fusion protein dimer, or pharmaceutical composition or drug combination of the present invention for the aforementioned methods (e.g., for treating diseases associated with B cells or autoantibodies, or immune system diseases (e.g., autoimmune diseases), or inflammation). The following examples are described to assist in understanding the present invention. The examples are not intended to, and should not be interpreted in any way, as limiting the scope of protection of the present invention.

[0257] These and other aspects and embodiments of the present invention are described in the accompanying drawings (a brief description of the drawings follows) and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may be made by those skilled in the art. Example

[0258] Example 1: TACI analysis and modification

[0259] The full-length TACI contains 293 amino acid residues, and there are protease cleavage sites at multiple locations in the extracellular domain, which makes the full-length TACI very easy to break after extracellular expression. According to reports, there are two cysteine-rich domains (CRDs) in the TACI extracellular domain, namely CRD1 and CRD2. The CRD2 domain alone has high binding activity with ligands. Therefore, in this application, we selected the CRD2 domain composed of amino acid residues 68-110 of TACI and the Fc portion of the human antibody IgG1 to construct a fusion protein (SEQ ID NO: 5), thereby improving the expression and fragmentation problem of TACI.

[0260] Because TACI proteins are prone to aggregation in neutral solutions, to further improve TACI's stability, we used Discovery Studio software to analyze surface hydrophobic groups, charged groups, and druggability risk sites (e.g., sites that pose aggregation risk) based on the crystal structure of TACI CRD2 (PDB ID: 1XU1). The results showed that amino acids 69, 72, 73, 74, 77, 78, 85, 102, and / or 103 of the TACI molecule pose potential aggregation risks. We also found that the protein structure of BCMA (6-42, PDB ID: 1XU2), a member of the TACI family, shares high structural homology with TACI, with essentially identical ligand-binding sites. Furthermore, the N-termini and C-termini of both proteins are not involved in ligand binding (Figure 1). More importantly, the Discovery Studio software analysis revealed that BCMA has a balanced surface charge distribution, making its aggregation risk significantly lower than that of TACI. Therefore, we designed a series of chimeric molecules of the extracellular domain of BCMA and TACI. On the basis of retaining the TACI and ligand binding sites, the N-terminus of TACI was replaced with the corresponding sequence of BCMA, and at the same time, the N-terminus and C-terminus of TACI were replaced with the corresponding sequence of BCMA and / or some of the druggability risk sites were replaced with amino acids to reduce the area of ​​the surface charge groups and hydrophobic groups of the TACI protein and reduce the aggregation risk of TACI. At the same time, ALPN-303 (US11274140B2) and Telitacicept (RC-18, CN101323643B) were constructed as controls. The sequence of wild-type TACI (uniprot: O14836, aa1-293) is shown in SEQ ID NO: 1 in this application. The sequence of wild-type BCMA (uniprot: Q02223, aa1-184) is shown in SEQ ID NO: 2 in this application.

[0261] Expression plasmid construction

[0262] Different TACI or TACI / BCMA chimeras were linked to the Fc portion of human IgG1 via GSGGGGS (SEQ ID NO: 39) and constructed into the pCDNA3.1 vector. In addition, ALPN-303 and Telitacicept (RC-18) for control were also constructed into the pCDNA3.1 vector and used to express the following proteins listed in Table 1:

[0263] Table 1. TACI or TACI / BCMA chimeric fusion proteins

[0264] The specific sequence information of the above protein molecules can be found in the sequence listing.

[0265] Expression and purification of fusion proteins

[0266] The vector containing the gene encoding the fusion protein was transfected into HEK293 cells using chemical transfection. Cultured HEK293 cells were transiently transfected using the chemical transfection reagent PEI according to the manufacturer's protocol.

[0267] First, prepare the expression plasmid DNA and transfection reagent in a clean bench. Add 5 mL of Opti-MEM medium (Gibco catalog number: 31985-070) to a 50 mL centrifuge tube, add 50 μg of the corresponding plasmid DNA, filter the Opti-MEM medium containing the plasmid through a 0.22 μm filter, then add 150 μg of PEI (1 g / L) and let it stand for 20 minutes. Gently pour the DNA / PEI mixture into 45 mL of HEK293 cells and mix thoroughly. After incubation at 37°C, 8% CO2 for 20 hours, add VPA to a final concentration of 2 mM and 2% (v / v) Feed, and continue incubation for 6 days.

[0268] After cell culture, the cell culture fluid was centrifuged at 13,000 rpm for 20 minutes, and the supernatant was collected and purified using prepacked Hitrap MabSelect Sure (GE, 11-0034-95) columns. The procedure was as follows: Before purification, the column was equilibrated with 5 column volumes of equilibration buffer (PBS buffer (Gibco, Cat. No. 70011-044)). The collected supernatant was passed through the column, and the column was then washed with 10 column volumes of equilibration buffer to remove nonspecifically bound proteins. The column was then rinsed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.3), and the eluate was collected. 60 μL of Tris (2M Tris) was added to each 1 mL of eluate, and the column was exchanged into PBS using ultrafiltration concentrators (MILLIPORE, Cat. No. UFC901096). The concentration was then determined. 100 μg of purified protein was taken and the concentration was adjusted to 1 mg / mL. The protein purity was determined using a gel filtration column SW3000 (TOSOH Product No.: 18675).

[0269] Purification results for TACI or TACI / BCMA chimeric fusion proteins and control samples are shown in Table 2 below. Expression levels of TACI or TACI / BCMA chimeric fusion proteins in HEK293 cells were all greater than 100 mg / mL, with most chimeric fusion proteins expressed at levels comparable to those in the control samples. The purity of TACI or TACI / BCMA chimeric fusion protein samples was greater than 95%, comparable to that of ALPN-303 and significantly superior to that of RC-18.

[0270] Table 2. Expression and purity of TACI or TACI / BCMA chimeric fusion proteins and control samples

[0271] Example 2: Determination of affinity between TACI or TACI / BCMA chimeric fusion protein and ligand

[0272] The equilibrium dissociation constant (KD) between the TACI or TACI / BCMA chimeric fusion protein and its ligand was determined using ForteBio interferometry.

[0273] ForteBio affinity determination was performed according to existing methods (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs, 2013. 5(2): p. 270-8). The affinity of candidate molecules for BAFF and APRIL was measured: the sensor was equilibrated offline in assay buffer for 20 minutes, followed by online monitoring for 120 seconds to establish a baseline. The candidate molecules were loaded onto an AHC sensor (Sartorius, 18-5060) for ForteBio affinity measurement. The loaded sensor was placed in a solution containing 100 nM BAFF or APRIL until plateau phase, after which the sensor was transferred to assay buffer for at least 2 minutes for association and dissociation rate measurements. Kinetic analysis was performed using a 1:1 binding model.

[0274] The experimental results are shown in Table 3. Compared with the control samples, the affinity of all TACI or TACI / BCMA chimeric fusion proteins to BAFF and APRIL was significantly better than that of RC-18 and comparable to that of ALPN-303.

[0275] Table 3. Affinity of TACI or TACI / BCMA chimeric fusion protein for BAFF and APRIL

[0276] Example 3: In vitro functional assay of TACI / BCMA chimeric fusion protein

[0277] Example 3.1 TACI / BCMA chimeric fusion protein blocks the binding of BAFF to IM-9 cells.

[0278] Samples (ALPN-303, RC-18, and TACI / BCMA chimeric fusion protein) were serially diluted two-fold starting at 200 nM and then three-fold diluted in FACS buffer, creating a total of 12 concentration gradients. Biotin Human BAFF (Acro, Cat#BAF-H82Q2-200 ug) was diluted to 0.2 μg / mL; the diluted samples were mixed with diluted Biotin human BAFF at a 1:1 ratio and incubated at 37°C for 30 min. IM-9 cells (Nanjing Kebai, Cat#CB60276) were counted and diluted to 2 × 10 6 Cells were centrifuged at 500 g for 5 minutes and the cell culture medium was removed. The incubated samples were then added to the U-shaped plate and the cells were resuspended in 100 μL / well. The cells were incubated at 4°C for 30 minutes. The supernatant was removed after centrifugation at 500 g for 5 minutes, and the cells were washed twice with FACS buffer. The cells were centrifuged at 500 g for 5 minutes, and the FACS buffer was removed. 50 μL of PE Streptavidin secondary antibody (BD, Cat#554061) (1:200 dilution in FACS buffer) was added to each well. The cells were incubated at 4°C in the dark for 30 minutes. The supernatant was removed after centrifugation at 500 g for 5 minutes, and the cells were washed three times with FACS buffer. The cells were resuspended in 200 μL of FACS buffer and analyzed by flow cytometry.

[0279] The results showed that ALPN-303, RC-18, and the TACI / BCMA chimeric fusion protein all inhibited the binding of BAFF to IM-9 cells. However, the TACI / BCMA chimeric fusion protein had significantly superior inhibitory activity against BAFF binding to IM-9 cells to RC-18 (IC50: 12.47±3.73nM) and comparable inhibitory activity to ALPN-303 (IC50: 1.72±0.48nM) (Table 4).

[0280] Example 3.2 TACI / BCMA chimeric fusion protein blocks the binding of APRIL to IM-9 cells.

[0281] Samples (ALPN-303, RC-18, and TACI / BCMA chimeric fusion protein) were serially diluted threefold in FACS buffer starting at 200 nM, for a total of 12 concentration gradients. Biotin Human APRIL (Acro, Cat# APL-H82F5-200UG) was diluted to 0.2 μg / mL; the diluted samples were mixed with diluted Biotin Human APRIL at a 1:1 ratio and incubated at 37°C for 30 min. IM-9 cells were counted and diluted to 2 × 10 6 Cells were centrifuged at 500 g for 5 minutes and the cell culture medium was removed. The incubated samples were then added to the U-shaped plate and the cells were resuspended in 100 μL / well. The cells were incubated at 4°C for 30 minutes. The supernatant was removed after centrifugation at 500 g for 5 minutes, and the cells were washed twice with FACS buffer. The cells were centrifuged at 500 g for 5 minutes, and the FACS buffer was removed. 50 μL of PE Streptavidin secondary antibody (BD, Cat#554061) (1:200 dilution in FACS buffer) was added to each well. The cells were incubated at 4°C in the dark for 30 minutes. The supernatant was removed after centrifugation at 500 g for 5 minutes, and the cells were washed three times with FACS buffer. The cells were resuspended in 200 μL of FACS buffer and analyzed by flow cytometry.

[0282] The results showed that ALPN-303, RC-18, and the TACI / BCMA chimeric fusion protein all inhibited the binding of APRIL to IM-9 cells. However, the TACI / BCMA chimeric fusion protein was significantly superior to RC-18 in inhibiting the binding of APRIL to IM-9 cells (IC50: 5.89±2.92nM) and comparable to the inhibitory activity of ALPN-303 (IC50: 0.35±0.29nM) (Table 4).

[0283] Example 3.3 TACI / BCMA chimeric fusion protein blocks BAFF / APRIL-induced NF-κB reporter signaling in Jurkat cells overexpressing human TACI

[0284] The cDNA encoding human TACI was cloned into the pLenti-IRES-puro vector (Invitrogen), and then lentiviral transfection was used to generate NF-κB reporter Jurkat cells (Jiman Bio, Cat#GM-C07855) overexpressing human TACI (i.e., TACI / NF-κB reporter Jurkat cells). A serial dilution assay was then used to assess the ability of the chimeric TACI / BCMA-Fc fusion protein to block BAFF / APRIL-induced TACI / NF-κB reporter Jurkat signaling.

[0285] Briefly, samples (ALPN-303, RC-18, and TACI / BCMA chimeric fusion protein) were serially diluted threefold starting at 1200 nM in RPMI-1640 + 10% FBS medium, for a total of 10 concentration gradients. Human BAFF (Acro, Cat#BAF-H52D4) + Human APRIL (Acro, Cat#APL-H52D1) was diluted to 20 nM in RPMI-1640 + 10% FBS medium; the diluted samples were mixed with a 20 nM human BAFF / APRIL mixture at a 1:1 ratio and incubated at 37°C for 30 min. TACI / NF-κB Reporter Jurkat cells were counted and diluted to 1 × 10 6 Cells were cultured at a concentration of 50 μL / well in a white flat-bottom 96-well plate. The incubated sample was then added to a white flat-bottom 96-well plate containing cells and resuspended at 50 μL / well. The cells were incubated at 37°C for 22 hours. After the incubation period, the luminescent signal produced by the cells was detected using the Bio-Lite Luciferase Assay System (Vazyme, #DD1201-03).

[0286] The results showed that ALPN-303, RC-18, and the TACI / BCMA chimeric fusion protein all inhibited BAFF / APRIL-TACI-induced NF-κB Reporter Jurkat signaling. However, the inhibitory activity of the TACI / BCMA chimeric fusion protein against BAFF / APRIL-TACI-induced NF-κB Reporter Jurkat signaling was significantly superior to that of RC-18 (IC50: 23.55±8.47nM) and comparable to that of ALPN-303 (IC50: 6.40±2.02nM) (Table 4).

[0287] Example 3.4 TACI / BCMA chimeric fusion protein blocks BAFF / APRIL-induced NF-κB Reporter Jurkat signaling overexpressing human BCMA

[0288] The cDNA encoding human BCMA was cloned into the pLenti-IRES-Neo vector (Invitrogen), and then lentiviral transfection was used to generate NF-κB Reporter Jurkat cells (Jiman Bio, Cat#GM-C07855) overexpressing human BCMA (i.e., BCMA / NF-κB Reporter Jurkat cells). A serial dilution assay was then used to test the ability of the chimeric TACI / BCMA-Fc fusion protein to block BAFF / APRIL-induced BCMA / NF-κB Reporter Jurkat signaling.

[0289] Briefly, samples (ALPN-303, RC-18, and TACI / BCMA chimeric fusion protein) were serially diluted threefold starting at 2400 nM in RPMI-1640 + 10% FBS medium, for a total of 10 concentration gradients. Human BAFF (Acro, Cat#BAF-H52D4) + Human APRIL (Acro, Cat#APL-H52D1) was diluted to 20 nM in RPMI-1640 + 10% FBS medium; the diluted samples were mixed with a 20 nM human BAFF / APRIL mixture at a 1:1 ratio and incubated at 37°C for 30 min. BCMA / NF-κB reporter Jurkat cells were counted and diluted to 2 × 10 6 Cells were cultured at a concentration of 50 μL / well in a white flat-bottom 96-well plate. The incubated sample was then added to a white flat-bottom 96-well plate containing cells and resuspended at 50 μL / well. The cells were incubated at 37°C for 5 h. After the incubation period, the luminescent signal generated by the cells was detected using the Bio-Lite Luciferase Assay System (Vazyme, #DD1201-03).

[0290] The results showed that ALPN-303, RC-18, and TACI / BCMA chimeric fusion proteins all inhibited BAFF / APRIL-BCMA-induced NF-κB Reporter Jurkat signaling. However, the inhibitory activity of TACI / BCMA chimeric fusion protein against BAFF / APRIL-BCMA-induced NF-κB Reporter Jurkat signaling was significantly superior to that of RC-18 (IC50: 53.25±8.81nM) and comparable to that of ALPN-303 (IC50: 4.06±0.53nM) (Table 4).

[0291] Example 3.5. Detection of the ability of TACI / BCMA chimeric fusion protein to block spleen cell proliferation

[0292] Mouse spleen cells were harvested, lysed, and the cell density was adjusted to 2 × 10 6 / mL, 50μL was added to each well, and the cells were plated at 1x10 5 Plate 100 μg of ALPN-303, RC-18, and TACI / BCMA chimeric fusion protein in RPMI-1640 medium with 10% FBS. Serial three-fold dilutions were performed starting at 1200 nM in RPMI-1640 medium with 10% FBS, for a total of nine concentrations. Human BAFF (Acro, Cat# BAF-H52D4) was diluted to 50 ng / mL in RPMI-1640 medium with 10% FBS. The diluted samples were mixed with diluted human BAFF at a 1:1 ratio and incubated at 37°C for 30 min.

[0293] Control wells were set up: IgG group (using IgG (Equitech-Bio, Cat#SLH56) as sample + cells + BAFF antigen) and blank group (RPMI-1640 + 10% FBS) group (culture medium + cells).

[0294] 50 μL / well of the incubated mixture was added to the cells and incubated at 37°C for 72 h. After 72 h of incubation, cell proliferation was detected using CellCount-LiteTM Luminescent Cell Viabil (Vazyme, #DD1101-02).

[0295] IC50 values ​​were calculated by curve fitting based on the readings at different concentrations. The results showed that ALPN-303, RC-18, and the TACI / BCMA chimeric fusion protein all inhibited BAFF-induced spleen cell proliferation. However, the TACI / BCMA chimeric fusion protein exhibited significantly superior inhibitory activity against BAFF-induced spleen cell proliferation than RC-18 (IC50 of 4.98±0.95 nM), and was comparable to the inhibitory activity of ALPN-303 (IC50 of 0.49±0.06 nM) (Table 4).

[0296] Table 4. In vitro functional activities of TACI / BCMA chimeric fusion proteins

[0297] Example 3.6. Detection of the ability of TACI / BCMA chimeric fusion protein to inhibit B cell proliferation

[0298] PBMCs were revived (AllCell, Cat#FPB004F-C), and B cells were isolated using the EasySep™ Human B cell Enrichment kit (STEMCELL, Cat#19054). The cells were counted and the cell density was adjusted to 2×10 6 / mL, and rhIL-4 (R&D, Cat#204-IL-050) and 1 μg / mL Anti-human IgM (Jakson, Cat#109-006-129) were added to a final concentration of 20 ng / mL. Samples (ALPN-303, RC-18, and TACI / BCMA chimeric fusion protein) were serially diluted threefold starting at 2400 nM in RPMI-1640 medium supplemented with 10% FBS, for a total of nine concentration gradients.

[0299] Human BAFF (Acro, Cat#BAF-H52D4) and human APRIL (Acro, Cat#APL-H52D1) were diluted to 20 nM in RPMI-1640 medium supplemented with 10% FBS. The diluted sample was mixed with a 20 nM human BAFF / APRIL mixture at a 1:1 ratio and incubated at 37°C for 30 min. Control wells were set up: IgG (Equitech-Bio, Cat#SLH56) was used as the sample. 50 μL / well of this mixture was added to the cells and incubated at 37°C for 72 h. After 72 h of incubation, cell proliferation was assessed using a CellCount-Lite™ Luminescent Cell Viabil (Vazyme, #DD1101-02) and a dose-effect curve was constructed.

[0300] The results showed that both ALPN-303 and TACI / BCMA chimeric fusion proteins could effectively inhibit BAFF / APRIL-induced B cell proliferation (Figure 4). The inhibition rate in the figure was calculated as experimental group / control IgG group × 100%.

[0301] Example 4: Stability determination of TACI / BCMA chimeric fusion protein

[0302] The thermal stability of the TACI / BCMA chimeric fusion protein was evaluated by dynamic light scattering (DLS), differential scanning calorimetry (DSC), and accelerated stability.

[0303] Dynamic Light Scattering (DLS) to assess thermal stability

[0304] A protein solution (1 mg / mL) was centrifuged at 13,000 g / min for 5 minutes and then added to a sample plate. The changes in protein particle size during a continuous temperature increase were measured using a DLS acquisition time of 5 seconds, 5 acquisitions, and an experimental temperature range of 25-85°C. After the experiment, the changes in sample particle size with temperature were analyzed.

[0305] Differential Scanning Calorimetry (DSC) to evaluate thermal stability

[0306] Dilute the antibody mutant sample to 0.5-1 mg / mL with PBS. Degas the diluted sample and PBS buffer. Then add the sample to the left sample plate and add PBS buffer to the corresponding position in the right reference plate. Start at 30°C and equilibrate for 10 minutes. Heat at a rate of 1°C / min to a final temperature of 90°C.

[0307] The results of DLS and DSC experiments are shown in Table 5. The experimental results show that the initial aggregation temperature of TACI / BCMA chimeric fusion proteins 99017 and 99018 is similar to the protein denaturation temperature T m 1 and T m Both were significantly superior to the control RC18 and the unmodified TACI CRD2 molecule 99001, and their thermal stability was comparable to that of ALPN-303.

[0308] Table 5. Drugability of TACI / BCMA chimeric fusion proteins

[0309] Accelerated stability assessment of TACI / BCMA chimeric fusion protein

[0310] The TACI / BCMA chimeric fusion protein was also transferred to a PBS solution (pH 7.4) using an ultrafiltration tube at a concentration of 10 mg / ml. The solution was then placed in a 50°C incubator for accelerated stability testing. After 4 days, samples were collected for purity analysis (SEC and CE-SDS). The results are shown in Table 6.

[0311] Experimental results showed that after 4 days at 50°C, the TACI / BCMA chimeric fusion proteins 099017 and 099018 exhibited the best stability, maintaining purity above 95% by SEC and above 98% by non-reducing CE-SDS. These proteins exhibited significantly better stability than the control RC-18 and the unmodified TACI CRD2 molecule 99001, as well as the control ALPN-303.

[0312] Table 6. CE-SDS evaluation of the forced stability of TACI / BCMA chimeric fusion proteins

[0313] Example 5: In vivo efficacy of TACI / BCMA chimeric fusion protein

[0314] Example 5.1. Pharmacological efficacy of TACI / BCMA chimeric fusion protein in a mouse KLH immune model

[0315] C57BL / 6N mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were selected and intraperitoneally injected with 0.2 mg KLH (Solarbiol, Cat#KB160) containing Alum adjuvant (Thermo, Cat#77161) on days 0 and 12. This experiment set up a blank control group (Blank, without any treatment), a model control IgG group, a 99001 group, a 99006 group and a 99017 group, with 5-7 mice in each group (3 mice in the blank control group). The mice were intraperitoneally injected with drugs on the 4th and 11th days (the model control IgG group was injected with an equal amount of IgG antibody (purchased from Equitech-Bio, Cat#SLH56)). On the 20th day, the spleens of the mice were collected for cell detection of B cell changes and blood was collected for detection of IgA (Invitrogen, Cat#EMIGA), IgM (Invitrogen, Cat#88-50470-88) and IgG (Invitrogen, Cat#88-50400-88) in the serum.

[0316] The results showed that compared with the control IgG group, the drug-treated groups were able to inhibit the proliferation of KLH-immunized splenocytes and B cells in the spleen (Table 7) as well as the levels of IgA, IgM, and IgG in the serum (Table 8). By calculating the inhibition rate of the drug-treated groups relative to the IgG group, the results showed that the 99017 group (TACI / BCMA chimeric fusion protein) had a better inhibitory effect than the 99001 group (unmodified TACI CRD2 molecule) and the 99006 group (unmodified BCMACRD2 molecule). The above data suggest that the modified TACI / BCMA chimeric fusion protein is significantly superior to the TACI or BCMAWT CRD2 fragment in terms of B cell and antibody levels.

[0317] Table 7 Number of spleen cells and B cells in spleen (n=5-7 / group)

[0318] * This means that there is a significant difference between 99017 and 99001 ( ** p<0.01, t test).

[0319] # This means that there is a significant difference between 99017 and 99006 ( ## p<0.01, ttest).

[0320] Table 8 Levels of IgA, IgM and IgG in serum (n=5-7 / group)

[0321] * This means that there is a significant difference between 99017 and 99001 ( ** p<0.01, t test).

[0322] # This means that there is a significant difference between 99017 and 99006 ( ### p<0.001, ttest).

[0323] Example 5.2. Efficacy of TACI / BCMA chimeric fusion protein in mouse BAFF stimulation model

[0324] Balb / c mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were selected and injected intraperitoneally with 10 μg of human BAFF protein (Sino Biological, Cat#10056-HNCHA) on days 0, 1, 2, and 3. This experiment set up a blank control group (Blank, without any treatment), a model control IgG group, an RC-18 group, an ALPN-303 group, and a TACI / BCMA chimeric fusion protein group, a total of 5 groups, with 5-7 mice in each group (3 mice in the blank control group). The mice were intraperitoneally injected on days 0 and 2 (the model control IgG group was injected with an equal amount of IgG antibody (purchased from Equitech-Bio, Cat#SLH56)). On day 4, the spleens of the mice were collected for cell detection, and ELISA kits were used to detect IgA (Invitrogen, Cat#EMIGA), IgM (Invitrogen, Cat#88-50470-88), and IgG (Invitrogen, Cat#88-50400-88) in the serum. The results showed that TACI / BCMA chimeric fusion protein could effectively inhibit the proliferation of spleen cells and B cells in the spleen induced by BAFF (Table 9). At the same time, TACI / BCMA chimeric fusion protein could effectively reduce the levels of IgA, IgM and IgG in the serum induced by BAFF (Table 10).

[0325] Table 9 Number of spleen cells and B cells in spleen (n=5-7 / group)

[0326] Table 10 Levels of IgA, IgM and IgG in serum (n=5-7 / group)

[0327] Example 5.3. Pharmacological efficacy of TACI / BCMA chimeric fusion protein in a mouse KLH immune model

[0328] C57BL / 6N mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were selected and intraperitoneally injected with 0.2 mg KLH (Solarbiol, Cat#KB160) containing Alum adjuvant (Thermo, Cat#77161) on days 0 and 12. This experiment set up 5 groups, including blank control group, model control IgG group, RC-18 group, ALPN-303 group and TACI / BCMA chimeric fusion protein group (99017 and 99018, respectively), with 5-7 mice in each group (3 mice in the blank control group). The mice were intraperitoneally injected on the 4th and 11th days (the model control IgG group was injected with an equal amount of IgG antibody (purchased from Equitech-Bio, Cat#SLH56)). On the 20th day, the spleens of the mice were taken for cell detection and blood was collected for detection of IgA (Invitrogen, Cat#EMIGA), IgM (Invitrogen, Cat#88-50470-88) and IgG (Invitrogen, Cat#88-50400-88) in the serum.

[0329] The results showed that compared with the control IgG group, both drug-treated groups were able to inhibit the proliferation of KLH-induced splenocytes, spleen B cells (Figure 5), and different B cell subsets (Table 11). However, compared with the RC-18 group, the TACI / BCMA chimeric fusion protein group had a superior inhibitory effect; compared with ALPN-303, the TACI / BCMA chimeric fusion protein was more effective in inhibiting the proliferation of MZ cells, GC B cells, and plasma cells. At the same time, the TACI / BCMA chimeric fusion protein effectively reduced the levels of IgA, IgM, and IgG in the serum produced by KLH immunization (Figure 6, Table 12).

[0330] Table 11 The proportion of different B cell subsets in total spleen cells (n = 5-7 / group)

[0331] * There is a significant difference between 99017 and ALPN-303 ( * p<0.05, ** p<0.01, ttest).

[0332] Table 12 Levels of IgA, IgM and IgG in serum (n=5-7 / group)

[0333] Example 5.4. Pharmacokinetic study of TACI / BCMA chimeric fusion protein in mice

[0334] The pharmacokinetics of the chimeric fusion protein were tested by intravenous injection (IV) in mice. Nine BALB / c mice weighing approximately 20 g were injected intravenously with 10 mg / kg of the test molecule. Blood samples were collected from the eye sockets 5 minutes, 0.5 hours, 2 hours, 6 hours, and on days 2, 4, 7, 14, and 21 after a single dose. Serum was collected by centrifugation after natural coagulation.

[0335] The antibody drug concentration in serum was determined as follows: Human APRIL-his protein (Acro Biosystems, Cat#APL-H52D1) was diluted to 1 μg / mL in coating solution (dissolve one packet of carbonate powder (Thermo, Cat#23282) in 400 mL of ultrapure water, mix thoroughly, and dilute to 500 mL). 100 μL was added to each well of a 96-well microtiter plate (Thermo, Cat#442404) and incubated overnight at 4°C. The coating solution was discarded, and the plates were washed three times with 1× PBST. 200 μL of blocking solution (2% BSA in PBST) was added to each well and blocked at room temperature for 1 hour. The blocking solution was discarded, and the plates were washed three times with 1× PBST. Diluted mouse serum was then added and incubated at room temperature for 2 hours. The plate was discarded and washed five times with 1× PBST. Diluted Goatx-human IgG-Fc-HRP (BETHYL, Cat#A80-104P) was added at 100 μL per well and incubated at room temperature for 1 hour. The plate was then discarded and washed five times with 1× PBST. 100 μL of TMB colorimetric solution (Solebol, Cat#PR1200) was added to each well and color was developed for 5-10 minutes. The color was stopped by adding 50 μL of stop solution (Solebol, Cat#C1058) per well. OD450nm and OD620nm values ​​were read on a microplate reader. The plasma concentrations of the test molecule in mice (10 mg / kg) at different time points are shown in Figure 7. Pharmacokinetic parameters were calculated using a non-compartmental model using Excel PK solver. Key drug exposure parameters (T1 / 2, Cmax, AUC0-t, AUC0-∞, CL, and Vss) are shown in Table 13. By analyzing the mouse pharmacokinetic parameters of the above-mentioned different molecules, the pharmacokinetics of TACI / BCMA chimeric fusion protein were significantly better than those of RC-18 and slightly better than those of ALPN-303.

[0336] Table 13 Pharmacokinetic parameters of T ACI / BCMA chimeric fusion protein in mice

[0337] Example 6. Toxicokinetic Study of TACI / BCMA Chimeric Fusion Protein in Cynomolgus Monkeys

[0338] Subcutaneous injection (sc) was performed in cynomolgus monkeys to test the TK of 99017 molecules. Two macaques (one male and one female) weighing approximately 3.5-4.5 kg were included in each dose group. Each group of macaques received five weekly subcutaneous injections of either 100 mg / kg or 200 mg / kg of the test molecule. Blood was collected 24 hours after the first dose (C1D1) and 24 hours after the fourth dose (C4D1). Blood was collected at 30 minutes, 2 hours, 6 hours, 10 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, and 168 hours. Serum was collected by centrifugation after natural coagulation.

[0339] The antibody drug concentration in serum was determined as follows: Human APRIL-his protein (Acro Biosystems, Cat# APL-H52D1) was diluted to 2 μg / mL in coating solution (dissolve one packet of carbonate powder in 400 mL of ultrapure water, mix well, and dilute to 500 mL). 100 μL was added to each well of a 96-well microtiter plate and incubated overnight at 4°C. The coating solution was discarded, and the plates were washed three times with 1× PBST. 300 μL of blocking solution (5% SM-1 in PBST) was added to each well and blocked at room temperature for 2 hours. The blocking solution was discarded, and the plates were washed three times with 1× PBST. Then, 100 μL of diluted cynomolgus macaque serum was added to each well and incubated at room temperature for 2 hours. The plate was discarded and washed three times with 1× PBST. Add diluted Human IgG Heavy and Light chain Monkey-Adsorbed Antibody (BETHYL, Cat#A80-319P), 100 μL per well, and incubate at room temperature for 1 hour. Discard the solution in the ELISA plate and wash 5 times with 1×PBST. Add 100 μL of TMB colorimetric solution to each well, color for 5-10 minutes, and add 100 μL of stop solution to each well to stop. Read the OD450nm and OD620nm values ​​on a microplate reader. The changes in blood drug concentrations at different time points after C1D1 and C4D1 in crab-eating monkeys given different doses of the test molecule are shown in Figure 8. The relevant kinetic parameters were calculated using the non-compartmental model of Phoenix WinNonlin 8.4, and the main drug exposure parameters (AUC0-t, Cmax, Tmax, T 1 / 2 , CI / F) are shown in Table 14.

[0340] The results are shown in Figure 8. The kinetics of 99017 at 200 mg / kg on C1D1 and C4D1 were higher than those at 100 mg / kg, showing a dose-dependent manner. The kinetic parameters calculated for different doses and different dosing frequencies were as follows: T 1 / 2 About 5-5.9 days, ALPN-303 disclosed 150mg / kg T1 / 2 The half-life of 99017 is 2.9 days (Dillon S, Evans L, Lewis K, et al. ALPN-303, an Enhanced, Potent Dual BAFF / APRIL Antagonist Engineered by Directed Evolution for the Treatment of Systemic Lupus Erythematosus (SLE) and Other B Cell-Related Diseases. Arthritis Rheumatol. 2021; 73 (suppl 9); https: / / www.alpineimmunesciences.com / wp-content / uploads / 2021 / 12 / ALPN-303_ACR-2021.pdf). Therefore, the half-life of 99017 is significantly better than that of ALPN-303.

[0341] Table 14. Toxicokinetic parameters in cynomolgus monkeys

[0342] Example 7. Pharmacokinetic and pharmacodynamic studies of TACI / BCMA chimeric fusion protein and YTE molecule in cynomolgus monkeys

[0343] The pharmacokinetics and pharmacodynamics of 99017 and 99025 were tested in cynomolgus monkeys using intravenous injection (IV). Three macaques were administered intravenously (3.5-4.5 kg) to each group. Each monkey received a 9 mg / kg dose of the test molecule. Blood samples were collected 5 minutes, 2 hours, 6 hours, 12 hours, 24 hours, 72 hours, 144 hours, 216 hours, 312 hours, 432 hours, 480 hours, 624 hours, 816 hours, and 984 hours after the single dose. After natural coagulation, the blood was centrifuged and serum was collected for the determination of antibody drug concentration. Blood samples were collected before a single dose and at 6, 9, 12, 18, 24, 32, and 40 days after administration. After natural coagulation, the blood was centrifuged to obtain serum. Serum IgA (Cat#05219205190), IgM (Cat#05220726190), and IgG (Cat#05220718190) were measured using Roche Diagnostic immunoglobulin assay kits. The percentage decrease in serum immunoglobulin levels at different time points after administration compared to pre-dose levels was calculated, as shown in Figure 10. The results showed that the TACI / BCMA chimeric fusion protein effectively reduced BAFF-induced serum IgA, IgM, and IgG levels.

[0344] The antibody drug concentration in serum was determined as follows: Human APRIL-his protein (Acro Biosystems, Cat# APL-H52D1) was diluted to 2 μg / mL in coating solution (dissolve one packet of carbonate powder in 400 mL of ultrapure water, mix thoroughly, and dilute to 500 mL). 100 μL was added to each well of a 96-well microtiter plate and incubated overnight at 4°C. The coating solution was discarded, and the plates were washed three times with 1× PBST. 300 μL of blocking solution (5% SM-1 in PBST) was added to each well and blocked for 2 hours at room temperature. The blocking solution was discarded, and the plates were washed three times with 1× PBST. Then, 100 μL of diluted cynomolgus macaque serum was added to each well and incubated for 2 hours at room temperature. The plate was discarded and washed three times with 1× PBST. Add diluted Human IgG Heavy and Light chain Monkey-AdsorbedAntibody (BETHYL, Cat#A80-319P), 100 μL per well, and incubate at room temperature for 1 hour. Discard the solution in the ELISA plate and wash 5 times with 1×PBST. Add 100 μL of TMB colorimetric solution to each well, develop for 5-10 minutes, and add 100 μL of stop solution to each well to stop. Read the OD450nm and OD620nm values ​​using a microplate reader. The changes in blood drug concentration at different time points in cynomolgus monkeys given the test molecule (administered to cynomolgus monkeys at 9 mg / kg) are shown in Figure 9.

[0345] The results are shown in Figures 9 and 10. Compared with 99017, 99025 has longer pharmacokinetics and better pharmacodynamics.

[0346] At the same time, compared with the data disclosed by ALPN-303 (Dillon S, Evans L, Lewis K, et al. ALPN-303, an Enhanced, Potent Dual BAFF / APRIL Antagonist Engineered by Directed Evolution for the Treatment of Systemic Lupus Erythematosus (SLE) and Other B Cell-Related Diseases. Arthritis Rheumatol. 2021; 73 (suppl 9); Lawrence S.Evans,Katherine E.Lewis,Daniel DeMonte,et al.Povetacicept,an Enhanced Dual APRIL / BAFF Antagonist That Modulates B Lymphocytes and Pathogenic Autoantibodies for the Treatment of Lupus and Other B Cell–Related Autoimmune Diseases. https: / / doi.org / 10.1002 / art.42462), 99017 and 99025 had better Cmax and AUC (Table 15), and the pharmacodynamics lasted longer.

[0347] Table 15. Comparison of Cmax and AUC in cynomolgus monkey pharmacokinetics

[0348] Sequence Listing:

Claims

1. A TACI / BCMA chimera comprising a chimeric protein in which the N-terminus and / or C-terminus of a functional fragment of the extracellular domain ECD of TACI is replaced by the N-terminus and / or C-terminus of BCMA.

2. The TACI / BCMA chimera of claim 1, comprising the following structure: BCMAN terminal amino acid-TACI portion, wherein the TACI portion is the extracellular domain ECD of TACI lacking the N-terminus or a functional fragment thereof, or BCMAN terminal amino acid-TACI part-BCMAC terminal amino acid, wherein the TACI part is the extracellular domain ECD of TACI lacking the N-terminus and C-terminus or a functional fragment thereof.

3. The TACI / BCMA chimera of claim 1 or 2, wherein the functional fragment of the extracellular region ECD of TACI comprises CRD2 of TACI and does not comprise CRD1 or any fragment of CRD1; Preferably, the CRD2 is the amino acid sequence of TACI corresponding to positions 71-104 shown in SEQ ID NO: 1, and the CRD1 is the amino acid sequence of TACI corresponding to positions 34-66 shown in SEQ ID NO: 1; Optionally, the extracellular ECD functional fragment of TACI further comprises a partial stem region of TACI and / or any amino acid sequence corresponding to positions 68-70 of SEQ ID NO: 1; Preferably, the functional fragment of the extracellular region ECD of TACI is or comprises the following fragment of TACI corresponding to the amino acid sequence shown in SEQ ID NO: 1: amino acid residues 68-110; for example, The extracellular domain ECD functional fragment of TACI comprises (i) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 31, 32 or 33; (ii) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 33 and has an amino acid sequence of Y102D; (iii) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 32 and has K77E, F78Y and Y102D; (iv) the amino acid sequence shown in SEQ ID NO: 31, 32 or 33; or (v) consisting of the sequence of any one of (i) to (iv).

4. The TACI / BCMA chimera of any one of claims 1 to 3, wherein the N-terminus of the functional fragment of the extracellular domain ECD of TACI refers to the N-terminal amino acid of TACI preceding amino acid Y79 of SEQ ID NO: 1; and / or The C-terminus of the extracellular ECD functional fragment of TACI refers to the C-terminal amino acid after the 99th amino acid, the 100th amino acid, the 101st amino acid, the 102nd amino acid, the 103rd amino acid, the 104th amino acid, the 105th amino acid, the 106th amino acid, the 107th amino acid, the 108th amino acid, the 109th amino acid or the 110th amino acid of TACI corresponding to SEQ ID NO: 1, preferably the C-terminal amino acid after the 99th or 105th amino acid.

5. The TACI / BCMA chimera of any one of claims 1 to 4, wherein the N-terminal amino acids of BCMA are selected from the amino acid sequence of BCMA corresponding to positions 1-13, 2-13, 3-13, 4-13, 5-13, 6-13, or 7-13 of SEQ ID NO: 2; preferably, the N-terminal amino acid sequence of BCMA comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 40-46; and / or The C-terminal amino acid sequence of BCMA is the amino acid sequence of BCMA corresponding to positions 37-47, 37-46, 37-45, or 37-44 of SEQ ID NO: 2, or the amino acid sequence corresponding to positions 43-44 or 43-46 of SEQ ID NO: 2; Optionally, the C-terminal amino acid of BCMA comprises a mutation, such as a substitution, that improves binding affinity, increases stability and / or improves druggability, for example, the mutation is a mutation at position 39 and / or position 42, such as a substitution, such as N42A or N42Q or R39D or N42A-R39D; Preferably, the C-terminal amino acid sequence of BCMA comprises the amino acid sequence shown in any one of SEQ ID NOs: 47-55, or consists of the amino acid sequence.

6. The TACI / BCMA chimera of any one of claims 1 to 5, wherein the TACI ECD functional fragment comprises an amino acid site mutation that reduces aggregation risk, such as a mutation such as a substitution at a druggability risk site, for example, the druggability risk site is selected from amino acids 69, 72, 73, 74, 77, 85, 102, or 103 corresponding to SEQ ID NO: 1, preferably, the amino acid at the druggability risk site is mutated to A or D, for example, the mutation is Y102D; Preferably, the TACI portion comprises the amino acid sequence shown in any one of SEQ ID NOs: 35, 31-34 or 36-38, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identical thereto, or consists of the amino acid sequence.

7. The TACI / BCMA chimera of any one of claims 1-6, wherein the TACI / BCMA chimera comprises the amino acid sequence set forth in any one of SEQ ID NOs: 73, 59-72, or 74-80, or comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identical thereto.

8. A fusion protein comprising the TACI / BCMA chimera according to any one of claims 1 to 7, and an Fc region; Preferably, the Fc region is human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc; Optionally, the Fc region comprises one or more of the following mutations: (i) Lysine K deleted at the C-terminus (K447del); (ii) mutations that reduce effector function mediated by the Fc region; (iii) mutations that reduce binding to Fcγ receptors, such as L234A / L235A mutations or L234A / L235E or L234A / L235E / G237A mutations; (iv) mutations that enhance the binding of the Fc fragment to FcRn, such as M252Y / S254T / T256E and / or M428L / N434S; For example, the Fc region comprises (i) the amino acid sequence of SEQ ID NO: 81 or 82, or an amino acid sequence that is at least 90% identical thereto, such as 95%, 96%, 97%, 99% or more identical thereto; (ii) the amino acid sequence shown in SEQ ID NO: 83 or 84, or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity thereto, optionally lacking the C-terminal lysine; (iii) an amino acid sequence as set forth in any one of SEQ ID NOs: 85-88 and 100-103, or an amino acid sequence that is at least 90% identical thereto, such as 95%, 96%, 97%, 98%, 99% or more identical thereto; (iv) an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 85 or 87 and comprises the mutations L234A / L235E / G237A; (v) an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 86 or 88 and comprises the mutations L234A / L235E / G237A and a C-terminal deleted lysine; (vi) an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 100 or 102 and comprises the mutations L234A / L235E / G237A and M252Y / S254T / T256E; (vii) an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 101 or 103 and comprises the mutations L234A / L235E / G237A, M252Y / S254T / T256E, and a C-terminal deleted lysine; or (viii) consisting of the amino acid sequence described in any one of (i) to (vii) above.

9. The fusion protein of claim 8, wherein the TACI / BCMA chimera is fused to Fc directly or through a linker, preferably, the C-terminus of the chimera is fused to the N-terminus of Fc directly or through a linker; Optionally, the linker is selected from (GSGGGGS) n , (GS) n 、(GSGGS) n 、(GGGGS) n or (GGGS) n , wherein n is an integer of at least 1, such as 1, 2, 3, 4 or 5; for example (GSGGGGS) n , wherein n=1-3, for example, the linker is the amino acid sequence shown in SEQ ID NO:

39.

10. The fusion protein of claim 8 or 9, wherein the fusion protein (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 10, 5-9 and 11-28; or (ii) comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 10, 5-9, and 11-28 A fusion protein dimer comprising a first monomer and a second monomer, wherein the first and second monomers respectively comprise the fusion protein chain according to any one of claims 8 to 10, preferably the first monomer and the second monomer are identical.

12. A polynucleotide encoding the TACI / BCMA chimera according to any one of claims 1 to 7, the fusion protein according to any one of claims 8 to 10, or the fusion protein dimer according to claim 11.

13. An expression vector comprising the polynucleotide according to claim 12, for example, the expression vector is a pCDNA expression vector, for example, a pCDNA3.1 expression vector.

14. A host cell comprising the polynucleotide of claim 12 or the expression vector of claim 13.

15. A method for preparing a TACI / BCMA chimera or a fusion protein or fusion protein dimer thereof, wherein the method comprises culturing the host cell of claim 14 under conditions suitable for the expression of the TACI / BCMA chimera or a fusion protein or fusion protein dimer thereof, and optionally recovering the TACI / BCMA chimera or a fusion protein or fusion protein dimer thereof from the host cell (or host cell culture medium).

16. A pharmaceutical composition comprising the TACI / BCMA chimera according to any one of claims 1 to 7, the fusion protein according to any one of claims 8 to 10, or the fusion protein dimer according to claim 11, and optionally a pharmaceutically acceptable excipient.

17. A drug combination comprising the TACI / BCMA chimera of any one of claims 1 to 7, the fusion protein of any one of claims 8 to 10, or the fusion protein dimer of claim 11, and one or more other therapeutic agents (e.g., cytokines, hormones, cytotoxic agents or inhibitors (e.g., cytostatic agents that affect T cell and / or B cell proliferation), antibodies or small molecule drugs, or immunomodulators (e.g., immunosuppressants)).

18. A method for preventing or treating a disease in a subject, such as a B cell or autoantibody-related disease or an immune system disease (such as an autoimmune disease) or inflammation, the method comprising administering to the subject the TACI / BCMA chimera of any one of claims 1 to 7, the fusion protein of any one of claims 8 to 10, the fusion protein dimer of claim 11, the pharmaceutical composition of claim 16, or the pharmaceutical combination of claim 17. For example, the B cell or autoantibody-related disease or immune system disease is an autoimmune disease mediated by B cells or autoantibodies; Optionally, the B cell or autoantibody-related disease or immune system disease or inflammation is a disease in which B cells in an individual abnormally proliferate or activate compared to a sample of a healthy individual, such as an autoimmune disease; Preferably, the disease is lupus such as systemic lupus erythematosus, rheumatoid arthritis, chronic kidney disease such as IgA nephropathy (IgAN) or membranous nephropathy, Sjögren's syndrome, myasthenia gravis, idiopathic thrombocytopenic purpura (ITP), warm antibody autoimmune hemolytic anemia (wAIHA), multiple sclerosis (MS), coronary heart disease (CAD) or thyroid eye disease; Optionally, the administration further comprises co-administration of one or more other therapeutic agents (e.g., cytokines, hormones, cytotoxic agents or inhibitors (e.g., cytostatic agents that affect T cell and / or B cell proliferation), antibodies or small molecule drugs or immunomodulators (e.g., immunosuppressants)).

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