Anti-april antibody, Anti-april / BAFF bispecific antibody, and preparation and use thereof

Abstract

Provided are an anti-APRIL antibody and an anti-APRIL / BAFF bispecific antibody. The bispecific antibody comprises an anti-APRIL antibody or an antigen-binding fragment thereof and an anti-BAFF antibody or an antigen-binding fragment thereof. The anti-APRIL / BAFF bispecific antibody can simultaneously block the binding of dual ligands APRIL and BAFF to BCMA, and also has excellent stability, thereby providing a new solution for preventing or treating APRIL and / or BAFF-related diseases.

Description

Anti-APRIL antibodies, anti-APRIL / BAFF bispecific antibodies and their preparation and application Technical Field

[0001] This invention relates to the field of antibody drug technology, specifically to anti-APRIL antibody, anti-APRIL / BAFF bispecific antibody, their preparation methods and applications. Background Technology

[0002] IgA nephropathy (IgAN) is the most common primary glomerular disease worldwide and also the most common glomerular disease in my country, accounting for 30%-40% of primary glomerulonephritis and affecting the kidney health of approximately 5 million people in my country. It is characterized by the deposition of large amounts of IgA aggregates within the glomeruli, and generally occurs in people under 35 years of age. IgAN can manifest as various clinical syndromes, including asymptomatic hematuria or proteinuria, chronic glomerulonephritis, rapidly progressive glomerulonephritis, or nephrotic syndrome. More than one-third of patients progress to end-stage renal disease (ESRD) within 20 years of onset, making it one of the leading causes of chronic kidney disease (CKD) and kidney failure.

[0003] A new member of the tumor necrosis factor (TNF) family, the proliferation-inducing ligand (APRIL), plays a crucial role in the pathogenesis of IgAN. Patients with the highest serum APRIL levels have a 10-fold higher risk of developing kidney failure than other patients. APRIL is a clinically validated target for IgAN. Another member of the TNF family, B-cell activation factor (BAFF), can induce B-cell proliferation and differentiation both in vivo and in vitro. BAFF overexpression in mice also leads to elevated serum IgA levels, suggesting its involvement in the induction of IgAN pathogenesis. Previous studies have shown elevated levels of both BAFF and APRIL in the serum of IgAN patients, and the degree of elevation is correlated with disease severity.

[0004] Therefore, it is necessary to develop antibodies that simultaneously target both BAFF and APRIL for the treatment, prevention, and diagnosis of IgAN and other diseases with similar mechanisms. Although fusion proteins targeting these two targets are already in development, unmet clinical needs remain. Summary of the Invention

[0005] The purpose of this invention is to provide an anti-APRIL antibody and an anti-APRIL / BAFF bispecific antibody, providing a new solution for the clinical prevention or treatment of APRIL and / or BAFF-related diseases.

[0006] The advantages of this invention are that the developed anti-APRIL / BAFF bispecific antibody can simultaneously block the binding of the two ligands APRIL and BAFF to their corresponding receptors, and its blocking effect is superior to that of the positive control sibeprenlimab; at the same time, the bispecific antibody also has excellent stability. It is reasonable to expect that it will have biological activity that inhibits the APRIL and / or BAFF signaling pathways, such as inhibiting B cell activation, and has better drug-like properties, thereby it can be developed into a clinical drug for the prevention or treatment of APRIL and / or BAFF-related diseases, such as autoimmune diseases.

[0007] In a first aspect of the invention, an anti-APRIL antibody or an antigen-binding fragment thereof is provided, wherein the heavy chain variable region comprises HCDR1-3 selected from the group consisting of:

[0008] 1) SEQ ID NO: 22-24;

[0009] 2) SEQ ID NO: 29-31;

[0010] Its light chain variable region includes LCDR1-3 selected from the following group:

[0011] 3) SEQ ID NO: 25, 27, 28 or SEQ ID NO: 26, 27, 28;

[0012] 4) SEQ ID NO: 32, 33, 35 or SEQ ID NO: 32, 34, 36.

[0013] In an optional example,

[0014] 1) The heavy chain variable region is selected from SEQ ID NO: 1, 3, 7, 9; and / or

[0015] 2) The light chain variable region is selected from SEQ ID NO: 2, 5, 8, 11; and / or

[0016] 3) The heavy chain variable region is selected from amino acid sequences that have at least 99%, 95%, 90%, 85%, or 80% identity with SEQ ID NO: 1, 3, 7, or 9; and the light chain variable region is selected from amino acid sequences that have at least 99%, 95%, 90%, 85%, or 80% identity with SEQ ID NO: 2, 5, 8, or 11.

[0017] In an alternative example, the heavy chain variable region and / or light chain variable region of 3) above have the same function as SEQ ID NO: 1, 3, 7, 9 or SEQ ID NO: 2, 5, 8, 11.

[0018] In one alternative embodiment, the anti-APRIL antibody comprises a heavy chain amino acid sequence selected from SEQ ID NO: 4 or 10, and a light chain amino acid sequence selected from SEQ ID NO: 6 or 12.

[0019] In a second aspect of the invention, an anti-APRIL / BAFF bispecific antibody is provided, comprising an anti-APRIL antibody or an antigen-binding fragment thereof and an anti-BAFF antibody or an antigen-binding fragment thereof.

[0020] In one alternative embodiment, it comprises the anti-APRIL antibody or its antigen-binding fragment as described in the first aspect; and / or, an anti-BAFF nanobody or its antigen-binding fragment having the following characteristics.

[0021] 1) The heavy chain variable region contains HCDR1-3 selected from the group consisting of SEQ ID NO: 37, 38, 39, or SEQ ID NO: 40, 38, 41; or,

[0022] 2) The heavy chain variable region is selected from SEQ ID NO: 13, 14, 42, 43, or selected from amino acid sequences having at least 99%, 95%, 90%, 85%, or 80% identity with SEQ ID NO: 13, 14, 42, 43. Preferably, the heavy chain variable region has the same function as the heavy chain variable regions selected from SEQ ID NO: 13, 14, 42, 43.

[0023] In one alternative example, it comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 15 and a light chain with an amino acid sequence as shown in SEQ ID NO: 6; or it comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 16 and a light chain with an amino acid sequence as shown in SEQ ID NO: 12.

[0024] In an alternative embodiment, the antibody or its antigen-binding fragment further comprises an Fc domain.

[0025] In one alternative, the antibody comprises a monomer or a dimer formed from monomers, the dimer being either homologous or heterologous.

[0026] In one alternative example, the antibody or its antigen-binding fragment is monovalent, bivalent, or polyvalent.

[0027] In one alternative example, the antibody or its antigen-binding fragment is selected from full-length antibodies, nanobodies, single-chain antibodies, scFv, Fv, Fd, Fab, F(ab')2 or F(ab').

[0028] In an alternative example, the Fc domain is selected from IgG1, IgG2, IgG3, or IgG4.

[0029] In an alternative example, the Fc domain contains a mutated amino acid. Examples include existing Fc mutation methods that can improve half-life or stability.

[0030] In an alternative example, the Fc structural domain is connected to the N-terminus or C-terminus of the heavy chain variable region.

[0031] In one alternative example, the connection is made via a peptide linker.

[0032] In one alternative, the antibody or its antigen-binding fragment is camel-derived, chimeric, human, or fully human.

[0033] In a third aspect of the invention, a polynucleotide molecule is provided, said polynucleotide molecule encoding the aforementioned anti-APRIL antibody or its antigen-binding fragment or anti-APRIL / BAFF bispecific antibody.

[0034] In an alternative example, preferably, the polynucleotide molecule is selected from SEQ ID NO: 18-21.

[0035] In a fourth aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide molecule described in the third aspect.

[0036] In one alternative example, the expression vector is a virus or a plasmid.

[0037] In one alternative example, the expression vector is selected from the group consisting of: pcDNA3.4, pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF, or pCHO 1.0.

[0038] In a fifth aspect of the invention, a host cell is provided, the host cell containing the expression vector described in the fourth aspect or integrating the polynucleotide molecule described in the third aspect.

[0039] In one alternative example, the host cell is selected from the group consisting of COS, CHO, NSO, sf9, sf21, DH5α, BL21(DE3), TG1, 293F, or 293E cells.

[0040] In a sixth aspect of the present invention, a method for preparing the anti-APRIL antibody or its antigen-binding fragment as described in the first aspect, or the anti-APRIL / BAFF bispecific antibody as described in the second aspect, is provided, the preparation method comprising the following steps:

[0041] 1) Culture the host cells described in the fifth aspect to obtain a culture containing the anti-APRIL antibody or its antigen-binding fragment described in the first aspect, or the anti-APRIL / BAFF bispecific antibody described in the second aspect;

[0042] 2) Isolate or recover the anti-APRIL antibody or its antigen-binding fragment or anti-APRIL / BAFF bispecific antibody from the culture;

[0043] 3) Optionally, purify and / or modify the anti-APRIL antibody or its antigen-binding fragment or anti-APRIL / BAFF bispecific antibody obtained in step 2).

[0044] In a seventh aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0045] The anti-APRIL antibody or its antigen-binding fragment as described in the first aspect, or the anti-APRIL / BAFF bispecific antibody as described in the second aspect, and a conjugate selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0046] In an eighth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising an effective amount of the anti-APRIL antibody or its antigen-binding fragment as described in the first aspect, or the anti-APRIL / BAFF bispecific antibody as described in the second aspect, or the immunoconjugate as described in the seventh aspect, and a pharmaceutically acceptable carrier or excipient.

[0047] In one alternative embodiment, the dosage form of the pharmaceutical composition includes a gastrointestinal dosage form or a parenteral dosage form.

[0048] In one alternative example, the parenteral dosage form includes intravitreal injection, intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, intracavitary injection, or respiratory administration.

[0049] In a ninth aspect of the invention, there is provided the use of the anti-APRIL antibody or its antigen-binding fragment as described in the first aspect, or the anti-APRIL / BAFF bispecific antibody as described in the second aspect, or the immunoconjugate as described in the seventh aspect, or the pharmaceutical composition as described in the eighth aspect, said use including any of the following applications:

[0050] 1) To prepare medicines for the prevention and / or treatment of diseases associated with BAFF and / or APRIL;

[0051] 2) To prepare drugs for the prevention and / or treatment of autoimmune diseases, inflammatory diseases or tumors;

[0052] 3) To prepare drugs for the prevention and / or treatment of BAFF and / or APRIL-mediated autoimmune diseases, inflammatory diseases or tumors;

[0053] 4) Prepare drugs that reduce immune response.

[0054] In a tenth aspect of the invention, a method for preventing / treating a disease is provided, the method comprising administering to a subject in need an anti-APRIL antibody or its antigen-binding fragment as described in the first aspect, or an anti-APRIL / BAFF bispecific antibody as described in the second aspect, or an immunoconjugate as described in the seventh aspect, or a pharmaceutical composition as described in the eighth aspect, wherein the disease is selected from:

[0055] 1) Diseases associated with BAFF and / or APRIL;

[0056] 2) Autoimmune diseases, inflammatory diseases, or tumors;

[0057] 3) BAFF and / or APRIL-mediated autoimmune diseases, inflammatory diseases, or tumors;

[0058] 4) Diseases with abnormal immune responses.

[0059] In one alternative example, the aforementioned diseases are selected from inflammatory arthritis (such as rheumatoid arthritis), systemic lupus erythematosus, myasthenia gravis, primary Sjögren's syndrome, kidney disease, neuromyelitis optica, optic neuritis, multiple sclerosis, B-cell neoplasms, connective tissue diseases, interstitial lung diseases, systemic scleroderma, diffuse scleroderma, immune thrombocytopenic purpura, myositis, polyangiitis, autoimmune hepatitis, chronic graft-versus-host disease, bronchial diseases, and rheumatic diseases. Preferably, the kidney diseases include IgAN (IgA nephropathy), lupus nephritis, and membranous glomerulonephritis.

[0060] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0061] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in this application, both endpoints of each numerical range and any value between the two endpoints may be used. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, any prior art methods, equipment, and materials similar to or equivalent to those described in the embodiments of this application may be used to implement this application, based on the prior art's mastery of the art and the description in this application. Unless otherwise specified, the instruments, materials, and reagents used in the embodiments are obtained through conventional means.

[0062] the term

[0063] Antibody

[0064] In this invention, the term "nanobody" refers to a single-domain antibody (VHH) consisting of only one variable domain of heavy chain, which is the smallest antigen-binding fragment with complete function.

[0065] The term "antigen-binding fragment" refers to a fragment derived from an antibody that can bind to an antigenic epitope, including but not limited to scFv, Fv, Fd, Fab, F(ab')2, or F(ab').

[0066] In this invention, the term "full-length antibody" refers to a heterotetraglycoprotein of approximately 150,000 Daltons with identical structural characteristics, comprising variable and constant regions, and consisting of two identical heavy chains (HC) and two identical light chains (LC). Each heavy chain has a heavy chain variable region (VH) at one end, followed by a heavy chain constant region, which consists of three domains: CH1, CH2, and CH3. Each light chain has a light chain variable region (VL) at one end and a light chain constant region at the other end, which includes a domain CL. The light chain constant region pairs with the CH1 domain of the heavy chain constant region, and the light chain variable region pairs with the heavy chain variable region. The constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). The heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant region includes κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of an antibody are covalently linked by interpeptide disulfide bonds formed between their hinge regions.

[0067] In this invention, the term "variable" refers to the fact that certain portions of the variable region in an antibody differ in sequence, resulting in the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the heavy chain and light chain variable regions, called complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called frame regions (FRs). The variable regions of the natural heavy and light chains each contain four FR regions, which are generally β-sheet configurations, linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are closely packed together through the FR regions and together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The CDRs of the heavy chain variable region (VH) and the light chain variable region (VL) are called HCDRs and LCDRs, respectively.

[0068] In this invention, the term "humanized antibody" refers to an antibody whose complementarity-determining region (CDR) is derived from a non-human species (e.g., rodents), while the remaining portions of the antibody molecule (including the frame region (FR) and constant region) are derived from humans. The frame region (FR) residues can be modified to maintain binding affinity. In this invention, the term "chimeric antibody" refers to an antibody whose variable region is derived from a non-human species (e.g., rodents), while the constant region is derived from humans.

[0069] In this invention, the term "frame region (FR)" refers to the portion of the antibody variable region excluding the hypervariable region, where the amino acid composition and sequence changes relatively little. The light and heavy chains of the antibody each have four FRs, referred to as FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Preferably, the FRs of this invention are human antibody FRs or their derivatives, wherein the derivatives of the human antibody FRs are substantially identical to naturally occurring human antibody FRs, i.e., the sequence homology reaches at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Those skilled in the art, upon knowing the amino acid sequence of the CDR, can determine the frames FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H sequences.

[0070] In this invention, the terms "antibody" and "binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. Typically, antibodies bind at a rate of less than approximately 10... -7 M, for example, less than approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 An antibody binds to an antigen with an equilibrium dissociation constant (KD) of M or less. The term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity between the antibody and the antigen can be determined using surface plasmon resonance (SPR) in a BIACORE instrument or using ELISA to determine the relative affinity of the antibody to the antigen.

[0071] Those skilled in the art can modify the antibodies or antigen-binding fragments of the present invention using techniques well known in the art, such as adding, deleting, and / or substituting one or more amino acid residues, thereby further improving or optimizing the performance (e.g., affinity) of the antibodies or antigen-binding fragments, and obtaining the modified results using conventional assay methods.

[0072] The antibodies or antigen-binding fragments of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, or any combination of the above substances.

[0073] The amino acid sequences or protein (peptide) structures in this invention are given in order from the amino terminus to the carboxyl terminus.

[0074] In this invention, the "-" in the protein (peptide) structure represents a peptide bond.

[0075] In this invention, "sequence identity" is a measure of identity between proteins at the amino acid level and between nucleic acids at the nucleotide level. Protein sequence identity can be determined by comparing the amino acid sequence at a given position in each sequence during alignment. Methods for sequence alignment are well known in the art, including BESTFIT, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. The BLAST algorithm calculates the sequence identity percentage and performs a statistical analysis of the similarity between two sequences. Software used for BLAST analysis is publicly available from the NCBI (National Center for Biotechnology Information) website. In some cases, the sequence identity percentage can be determined as the percentage of amino acid residues (or nucleotide residues) in the candidate sequence that are identical to those in the reference sequence after alignment and the introduction of gaps (if necessary) to achieve the maximum sequence identity percentage. Those skilled in the art can determine suitable algorithms and parameters for sequence alignment.

[0076] Nucleic acid encoding and expression vector

[0077] This invention also provides polynucleotide molecules encoding the aforementioned antibodies or their antigen-binding fragments. The polynucleotides of this invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. In this invention, the term "expression vector" refers to a vector carrying an expression cassette for expressing a specific target protein or other substance, such as plasmids, viral vectors (e.g., adenovirus, retrovirus), bacteriophages, yeast plasmids, or other vectors. For example, conventional expression vectors in the art containing suitable regulatory sequences, such as promoters, terminators, enhancers, etc., include, but are not limited to: viral vectors (e.g., adenovirus, retrovirus), plasmids, bacteriophages, yeast plasmids, or other vectors. The expression vectors preferably include pDR1, pcDNA3, pDHFR, or pTT5.

[0078] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0079] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0080] In this invention, the term "host cell" refers to any type of host cell conventional in the art, as long as it enables the vector to replicate stably and the carried polynucleotide molecules to be effectively expressed. The host cells include prokaryotic expression cells and eukaryotic expression cells, and preferably include: COS, CHO, NSO, sf9, sf21, DH5α, BL21(DE3), TG1, 293F, or 293E cells.

[0081] The preparation of antibodies using biological materials through DNA recombination and molecular cloning techniques is well-known in the field. For example, the variable region of the heavy chain or the variable region of the light chain is spliced ​​to its corresponding antibody constant region sequence. After amplifying the full-length fragments of the antibody heavy and light chains, the fragments are introduced into a suitable vector, which is then transfected into host cells. The cells are cultured, the cell culture is collected, and finally purified to obtain the antibody.

[0082] APRIL

[0083] APRIL, a proliferation-inducing ligand, is a type II transmembrane protein that binds to two TNF receptors—B cell maturation antigen (BCMA) and the transmembrane activator and calmotropic ligand interactor (TACI). APRIL plays a role in B cell signaling and drives B cell proliferation and survival in humans and mice. APRIL is primarily expressed by immune cell subsets such as monocytes, macrophages, dendritic cells, neutrophils, B cells, and T cells, many of which also express BAFF. In addition, APRIL can also be expressed by non-immune cells, such as osteoclasts, epithelial cells, and various tumor tissues; high levels of APRIL mRNA expression have been found in primary human tumors such as colon cancer and lymphoma. Due to its role in B cell biology, APRIL also plays a role in many autoimmune and inflammatory diseases. Therefore, antagonizing APRIL is a therapeutic strategy for many such diseases. For example, Otsuka Pharmaceutical developed Sibeprenlimab, a humanized IgG2 monoclonal antibody against APRIL, which is under investigation for IgA nephropathy. Clinical data suggest that Sibeprenlimab can significantly reduce the urine protein-to-creatinine ratio (uPCR), slow the rate of renal function decline, and reduce Gd-IgA1 and APRIL levels.

[0084] BAFF

[0085] B-cell activating factor BAFF is a cytokine belonging to the TNF ligand superfamily, acting as a ligand for receptors BAFF-R, TACI, and BCMA. The interaction between BAFF and its receptor triggers signals crucial for the formation and maintenance of B cells. Excessive BAFF levels can lead to abnormally high antibody production, exhibiting autoimmunity. Autoimmune diseases, including lupus and rheumatoid arthritis, are caused by excessive levels of BAFF in the body. BAFF can exist in three forms: membrane-bound (mbBAFF), soluble trimeric BAFF (sBAFF), and a polymeric form composed of 60 BAFF monomers.

[0086] "Diseases associated with BAFF and / or APRIL" refer to diseases or symptoms caused by, mediated by, or abnormally expressed by BAFF and / or APRIL, such as autoimmune diseases, inflammatory diseases, tumors, or cancers. Autoimmune diseases include, but are not limited to, lupus nephritis, glomerulonephritis, systemic lupus erythematosus (SLE), rheumatoid arthritis, connective tissue diseases, interstitial lung diseases, systemic scleroderma, immune thrombocytopenic purpura, multiple sclerosis (MS), myositis, polyangiitis, IgA nephropathy, myasthenia gravis, primary Sjögren's syndrome, neuromyelitis optica, and optic neuritis; tumors or cancers include, but are not limited to, non-Hodgkin's lymphoma, B-cell chronic lymphocytic leukemia, and multiple myeloma.

[0087] Belimumab (belimumab, ) is a marketed anti-BAFF human IgG1γ monoclonal antibody. Developed by GSK, it is marketed for indications including lupus erythematosus and lupus nephritis. Other indications under development include connective tissue diseases, interstitial lung diseases, immune thrombocytopenic purpura, systemic scleroderma, diffuse scleroderma, autoimmune hepatitis, bronchial diseases, leukemia, nephrotic syndrome, idiopathic membranous nephropathy, rheumatic diseases, and chronic graft-versus-host disease.

[0088] Pharmaceutical Compositions and Applications

[0089] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the aforementioned antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 4-8, preferably about 5-8, 5-7, or 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavitary injection.

[0090] In this invention, the term "pharmaceutical composition" refers to a pharmaceutical formulation composition in which the antibody or its antigen-binding fragment can be combined with a pharmaceutically acceptable carrier to achieve a more stable therapeutic effect. These formulations can ensure the conformational integrity of the amino acid core sequence of the protein disclosed in this invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation).

[0091] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody or antigen-binding fragment thereof described above, and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the antibody or antigen-binding fragment thereof of the present invention can also be used with other therapeutic agents.

[0092] When using a pharmaceutical composition, a safe and effective amount of the antibody or its antigen-binding fragment is administered to a mammal. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0093] In this invention, the term "effective amount" refers to the amount or dose in which the pharmaceutical composition of this invention produces the desired effect in a treated individual after administration to a subject, including improvement of the individual's condition. The term "subject" includes, but is not limited to, mammals, such as humans, non-human primates, rats, and mice. Attached Figure Description

[0094] Figure 1A shows the binding activity of the anti-APRIL monoclonal antibody APRIL-hu30H7 to human APRIL;

[0095] Figure 1B shows the binding activity of the anti-APRIL monoclonal antibody APRIL-hu112A2B6 with human APRIL;

[0096] Figure 1C shows the blocking activity of the anti-APRIL monoclonal antibody APRIL-hu30H7 against the binding of APRIL to BCMA;

[0097] Figure 1D shows the blocking activity of the anti-APRIL monoclonal antibody APRIL-hu112A2B6 against the binding of APRIL to BCMA;

[0098] Figure 2 shows the cell-blocking activity of the anti-APRIL monoclonal antibody against APRIL and TACI;

[0099] Figure 3A shows the binding activity of anti-BAFF / APRIL bispecific antibodies with different structures to human BAFF;

[0100] Figure 3B shows the blocking activity of the anti-BAFF / APRIL bispecific antibody against BCMA and APRIL;

[0101] Figure 3C shows the bridging activity of the anti-BAFF / APRIL bispecific antibody with BAFF and APRIL;

[0102] Figure 4A shows the binding activity of the anti-BAFF / APRIL bispecific antibody to human BAFF;

[0103] Figure 4B shows the binding activity of the anti-BAFF / APRIL bispecific antibody to human APRIL;

[0104] Figure 5 shows the cell-blocking activity of the anti-BAFF / APRIL bispecific antibody against APRIL and BAFF and BCMA;

[0105] Figure 6A shows the UPLC-SEC pattern of the anti-BAFF / APRIL bispecific antibody BAFF-hu1A2-APRIL-hu30H7;

[0106] Figure 6B shows the UPLC-SEC pattern of the anti-BAFF / APRIL bispecific antibody BAFF-hu3B2-APRIL-hu112A2B6;

[0107] Figure 6C shows the iCIEF pattern of the anti-BAFF / APRIL bispecific antibody BAFF-hu1A2-APRIL-hu30H7;

[0108] Figure 6D shows the iCIEF pattern of the anti-BAFF / APRIL bispecific antibody BAFF-hu3B2-APRIL-hu112A2B6;

[0109] Figures 7A and 7B show the affinity of the anti-BAFF / APRIL bispecific antibody BAFF-hu1A2-APRIL-hu30H7 for human ARPIL and BAFF, respectively.

[0110] Figures 8A-8C show the blocking activities of the anti-BAFF / APRIL bispecific antibody BAFF-hu1A2-APRIL-hu30H7 against APRIL and BCMA, APRIL and TACI, and BAFF and BCMA, respectively.

[0111] Figures 9A-9C show that the anti-BAFF / APRIL bispecific antibody BAFF-hu1A2-APRIL-hu30H7 inhibits APRIL, BAFF, and APRIL and BAFF simultaneously mediated downstream BCMA receptor signaling.

[0112] Figures 10A and 10B show the affinity of the anti-BAFF / APRIL bispecific antibody BAFF-hu1A2-APRIL-hu30H7 for FcRn under acidic and neutral conditions, respectively; and

[0113] Figures 11A-11D show the levels of serological markers and autoantibodies after B cell activation, respectively. Detailed Implementation

[0114] The sequence of the present invention is shown in the table below:

[0115] The antibody sequences of this invention all adopt the Kabat system numbering rules.

[0116] Experimental materials

[0117] Balb / c mice: Brand: Vital Rivers, strain code 211.

[0118] pcDNA 3.4: Brand Thermo Fisher, product number A14697.

[0119] HEK-293F: Brand: Thermo Fisher, Product No.: A14527.

[0120] 0.45μm filter: brand Millipore, part number SLHV013SL.

[0121] Hitrap Mabselect Sure affinity chromatography column: Brand: Sitopan, Product No. 11003493.

[0122] Waters MassPREP™ Micro Desalting Column: Brand: Waters, Model: 186004032.

[0123] E. coli Top 10 competent cells: Brand: Shenggong, Product No.: B528412.

[0124] Endotoxin-free plasmid extraction kit: Brand: Tiangen, Product No.: DP117.

[0125] DNA Purification and Recovery Kit: Brand: Tiangen, Product No.: DP214.

[0126] ClonExpress™ II One Step Cloning Kit: Brand: Novizan, Product No.: C112.

[0127] HSDNA Polymerase: Brand: Takara, Product Code: R010B.

[0128] Coating solution: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L with double distilled water.

[0129] PBST: PBS + 0.05% Tween 20.

[0130] Tween 20: Brand Aladdin, item number T104863.

[0131] ELISA blocking solution: PBST + 1% BSA.

[0132] BSA: Brand Shenggong, Item No. A60332.

[0133] TMB: Brand BD Biosciences, catalog number 555214.

[0134] Termination solution: 2M sulfuric acid solution.

[0135] Mouse anti-Flag tag - HRP antibody: Brand: Beyotime, Product No.: AF2855.

[0136] Recombinant human BCMA protein: Brand: Kaika Biotechnology, Product No.: BCM-HM217.

[0137] Recombinant human BAFF protein: Brand: Kaika Biotechnology, Product No.: BAF-HM112.

[0138] Goat anti-human IgG (Fc specific)-HRP antibody: brand Merck, catalog number A0170.

[0139] Streptavidin HRP: Brand BD Biosciences, Product No. 554066.

[0140] Lipofectamine 3000 transfection reagent: Brand: Thermo Fisher, Product No.: L3000015.

[0141] BCMA NFκB-Luc HEK293 cells: Brand COBIOER, catalog number CBP74072.

[0142] ONE-Glo TM Luciferase Assay System: Brand Promega, Product No. E6120.

[0143] 0.25% Pancreatic Enzyme-EDTA: Brand GIBCO, Product No. 25200-072.

[0144] FBS: Brand GIBCO, Item No. 10099-141.

[0145] Pen Strep: Brand GIBCO, Product No. 1514022.

[0146] Sodium pyruvate: Brand GIBCO, Product No. 11360-070.

[0147] GlutaMAX: Brand GIBCO, Product No. 35050-061.

[0148] Goat anti-human FITC secondary antibody: Brand Thermo Fisher, Product No. MA1-10379.

[0149] Goat anti-human PE secondary antibody: Brand: Jackson ImmunoResearch, Product No. 109-115-098.

[0150] MTS / PMS: Brand Promega, Product No. G5421.

[0151] Example 1. Screening of APRIL-binding mouse monoclonal antibodies

[0152] 1. Preparation of antigens for immunization of mice

[0153] The amino acid sequence (SEQ ID NO: 17) of the human APRIL extracellular domain and TNC fusion protein (APRIL-TNC-FLAG) was obtained by fusing the human APRIL extracellular domain and TNC. The protein was expressed and used as an immunogen to immunize mice, stimulating the mice to produce antibodies with high affinity for the APRIL extracellular domain.

[0154] 2. Hybridoma preparation and screening

[0155] Lymphocytes and myeloma cells SP2 / 0 from the spleens of multiple mice were fused to form hybridoma cells. The affinity of the hybridoma supernatant for APRIL antigen was detected by ELISA, and the neutralizing activity of the hybridoma supernatant for APRIL was screened by ELISA blocking, identifying hybridoma clones with high ELISA binding and blocking activity.

[0156] Example 2. Construction and purification of anti-APRIL monoclonal antibody

[0157] Hybridoma clones were sequenced to obtain the heavy chain variable region amino acid sequence (SEQ ID NO: 1) and light chain variable region amino acid sequence (SEQ ID NO: 2) of anti-APRIL mouse monoclonal antibody 30H7. Homology comparison was performed between the heavy chain variable region of the anti-APRIL mouse monoclonal antibody 30H7 and the germline sequence of the heavy chain variable region of the human antibody. The corresponding CDRs were replaced, and a reversion mutation that maintains activity was introduced into the frame region to obtain the amino acid sequence of the humanized heavy chain variable region of 30H7 (SEQ ID NO: 3). This sequence was then linked to the human heavy chain constant region to obtain the APRIL-hu30H7 heavy chain amino acid sequence (SEQ ID NO: 4). Homology comparison was performed between the light chain variable region of the anti-APRIL mouse monoclonal antibody 30H7 and the germline sequence of the light chain variable region of the human antibody. The corresponding CDRs were replaced, and a reversion mutation that maintains activity was introduced into the frame region. In order to improve the chemical stability of the antibody, an N28S mutation was introduced into L-CDR1 to obtain the amino acid sequence of the humanized light chain variable region of 30H7 (SEQ ID NO: 5). This sequence was then linked to the human light chain constant region to obtain the APRIL-hu30H7 light chain amino acid sequence (SEQ ID NO: 6).

[0158] Hybridoma clones were sequenced to obtain the heavy chain variable region amino acid sequence (SEQ ID NO: 7) and light chain variable region amino acid sequence (SEQ ID NO: 8) of anti-APRIL mouse monoclonal antibody 112A2B6. Homology comparison was performed between the heavy chain variable region of the anti-APRIL mouse monoclonal antibody 112A2B6 and the germline sequence of the heavy chain variable region of the human antibody. The corresponding CDRs were replaced, and a reversion mutation to maintain activity was introduced into the frame region, yielding the amino acid sequence of the humanized heavy chain variable region of 112A2B6 (SEQ ID NO: 9). This sequence was then linked to the human heavy chain constant region to obtain the APRIL-hu112A2B6 heavy chain amino acid sequence (SEQ ID NO: 10). Homology comparison was performed between the light chain variable region of the anti-APRIL mouse monoclonal antibody 112A2B6 and the germline sequence of the light chain variable region of the human antibody. The corresponding CDRs were replaced, and a reversion mutation to maintain activity was introduced into the frame region. Furthermore, to improve antibody chemical stability, an N56S mutation was introduced at L-CDR2, and a C91S mutation was introduced at L-CDR3 to delete free cysteine, yielding the amino acid sequence of the humanized light chain variable region of 112A2B6 (SEQ ID NO: 9). NO: 11), and linked with the human light chain constant region to obtain the APRIL-hu112A2B6 light chain amino acid sequence (SEQ ID NO: 12).

[0159] The nucleotide sequence of the anti-APRIL monoclonal antibody was subcloned into the vector pcDNA3.4. The recombinant plasmid was extracted and co-transfected into 293F cells and / or CHO cells. After 5-7 days of cell culture, the culture medium was centrifuged at high speed, filtered through a 0.22 μm filter, and loaded onto a Hitrap Mabselect Sure affinity chromatography column. The protein was eluted in one step with 100 mM citric acid, pH 3.5 elution buffer. The target sample was recovered and dialyzed to PBS at pH 7.4 to obtain the purified anti-APRIL monoclonal antibody protein.

[0160] Example 3. Affinity detection of anti-APRIL monoclonal antibody

[0161] The binding activity of anti-APRIL monoclonal antibody to human APRIL was determined by ELISA. The specific detection method is as follows: Dilute human APRIL (APRIL-TNC-FLAG, SEQ ID NO: 17) with coating buffer to 1.2 μg / mL, and add 100 μL / well to the microplate. Incubate overnight at 4°C. Remove the coating buffer (remove any residual droplets with absorbent paper). Add 200 μL / well of blocking buffer to the microplate and incubate at room temperature for 2 hours. Remove the blocking buffer (remove any residual droplets with absorbent paper), and then dilute the anti-APRIL monoclonal antibody with blocking buffer to 200 nM. Perform 4-fold dilutions to create 12 concentration gradients, and add 100 μL / well sequentially to the blocked microplate. Incubate at 37°C for 1 hour. Wash the plate three times with PBST (remove any residual droplets with absorbent paper). Dilute the HRP-labeled goat anti-human Fc antibody 1:3000 with blocking buffer and add 100 μL / well to the plate. Incubate at 37°C for 30 minutes. Wash the plate three times with PBST (remove any residual droplets with absorbent paper). Add 100 μL / well of TMB and incubate at room temperature in the dark for 5 minutes. Add 50 μL / well of stop solution to stop the substrate colorimetric reaction. Read the OD value at 450 nm using a microplate reader and calculate the EC50. 50 .

[0162] The experimental results are shown in Figures 1A and 1B. Both the anti-APRIL monoclonal antibodies APRIL-hu30H7 and APRIL-hu112A2B6 can bind to human APRIL, EC... 50 The values ​​are 0.088 nM and 0.047 nM, respectively.

[0163] Simultaneously, the blocking activity of the anti-APRIL monoclonal antibody against the binding of APRIL to BCMA was determined using ELISA. The specific detection method is as follows: Dilute human BCMA with coating buffer to 200 ng / mL, add 100 μL / well to the microplate, and incubate overnight at 4°C. Remove the coating buffer (remove any residual droplets with absorbent paper), add 200 μL / well of blocking buffer to the microplate, and incubate at room temperature for 2 hours. Remove the blocking buffer (remove any residual droplets with absorbent paper), dilute human APRIL with blocking buffer to 50 ng / mL, and then dilute the anti-APRIL monoclonal antibody with this solution to 500 nM. Create 12 concentration gradients by 4-fold dilution, and add 100 μL / well sequentially to the blocked microplate, incubating at 37°C for 1 hour. Wash the plate three times with PBST (remove any residual droplets with absorbent paper). Dilute the HRP-labeled anti-Flag antibody 1:3000 with blocking buffer and add 100 μL / well to the plate. Incubate at 37°C for 30 minutes. Wash the plate three times with PBST (remove any residual droplets with absorbent paper). Add 100 μL / well of TMB and incubate at room temperature in the dark for 5 minutes. Add 50 μL / well of stop solution to stop the substrate colorimetric reaction. Read the OD value at 450 nm using a microplate reader and calculate the IC50.50 .

[0164] The results are shown in Figures 1C and 1D. Both the anti-APRIL monoclonal antibodies APRIL-hu30H7 and APRIL-hu112A2B6 can block the binding of APRIL to BCMA, IC50... 50 The values ​​are 1.223 nM and 1.927 nM, respectively.

[0165] Example 4. Cell-blocking activity of anti-APRIL monoclonal antibody against APRIL and TACI

[0166] The reporter gene drug target model TACI NFκB-Luc HEK293 cells can mimic the signal transduction process of the nuclear factor-activated B cell κ-light chain enhancement (NF-κB) cascade reaction after the binding of ligands APRIL and / or BAFF to TACI in vivo. When the reaction between the ligands and TACI is blocked, the effect of the blocking drug can be verified by fluorescence reaction.

[0167] Construction of the TACI NFκB-Luc HEK293 cell line: The packaged viral vector pLVX-TACI (gene number O14836-1) was reacted with NFκB-Luc HEK293 cells using Lipofectamine 3000 transfection reagent. The cells were cultured and passaged in antibiotic-containing medium until they reached stable growth to obtain the TACI NFκB-Luc HEK293 cell population. The cell population was then diluted to 2-3 cells / mL and added to 96-well plates for single-clone sorting to obtain the TACI NFκB-Luc HEK293 cell line. TACI NFκB-Luc HEK293 cells were diluted with culture medium at a concentration of 30,000 cells / 100 μL / well and added to 96-well plates. Simultaneously, human APRIL was diluted to 80 ng / mL with culture medium. Anti-APRIL monoclonal antibody and the positive control sibeprenlimab were then diluted to 50 nM with culture medium containing human APRIL. Nine concentration gradients were created by four-fold dilution and added to 96-well plates containing cells. After incubation at 37°C for 6 hours, ONE-Glo... TM The Luciferase Assay System was used for detection, and the Luminescence value was read using a microplate reader to calculate the IC50. 50 .

[0168] The results are shown in Figure 2. Both the anti-APRIL monoclonal antibodies APRIL-hu30H7 and APRIL-112A2B6 can block the binding of APRIL ligand to TACI, IC50. 50The concentrations were 0.956 nM and 0.946 nM, respectively. The positive control sibeprenlimab (Sheila A. sibeprenlimab, which neutralizes A PRoliferation Inducing Ligand (APRIL), as a new approach to treating IgA nephropathy[J], Expert Opin Biol Ther, 2024; 24:335-338) also showed significant blocking effects, with IC50 values ​​of 0.956 nM and 0.946 nM, respectively. 50 The concentration was 3.337 nM. However, the positive antibody showed a weaker blocking effect compared to the anti-APRIL monoclonal antibody.

[0169] Example 5. Construction and screening of anti-BAFF / APRIL bispecific antibodies

[0170] 5.1 Obtaining anti-BAFF nanobodies

[0171] Camels were immunized with human BAFF antigen (gene number Q9Y275-1, Ala134-Leu285). Camel serum was collected and its titer was detected by ELISA. After the titer was verified to be qualified, peripheral blood was collected from camels and peripheral blood mononuclear cells (PBMCs) were isolated to obtain B lymphocytes. Total RNA was extracted from PBMCs by TriZol method, and cDNA was prepared by reverse transcription. Antibody genes were amplified to prepare phage display antibody library. After multiple rounds of screening and titer identification, positive clone sequences were analyzed and grouped by HCDR3 to obtain camel-derived VHH domain BAFF-1A2 (SEQ ID NO: 42) and BAFF-3B2 (SEQ ID NO: 43).

[0172] The camel-derived chimeric anti-BAFF nanobody was humanized. The CDR loop structure of the VHH domain was determined. The closest homologous sequences for each V / J region of the VHH domain were found in the human phylogenetic sequence database. The human phylogenetic lineage that best matches the VHH domain and the minimum amount of reversion mutations were screened. The CDR region of the chimeric antibody was constructed onto the human backbone region. Using sequence and structural features, the amino acid positions in the backbone region that maintain the CDR function were determined. Reversion mutations were then performed on important sequence positions to optimize the amino acids at risk sites. The VHH domain amino acid sequences of anti-BAFF humanized nanobody 1A2 (SEQ ID NO: 13) and anti-BAFF humanized nanobody 3B2 (SEQ ID NO: 14) were obtained. The human Fc domain (SEQ ID NO: 44) was linked to the C-terminus of VHH through the adapter L1 (SEQ ID NO: 45) to obtain hBAFF-1A2-Fc and hBAFF-3B2-Fc.

[0173] The nucleotide sequence of the anti-BAFF nanobody was subcloned into the vector pcDNA3.4. The recombinant plasmid was extracted and co-transfected into 293F cells and / or CHO cells. After 5-7 days of cell culture, the culture medium was centrifuged at high speed, filtered through a 0.22 μm filter, and loaded onto a Hitrap Mabselect Sure affinity chromatography column. Protein was eluted in one step with 100 mM citric acid, pH 3.5 elution buffer. The target sample was recovered and dialyzed to pH 7.4 PBS. The purified humanized anti-BAFF nanobody was then subjected to ELISA affinity assay.

[0174] ELISA assay for the binding activity of humanized anti-BAFF nanobody to human BAFF: Dilute recombinant human BAFF to 200 ng / mL with coating buffer, add 100 μL / well to the plate, and incubate overnight at 4°C. Remove the coating buffer (remove any residual droplets with absorbent paper), add 200 μL / well of blocking buffer to the plate, and incubate at room temperature for 2 hours. Remove the blocking buffer (remove any residual droplets with absorbent paper), then dilute the humanized anti-BAFF nanobody and positive control Belimumab to 10 μg / mL with blocking buffer, and then perform 4-fold dilutions to form 12 concentration gradients. Add 100 μL / well sequentially to the blocked plate and incubate at 37°C for 1 hour. Wash the plate three times with PBST (remove any residual droplets with absorbent paper). Dilute the HRP-labeled goat anti-human Fc antibody 1:3000 with blocking buffer and add 100 μL / well to the plate. Incubate at 37°C for 30 minutes. Wash the plate three times with PBST (remove any residual droplets with absorbent paper). Add 100 μL / well of TMB and incubate at room temperature in the dark for 5 minutes. Add 50 μL / well of stop solution to stop the substrate colorimetric reaction. Read the OD value at 450 nm using a microplate reader and calculate the EC50.50 .

[0175] The results showed that humanized anti-BAFF nanobodies hBAFF-1A2-Fc, hBAFF-3B2-Fc, and the positive antibody Belimumab (reference: Christine K, Dehlia C, Laure W, et al. Inhibition of Membrane-Bound BAFF by the Anti-BAFF Antibody Belimumab[J]. FRONT IMMUNOL. 2018; 9:2698.) could all bind to human BAFF, EC 50 The values ​​are 0.035 nM, 0.038 nM, and 0.080 nM, respectively.

[0176] 5.2 Preparation of anti-BAFF / APRIL bispecific antibody

[0177] The VHH domains of 1A2 and / or 3B2 were ligated to the N-terminus and / or C-terminus of the APRIL-hu30H7 heavy chain via adapter L2 (SEQ ID NO: 46), and L234A / L235A mutations and M252Y / S254T / T256E mutations (SEQ ID NO: 51) were introduced into the Fc domain to obtain the amino acid sequence of the BAFF-hu1A2-APRIL-hu30H7 heavy chain (SEQ ID NO: 15). The nucleotide sequence of the anti-BAFF / APRIL bispecific antibody was subcloned into the vector pcDNA3.4, and the recombinant plasmid was extracted and co-transfected into 293F cells and / or CHO cells. After 5-7 days of cell culture, the culture medium was centrifuged at high speed, filtered through a 0.22 μm filter membrane, and then loaded into a Hitrap Mabselect Sure affinity chromatography column. The protein was eluted in one step with 100 mM citric acid and pH 3.5 elution buffer. The target sample was recovered and dialyzed to PBS at pH 7.4 to obtain purified anti-BAFF / APRIL bispecific antibody protein.

[0178] 5.3 Screening of anti-BAFF / APRIL bispecific antibodies

[0179] The binding activity of anti-BAFF / APRIL bispecific antibodies with different structures to human BAFF was determined by ELISA, with the anti-BAFF monoclonal antibody Belimumab used as a positive control. The specific detection method is as follows: Dilute recombinant human BAFF to 1 μg / mL with coating buffer, add 100 μL / well to the microplate, and incubate overnight at 4°C. Remove the coating buffer (remove any residual droplets with absorbent paper), add 200 μL / well of blocking buffer to the microplate, and incubate at room temperature for 2 hours. Remove the blocking buffer (remove any residual droplets with absorbent paper), then dilute the anti-BAFF / APRIL bispecific antibody and the positive control Belimumab to 200 nM with blocking buffer, creating 12 concentration gradients through 4-fold dilutions, and add 100 μL / well sequentially to the blocked microplate, incubating at 37°C for 1 hour. Wash the plate three times with PBST (remove any residual droplets with absorbent paper). Dilute the HRP-labeled goat anti-human Fc antibody 1:3000 with blocking buffer and add 100 μL / well to the plate. Incubate at 37°C for 30 minutes. Wash the plate three times with PBST (remove any residual droplets with absorbent paper). Add 100 μL / well of TMB and incubate at room temperature in the dark for 5 minutes. Add 50 μL / well of stop solution to stop the substrate colorimetric reaction. Read the OD value at 450 nm using a microplate reader and calculate the EC50. 50 .

[0180] As shown in Figure 3A, both BAFF-hu1A2-APRIL-hu30H7 and BAFF-hu3B2-APRIL-hu30H7, whose anti-BAFF-VHH domains are attached to the N-terminus of the APRIL-hu30H7 heavy chain, can bind to human BAFF, EC 50 The concentrations were 0.045 nM and 0.042 nM, respectively; the anti-BAFF-VHH domains of APRIL-hu30H7-BAFF-hu1A2 and APRIL-hu30H7-BAFF-hu3B2, which are connected to the C-terminus of the APRIL-hu30H7 heavy chain, can both bind to human BAFF, EC 50 The concentrations were 0.088 nM and 0.084 nM, respectively; the positive control Belimumab bound to human BAFF in EC... 50 The value was 0.052 nM, indicating that the anti-BAFF-VHH domain, when linked to the N-terminus of APRIL-hu30H7, binds more strongly to human BAFF and is superior to the positive control.

[0181] Simultaneously, the blocking activity against APRIL-BCMA binding after the anti-BAFF-VHH domain is linked to the N-terminus of the APRIL-hu30H7 heavy chain was determined by ELISA. The specific detection method is as follows: Dilute human BCMA to 200 ng / mL with coating buffer, add 100 μL / well to the microplate, and incubate overnight at 4°C. Remove the coating buffer (remove any residual droplets with absorbent paper), add 200 μL / well of blocking buffer to the microplate, and incubate at room temperature for 2 hours. Remove the blocking buffer (remove any residual droplets with absorbent paper), dilute human APRIL to 50 ng / mL with the blocking buffer, and then dilute the anti-BAFF / APRIL bispecific antibody and the positive control sibeprenlimab to 500 nM with this solution, creating 12 concentration gradients through 4-fold dilutions. Add 100 μL / well sequentially to the blocked microplate and incubate at 37°C for 1 hour. Wash the plate three times with PBST (remove any residual droplets with absorbent paper). Dilute the HRP-labeled anti-Flag antibody 1:3000 with blocking buffer and add 100 μL / well to the plate. Incubate at 37°C for 30 minutes. Wash the plate three times with PBST (remove any residual droplets with absorbent paper). Add 100 μL / well of TMB and incubate at room temperature in the dark for 5 minutes. Add 50 μL / well of stop solution to stop the substrate colorimetric reaction. Read the OD value at 450 nm using a microplate reader and calculate the EC50. 50 .

[0182] The results are shown in Figure 3B. The anti-BAFF-VHH domains linked to the N-terminus of the APRIL-hu30H7 heavy chain, including BAFF-hu1A2-APRIL-hu30H7 and BAFF-hu3B2-APRIL-hu30H7, as well as the positive control sibeprenlimab, effectively blocked the binding of APRIL to BCMA. 50 The values ​​were 0.813 nM, 0.954 nM, and 1.579 nM, respectively, and the blocking activity of the anti-BAFF / APRIL bispecific antibody was superior to that of the positive control.

[0183] Simultaneously, the activity of the anti-BAFF / APRIL bispecific antibody in binding to both human BAFF and human APRIL was determined using a bridging ELISA method. The specific detection method is as follows: The coating buffer was diluted with human BAFF to 1 μg / mL, and 100 μL / well was added to each well of the ELISA plate and incubated overnight at 4°C. The coating buffer was removed (residual droplets were removed with absorbent paper), and 200 μL / well of the blocking buffer was added to each well of the ELISA plate and incubated at room temperature for 2 hours. The blocking buffer was then removed (residual droplets were removed with absorbent paper), and the anti-BAFF / APRIL bispecific antibody, anti-BAFF nanobody, and anti-APRIL monoclonal antibody were diluted with the blocking buffer to 200 nM, forming 12 concentration gradients (4-fold dilutions). 100 μL / well of each gradient was added sequentially to the blocked ELISA plate and incubated at 37°C for 1 hour. Wash the plate three times with PBST (remove any residual droplets with absorbent paper). Dilute the biotin-labeled human APRIL to 500 ng / ml with blocking buffer, and add 100 μL / well to the plate. Incubate at 37°C for 1 hour. Wash the plate three times with PBST (remove any residual droplets with absorbent paper). Dilute the HRP-labeled streptavidin 1:1000 with blocking buffer, and add 100 μL / well to the plate. Incubate at 37°C for 30 minutes. Wash the plate three times with PBST (remove any residual droplets with absorbent paper). Add 100 μL / well of TMB, incubate at room temperature in the dark for 5 minutes, and add 50 μL / well of stop solution to terminate the substrate colorimetric reaction. Read the OD value at 450 nm using a microplate reader and calculate the EC50. 50 .

[0184] The results are shown in Figure 3C. Both the anti-BAFF / APRIL bispecific antibodies BAFF-hu1A2-APRIL-hu30H7 and BAFF-hu3B2-APRIL-hu30H7 can simultaneously bind to human BAFF and human APRIL, EC... 50 The concentrations were 0.041 nM and 0.043 nM, respectively. The anti-BAFF nanobodies BAFF-hu1A2 and BAFF-hu3B2, and the anti-APRIL monoclonal antibody APRIL-hu30H7, could not simultaneously bind to human BAFF and human APRIL.

[0185] Example 6. ELISA binding activity of anti-BAFF / APRIL bispecific antibody

[0186] Based on the above results, the VHH domain of 3B2 was linked to the N-terminus of the APRIL-hu112A2B6 heavy chain via a linker, and L234A / L235A and M252Y / S254T / T256E mutations (SEQ ID NO: 51) were introduced into the Fc domain to obtain the amino acid sequence of the BAFF-hu3B2-APRIL-hu112A2B6 heavy chain (SEQ ID NO: 16). The binding activities of BAFF-hu1A2-APRIL-hu30H7 and BAFF-hu3B2-APRIL-hu112A2B6 with human BAFF and human APRIL were determined by ELISA. The specific detection method is as follows: the coating buffer was diluted to 1 μg / mL for human BAFF and 1.2 μg / mL for human APRIL, and 100 μL / well was added to the microplate, respectively, and incubated overnight at 4°C. Remove the coating solution (remove any residual droplets with absorbent paper). Add 200 μL of blocking solution to each well of the ELISA plate and incubate at room temperature for 2 hours. Remove the blocking solution (remove any residual droplets with absorbent paper). Dilute the anti-BAFF / APRIL bispecific antibody to 200 nM with the blocking solution, creating a 4-fold dilution to form 12 concentration gradients. Add 100 μL of each gradient to each well of the plate and incubate at 37°C for 1 hour. Wash the plate three times with PBST (remove any residual droplets with absorbent paper). Dilute the HRP-labeled goat anti-human Fc antibody 1:3000 with the blocking solution and add 100 μL of each gradient to each well. Incubate at 37°C for 30 minutes. Wash the plate three times with PBST (remove any residual droplets with absorbent paper), add 100 μL of TMB per well, incubate at room temperature in the dark for 5 minutes, then add 50 μL of stop solution per well to terminate the substrate colorimetric reaction. Read the OD value at 450 nm using a microplate reader and calculate the EC50. 50 .

[0187] The results are shown in Figures 4A and 4B. The bispecific antibodies BAFF-hu1A2-APRIL-hu30H7 and BAFF-hu3B2-APRIL-hu112A2B6 can effectively bind to human BAFF, EC2 50 The concentrations were 0.061 nM and 0.041 nM, respectively. The bispecific antibody BAFF-hu3B2-APRIL-hu112A2B6 showed superior binding activity to human BAFF compared to the bispecific antibody BAFF-hu1A2-APRIL-hu30H7. Both bispecific antibodies BAFF-hu1A2-APRIL-hu30H7 and BAFF-hu3B2-APRIL-hu112A2B6 effectively bound to human APRIL, EC50. 50 The concentrations were 0.042 nM and 0.027 nM, respectively, and both bispecific antibodies showed superior activity in binding to human APRIL.

[0188] Example 7. Cell-blocking activity of anti-BAFF / APRIL bispecific antibody against APRIL / BAFF and BCMA

[0189] The reporter gene drug target model BCMA NFκB-Luc HEK293 cells can mimic the signal transduction process of the nuclear factor kappa-B (NF-κB) enhancement cascade reaction of B cells activated by nuclear factor after the binding of ligands APRIL and / or BAFF to BCMA in vivo. When the ligand-BCMA reaction is blocked, the effect of the blocking drug can be verified by fluorescence reaction. The specific method is as follows: BCMA NFκB-Luc HEK293 cells were diluted with culture medium at a concentration of 30,000 cells / 100 μL / well and added to 96-well plates. Simultaneously, human APRIL and human BAFF were diluted to 80 ng / mL with culture medium. Anti-BAFF / APRIL bispecific antibody and the positive control sibeprenlimab were diluted to 50 nM with culture medium containing human APRIL and human BAFF, creating nine concentration gradients through four-fold dilutions. These were added to 96-well plates containing cells and incubated at 37°C for 6 hours. Then, ONE-Glo... TM The Luciferase Assay System was used for detection, and the Luminescence value was read using a microplate reader to calculate the IC50. 50 .

[0190] The results are shown in Figure 5. Both the anti-BAFF / APRIL bispecific antibodies BAFF-hu1A2-APRIL-hu30H7 and BAFF-hu3B2-APRIL-hu112A2B6 can block the simultaneous binding of the dual ligands APRIL and BAFF to BCMA, with an IC50 concentration of [missing information]. 50 The concentrations were 0.513 nM and 0.605 nM, respectively. The positive control, sibeprenlimab, also showed significant blocking activity, with an IC50 concentration of 0.513 nM and 0.605 nM. 50 The value was 1.217 nM. However, the positive antibody showed poorer blocking activity compared to the anti-BAFF / APRIL bispecific antibody, indicating that the bispecific antibody targeting both APRIL and BAFF is superior in blocking the binding of BCMA to its ligand.

[0191] Example 8. Stability study of anti-BAFF / APRIL bispecific antibody

[0192] The purity of the anti-BAFF / APRIL bispecific antibody protein was determined by separating it from impurity proteins using size exclusion chromatography (SEC) in ultra-high performance liquid chromatography (UPLC). The proteins of the anti-BAFF / APRIL bispecific antibodies BAFF-hu1A2-APRIL-hu30H7 and BAFF-hu3B2-APRIL-hu112A2B6 were concentrated to 100 mg / mL and incubated at 37°C for 7 days. UPLC-SEC was then measured. The UPLC-SEC chromatograms are shown in Figures 6A and 6B. After 7 days at 37°C, the protein purity of BAFF-hu1A2-APRIL-hu30H7 decreased from 98.84% to 97.65%; the protein purity of BAFF-hu3B2-APRIL-hu112A2B6 decreased from 96.84% to 95.08% after the same period. This indicates that the anti-BAFF / APRIL bispecific antibody has a stable structure.

[0193] Imaged capillary isoelectric focusing (iCIEF) was used to separate anti-BAFF / APRIL bispecific antibodies from their charge variants based on differences in protein isoelectric point (pI), thereby determining the purity of the antibody molecules. The proteins of the anti-BAFF / APRIL bispecific antibodies BAFF-hu1A2-APRIL-hu30H7 and BAFF-hu3B2-APRIL-hu112A2B6 were concentrated to 100 mg / mL and incubated at 37°C for 7 days. iCIEF was then measured. As shown in Figures 6C and 6D, the iCIEF spectra of BAFF-hu1A2-APRIL-hu30H7 protein increased from 14.22% to 22.83% after being incubated at 37°C for 7 days. Similarly, the increase in isoformation of BAFF-hu3B2-APRIL-hu112A2B6 protein between pI 8.166 and 8.003 increased from 17.23% to 32.29% after the same incubation period. This indicates that the anti-BAFF / APRIL bispecific antibodies were relatively stable in the 37°C forced degradation experiment.

[0194] Example 9. Biacore determination of the affinity of anti-BAFF / APRIL bispecific antibody for antigen.

[0195] The binding / dissociation kinetics and equilibrium dissociation constant of the anti-BAFF / APRIL bispecific antibody BAFF-hu1A2-APRIL-hu30H7 to human APRIL and BAFF were determined using surface plasmon resonance (SPR). The anti-APRIL monoclonal antibody sibeprenlimab and the anti-BAFF monoclonal antibody belimumab were used as positive controls. The specific detection method is as follows: The affinity of BAFF-hu1A2-APRIL-hu30H7 for APRIL was determined using a Protein A chip. The antibody concentration was diluted to 6.5 nM with HBS-EP+Buffer, and the APRIL antigen was diluted to a maximum concentration of 125 nM with HBS-EP+Buffer. Eight 2-fold dilutions were performed, with a 0 concentration point set. 6M guanidine hydrochloride solution was used as the regeneration buffer. The sample was injected onto a Biacore 8K with the following parameters: binding time 120 s, dissociation time 600 s, flow rate 30 μL / min, and regeneration contact time 30 s. The affinity of BAFF-hu1A2-APRIL-hu30H7 for BAFF was determined using the CM5 chip capture method. pH Scouting was run to explore suitable pH coupling conditions, and the optimal coupling condition was determined to be pH 4.0. BAFF-hu1A2-APRIL-hu30H7 was diluted to 35 μg / mL with Acetate 4.0, and belimumab was diluted to 25 μg / mL. The coupling program was run with a coupling target of 10000 RU. BAFF antigen was diluted using HBS-EP+Buffer to a maximum concentration of 500 nM, with eight 2-fold dilutions and a 0 concentration point set. 10 mM glycine (pH 1.5) was used as the regeneration buffer. The sample was injected into a Biacore 8K with the following parameters: binding time 150 s, dissociation time 1500 s, flow rate 45 μL / min, and regeneration contact time 30 s. Data were analyzed using Biacore 8K Evaluation Software.

[0196] The binding affinity with APRIL is shown in Table 1. BAFF-hu1A2-APRIL-hu30H7 and sibeprenlimab have essentially the same KD for APRIL, with KD values ​​of 5.05E-10M and 2.85E-10M, respectively. The binding affinity with BAFF is shown in Table 2. BAFF-hu1A2-APRIL-hu30H7 and belimumab have essentially the same KD for BAFF, with KD values ​​of 2.44E-9M and 3.14E-9M, respectively. These results indicate that BAFF-hu1A2-APRIL-hu30H7 can effectively bind to both APRIL and BAFF, and its affinity for both APRIL and BAFF is comparable to that of positive monoclonal antibodies.

[0197] Table 1. Binding and dissociation kinetic parameters and equilibrium dissociation constant of BAFF-hu1A2-APRIL-hu30H7 to APRIL Note: K on : Combination constant; K off KD: Dissociation constant; Equilibrium dissociation constant (affinity)

[0198] Table 2. Binding and dissociation kinetic parameters and equilibrium dissociation constant of BAFF-hu1A2-APRIL-hu30H7 to BAFF Note: K on : Combination constant; K off KD: Dissociation constant; KD: Equilibrium dissociation constant (affinity).

[0199] Example 10. Affinity of anti-BAFF / APRIL bispecific antibody

[0200] The binding activity of the anti-BAFF / APRIL bispecific antibody BAFF-hu1A2-APRIL-hu30H7 to human APRIL and human BAFF was determined by ELISA. The anti-APRIL monoclonal antibody sibeprenlimab and the anti-BAFF monoclonal antibody belimumab served as positive controls. The specific detection method is as follows: The recombinant antigens were diluted to appropriate concentrations with coating buffer (human APRIL recombinant antigen diluted to 1.2 μg / mL, human BAFF recombinant antigen diluted to 1 μg / mL), and the antigen solutions were transferred to microplates (100 μL / well) using a multichannel pipette and incubated overnight at 4°C. The plates were washed once with PBST, and then blocked with PBST containing 1% BSA (200 μL / well) and incubated at room temperature for 2 h. The blocking solution was discarded, and the microplates were stored at 4°C for later use. The antibodies were diluted with PBST containing 1% BSA to an initial concentration of 50 nM, and then serially diluted 11 times in a 4-fold gradient. The diluted antibody was transferred to the blocked microplates (100 μL / well) and incubated at room temperature for 1 h. The plates were washed three times with PBST, and then the HRP-labeled goat anti-human antibody was diluted 3000-fold with PBST containing 1% BSA. This solution was transferred to microplates (100 μL / well) and incubated at room temperature for 30 min. The plates were washed three times with PBST, and TMB chromogenic solution (100 μL / well) was added to the microplates. After incubation at room temperature for 1–5 min, stop solution (50 μL / well) was added to terminate the chromogenic reaction. The OD value at 450 nm was read using a microplate reader. The obtained data were analyzed using GraphPad Prism 9 and EC was calculated. 50 .

[0201] The results are shown in Figures 7A and 7B. BAFF-hu1A2-APRIL-hu30H7 can effectively bind to human ARPIL and BAFF, EC 50 The values ​​were 0.079 nM and 0.059 nM, respectively (Table 3). The affinity of BAFF-hu1A2-APRIL-hu30H7 and the positive antibody sibeprenlimab to APRIL was basically the same, while the affinity of BAFF-hu1A2-APRIL-hu30H7 to BAFF was better than that of the positive antibody belimumab.

[0202] Table 3. Affinity of BAFF-hu1A2-APRIL-hu30H7 with the antigen

[0203] Example 11. ELISA blocking activity of anti-BAFF / APRIL bispecific antibody against antigen and its receptor.

[0204] Both APRIL and BAFF can bind to BCMA and TACI to regulate B cell function and survival. The inventors used a blocking ELISA to examine the blocking ability of the anti-BAFF / APRIL bispecific antibody BAFF-hu1A2-APRIL-hu30H7 against APRIL-BCMA, APRIL-TACI, and BAFF-BCMA. The specific method is as follows:

[0205] APRIL receptor binding blocking: Dilute the recombinant protein to an appropriate concentration with coating buffer (BCMA-Fc to 200 ng / mL, TACI-Fc to 1 μg / mL). Transfer the antigen solution to microplates (100 μL / well) using a multichannel pipette and incubate overnight at 4°C. Wash the plate once with PBST, then block with PBST containing 1% BSA (200 μL / well) and incubate at room temperature for 2 h. Discard the blocking solution and store the microplates at 4°C. Dilute the APRIL protein to 50 ng / mL with PBST containing 1% BSA. Dilute the antibody to be tested with this solution, starting at 500 nM, and serially dilute 11 times in a 3-fold gradient, incubating at room temperature for 1 h. Transfer the dilution to the BCMA-Fc-coated microplates (100 μL / well) and incubate at 37°C for 1 h. Dilute the APRIL protein to 300 ng / mL with PBST containing 1% BSA. Dilute the antibody to be tested with this solution. The initial antibody concentration is 100 nM. Perform 11 serial dilutions at a 2-fold gradient and incubate at room temperature for 30 min. Transfer the diluted solution to the TACI-Fc-coated microplates (100 μL / well) and incubate at 37°C for 1 h. Wash the plate three times with PBST. Then, dilute the HRP-labeled streptavidin 2000-fold with PBST containing 1% BSA. Transfer this solution to the microplates (100 μL / well) and incubate at room temperature for 30 min. Wash the plate three times with PBST. Add TMB chromogenic solution (100 μL / well) to the microplates and incubate at room temperature for 1–5 min. Add stop solution (50 μL / well) to terminate the chromogenic reaction. Read the OD value at 450 nm using a microplate reader. Analyze the obtained data using GraphPad Prism 9 and calculate EC. 50 .

[0206] BAFF receptor binding blocking: Dilute BCMA-Fc to 1 μg / mL with coating buffer, and transfer the antigen solution to microplates (100 μL / well) using a multichannel pipette. Incubate overnight at 4°C. Wash the plate once with PBST, and then block with PBST containing 1% BSA (200 μL / well), incubating at room temperature for 2 h. Discard the blocking solution and store the microplates at 4°C. Dilute BAFF protein to 500 ng / mL with PBST containing 1% BSA. Dilute the antibody to be tested with this solution, starting at 500 nM, and perform 11 serial dilutions at a 3-fold gradient, incubating at room temperature for 1 h. Transfer the diluted solution to the BCMA-Fc-coated microplates (100 μL / well) and incubate at 37°C for 1 h. The plate was washed three times with PBST. Then, the HRP-labeled streptavidin was diluted 2000-fold with PBST containing 1% BSA. This solution was transferred to microplates (100 μL / well) and incubated at room temperature for 30 min. The plate was washed three times with PBST. TMB chromogenic solution (100 μL / well) was added to the microplate, and the plate was incubated at room temperature for 1–5 min. Finally, stop solution (50 μL / well) was added to terminate the chromogenic reaction. OD values ​​at 450 nm were read using a microplate reader. The obtained data were analyzed using GraphPad Prism 9 and EC was calculated. 50 .

[0207] The results are shown in Figures 8A-8C. BAFF-hu1A2-APRIL-hu30H7 effectively blocked the in vitro binding of APRIL to BCMA, APRIL to TACI, and BAFF to BCMA. 50 The concentrations were 3.396 nM, 7.498 nM, and 10.1 nM, respectively (Table 4). Compared with the control, the blocking activity of BAFF-hu1A2-APRIL-hu30H7 was significantly better than that of the positive monoclonal antibody control.

[0208] Table 4. Blocking ability of BAFF-hu1A2-APRIL-hu30H7 against TACI / BAFF binding to BCMA and TACI - IC 50 (nM)

[0209] Example 12. Cell blocking activity of anti-BAFF / APRIL bispecific antibody

[0210] The reporter gene drug target model BCMA-NF-κB reporter cell can mimic the signal transduction process of the nuclear factor-activated B cell κ-light chain enhancement (NF-κB) cascade reaction after the binding of ligands APRIL and / or BAFF to BCMA in vivo. When the ligand-BCMA reaction is blocked, the effect of the blocking drug can be verified by fluorescence reaction. The specific method is as follows: Cells are resuspended in MEM analytical medium (containing 10% FBS, 1% NEAA, 1mM sodium pyruvate, and 1% PenStrip), and the cell density is adjusted to 3×10⁶ cells / year. 5 Cells / mL. Add 100 μL / well to the center well of a 96-well plate. Incubate overnight in a CO2 cell culture incubator at 37°C. Prepare the following solutions using MEM analytical medium: containing 80 ng / mL APRIL, containing 80 ng / mL BAFF, and containing both 340 ng / mL BAFF and 40 ng / mL APRIL. Dilute the test antibody with the above solutions containing different ligands, with an initial antibody concentration of 50 nM, and perform nine serial dilutions at a 4-fold gradient. A blank control with an antibody concentration of 0% is also included. Incubate for 1 h in a CO2 cell culture incubator at 37°C. Remove the cell-coated 96-well plate and discard 90 μL. Add 100 μL of the incubated mixture to each well of the cell-coated plate. Incubate for 6 h in a CO2 cell culture incubator at 37°C. Add 100 μL of Bio-Lite Luciferase Assay System to each well, react at room temperature for 15 min, and then measure the luminescence value using a microplate reader.

[0211] The results, shown in Figures 9A-9C, indicate that BAFF-hu1A2-APRIL-hu30H7 can inhibit downstream signaling of the BCMA receptor mediated by APRIL, BAFF, and both APRIL and BAFF. 50 The concentrations were 0.3176 nM, 0.1361 nM, and 1.024 nM, respectively (Table 5). In APRIL- or BAFF-mediated cell activity, the blocking activity of BAFF-hu1A2-APRIL-hu30H7 was comparable to that of the corresponding positive monoclonal antibody; in cell activity mediated by both APRIL and BAFF, the inhibitory activity of BAFF-hu1A2-APRIL-hu30H7 was superior to the combination of the APRIL monoclonal antibody sibeprenlimab and the BAFF monoclonal antibody belimumab.

[0212] Table 5. Cell blocking activity of BAFF-hu1A2-APRIL-hu30H7 based on reporter gene - IC50 (nM)

[0213] Example 13. Affinity of anti-BAFF / APRIL bispecific antibody to FcRn

[0214] To prolong the half-life, an M252Y / S254T / T256E mutation was introduced into the Fc constant region of the anti-BAFF / APRIL bispecific antibody BAFF-hu1A2-APRIL-hu30H7. This mutation enhances the binding activity with FcRn under acidic conditions. Using an IgG1 antibody without the M252Y / S254T / T256E mutation as a control antibody, the ability of BAFF-hu1A2-APRIL-hu30H7 to bind FcRn under acidic (pH 5.8) and neutral (pH 7.4) conditions was detected by ELISA. The specific detection method is as follows: the antibody to be tested was diluted to 5 μg / mL with coating buffer, and the solution was transferred to microplates (100 μL / well) using a multichannel pipette and incubated overnight at 4°C. Wash the plate once with PBST (pH 5.8), then block with PBST (pH 5.8) containing 1% BSA (200 μL / well) and incubate at room temperature for 2 h. Discard the blocking solution and store the microplate at 4 °C. Dilute the biotinylated FcRn with PBST (pH 5.8) containing 1% BSA to an initial concentration of 200 nM, and perform 12 serial dilutions at a 2-fold gradient. Transfer the diluted solution to the previously blocked microplate (100 μL / well) and incubate at room temperature for 2 h. Wash the plate three times with PBST (pH 5.8), then dilute the HRP-labeled streptavidin 3000-fold with PBST (pH 5.8) containing 1% BSA, and transfer this solution to the microplate (100 μL / well) and incubate at room temperature for 1 h. Wash the plate three times with PBST (pH 5.8). Add TMB chromogenic solution (100 μL / well) to each well of the microplate and incubate at room temperature for 1–5 min. Then add stop solution (50 μL / well) to terminate the chromogenic reaction. Read the OD value at 450 nm using a microplate reader. Analyze the data using GraphPad Prism 9 and calculate EC. 50 .

[0215] Dilute the antibody to be tested to 100 ng / mL with coating buffer and transfer the solution to microplates (100 μL / well) using a multichannel pipette, incubating overnight at 4°C. Wash the plate once with PBST (pH 7.4), then block with PBST (pH 7.4) containing 1% BSA (200 μL / well), incubating at room temperature for 2 h. Discard the blocking solution and store the microplates at 4°C. Dilute the biotinylated FcRn with PBST (pH 7.4) containing 1% BSA to an initial concentration of 250 nM, and perform 12 serial dilutions at a 2-fold gradient. Transfer the diluted solution to the blocked microplates (100 μL / well) and incubate at room temperature for 2 h. The plate was washed three times with PBST (pH 7.4). Then, the HRP-labeled streptavidin was diluted 3000-fold with PBST (pH 7.4) containing 1% BSA. This solution was transferred to microplates (100 μL / well) and incubated at room temperature for 1 h. The plate was washed three times with PBST (pH 7.4). TMB chromogenic solution (100 μL / well) was added to the microplate, and the plate was incubated at room temperature for 5 min. Stop solution (50 μL / well) was then added to terminate the chromogenic reaction. OD values ​​at 450 nm were read using a microplate reader. The obtained data were analyzed using GraphPad Prism 9 and EC was calculated. 50 .

[0216] As shown in Figures 10A and 10B, BAFF-hu1A2-APRIL-hu30H7 can bind to FcRn under both acidic and neutral conditions, with significantly better affinity under acidic conditions than under neutral conditions. Compared to control IgG1, BAFF-hu1A2-APRIL-hu30H7 shows significantly enhanced binding to FcRn under acidic conditions, while its binding to FcRn is essentially the same under neutral conditions.

[0217] Example 14. Inhibitory effect of anti-BAFF / APRIL bispecific antibody on B6-hAPRIL-LTgBAFF mouse model

[0218] To evaluate the inhibitory effect of BAFF-hu1A2-APRIL-hu30H7 on BAFF and APRIL-induced B cell activation, differentiation, and / or survival in animals, and to verify its potential to control B cells by blocking the pathogenesis of autoimmune diseases driven by these two cytokines, the inventors conducted in vivo pharmacodynamic experiments. The experiments used B6-hAPRILTg(hBAFF) transgenic mice (humanized APRIL / BAFF mice, background C57BL / 6, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.). This model, through gene editing technology, inserts human APRIL and BAFF genes into the mouse genome, spontaneously inducing lupus-like lesions, manifested as abnormal expression of indicators such as anti-double-stranded DNA antibody (anti-dsDNA), immunoglobulin A (IgA), and immunoglobulin G (IgG) after B cell activation, as well as symptoms such as splenomegaly. Therefore, the inventors used this model to investigate and evaluate the in vivo efficacy of BAFF-hu1A2-APRIL-hu30H7.

[0219] Female B6-hAPRIL-hu30H7 mice, aged 6-7 weeks, were acclimatized for 5-7 days. Peripheral blood serum was collected, and anti-dsDNA levels were detected by ELISA. Based on anti-dsDNA levels and body weight, the mice were divided into four groups of six mice each: a model control group (G2), a low-dose BAFF-hu1A2-APRIL-hu30H7 test product group (G3, 5 mpk), a high-dose BAFF-hu1A2-APRIL-hu30H7 test product group (G4, 10 mpk), and a Teletacicept positive control group (G5, 10 mpk). A healthy control group of C57BL / 6 mice (G1) was also included. Grouping and administration methods are shown in Table 6. Administration was twice weekly for four weeks. Mice were weighed weekly, and serum was collected at week 4 for ELISA detection of anti-dsDNA, IgA, IgM, and IgG.

[0220] Table 6. Grouping and Administration Methods of B6-hAPRILTg (hBAFF) Mouse Model

[0221] The results of serological markers and autoantibody levels after B cell activation are shown in Figures 11A-11D. Compared with the healthy control group G1 (C57BL / 6), the serum levels of anti-dsDNA, mIgG, mIgM, and mIgA in the model control group G2 (B6-hAPRIL / hBAFF, Vehicle) mice were significantly increased. Compared with the model control group G2 (B6-hAPRIL / hBAFF, Vehicle), the serum levels of anti-dsDNA, mIgG, mIgM, and mIgA in the low-dose administration group G3 (B6-hAPRIL / hBAFF, BAFF-hu1A2-APRIL-hu30H7, 5mpk), the high-dose administration group G4 (B6-hAPRIL / hBAFF, BAFF-hu1A2-APRIL-hu30H7, 10mpk), and the positive control group G5 (B6-hAPRIL / hBAFF, Telitacicept, 10mpk) were significantly decreased. Compared with the positive control group G5 (B6-hAPRIL / hBAFF, Telitacicept, 10 mpk), the serum levels of anti-dsDNA, mIgG, mIgM, and mIgA were significantly reduced in the low-dose group G3 (B6-hAPRIL / hBAFF, BAFF-hu1A2-APRIL-hu30H7, 5 mpk) and the high-dose group G4 (B6-hAPRIL / hBAFF, BAFF-hu1A2-APRIL-hu30H7, 10 mpk). Among them, the high-dose group G4 showed a more significant reduction in anti-dsDNA and mIgG levels, exhibiting a more pronounced reduction than Telitacicept.

[0222] in conclusion

[0223] As can be seen from the above results, the bispecific antibody provided by the present invention can bind to APRIL and BAFF simultaneously, and can simultaneously block the activation, differentiation and survival signals of B cells activated by the two ligands, thereby inhibiting the B cell-mediated autoimmune response.

Claims

1. An anti-APRIL antibody or its antigen-binding fragment, characterized in that, Its heavy chain variable region contains HCDR1-3 selected from the following groups: 1) SEQ ID NO: 22-24; 2) SEQ ID NO: 29-31; Its light chain variable region includes LCDR1-3 selected from the following group: 3) SEQ ID NO: 25, 27, 28 or SEQ ID NO: 26, 27, 28; 4) SEQ ID NO: 32, 33, 35 or SEQ ID NO: 32, 34, 36.

2. The anti-APRIL antibody or its antigen-binding fragment as described in claim 1, characterized in that, 1) The heavy chain variable region is selected from SEQ ID NO: 1, 3, 7, 9; and / or 2) The light chain variable region is selected from SEQ ID NO: 2, 5, 8, 11; and / or 3) The heavy chain variable region is selected from amino acid sequences that have at least 99%, 95%, 90%, 85%, or 80% identity with SEQ ID NO: 1, 3, 7, or 9; and the light chain variable region is selected from amino acid sequences that have at least 99%, 95%, 90%, 85%, or 80% identity with SEQ ID NO: 2, 5, 8, or 11.

3. The anti-APRIL antibody or its antigen-binding fragment as described in claim 1, characterized in that, It contains a heavy chain amino acid sequence selected from SEQ ID NO: 4 or 10, and a light chain amino acid sequence selected from SEQ ID NO: 6 or 12.

4. A bispecific antibody against APRIL / BAFF, characterized in that, It contains anti-APRIL antibody or its antigen-binding fragment and anti-BAFF antibody or its antigen-binding fragment.

5. The anti-APRIL / BAFF bispecific antibody as described in claim 4, characterized in that, Include: The anti-APRIL antibody or its antigen-binding fragment as described in any one of claims 1-3; and / or, Anti-BAFF nanobodies or their antigen-binding fragments possess the following characteristics: 1) The heavy chain variable region contains HCDR1-3 selected from the group consisting of SEQ ID NO: 37, 38, 39, or SEQ ID NO: 40, 38, 41; or, 2) The heavy chain variable region is selected from SEQ ID NO: 13, 14, 42, 43, or from an amino acid sequence that has at least 99%, 95%, 90%, 85%, or 80% identity with SEQ ID NO: 13, 14, 42, 43.

6. The anti-APRIL / BAFF bispecific antibody as described in claim 4, characterized in that, It comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 15 and a light chain with an amino acid sequence as shown in SEQ ID NO: 6; or it comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 16 and a light chain with an amino acid sequence as shown in SEQ ID NO:

12.

7. The anti-APRIL / BAFF bispecific antibody as described in claim 4, characterized in that, It also includes the Fc structure field.

8. The anti-APRIL / BAFF bispecific antibody as described in claim 4, characterized in that, The bispecific antibody comprises a monomer or a dimer formed from the monomer, the dimer being either homologous or heterologous.

9. The anti-APRIL / BAFF bispecific antibody as described in claim 4, characterized in that, The antibody or its antigen-binding fragment is selected from full-length antibodies, nanobodies, single-chain antibodies, scFv, Fv, Fd, Fab, F(ab')2 or F(ab').

10. A polynucleotide molecule, characterized in that, The polynucleotide molecule encodes the anti-APRIL antibody or its antigen-binding fragment as described in any one of claims 1-3 or the anti-APRIL / BAFF bispecific antibody as described in any one of claims 4-9; preferably, the polynucleotide molecule is selected from SEQ ID NO: 18-21.

11. An expression carrier, characterized in that, The expression vector contains the polynucleotide molecule as described in claim 10.

12. A host cell, characterized in that, The host cell contains the expression vector as described in claim 11 or its genome is integrated with the polynucleotide as described in claim 10.

13. A method for preparing anti-APRIL antibody or its antigen-binding fragment or anti-APRIL / BAFF bispecific antibody, characterized in that, Including the following steps: 1) Culturing the host cells of claim 12 to obtain a culture containing the anti-APRIL antibody or its antigen-binding fragment or anti-APRIL / BAFF bispecific antibody; 2) Isolate or recover the anti-APRIL antibody or its antigen-binding fragment or anti-APRIL / BAFF bispecific antibody from the culture; 3) Optionally, purify and / or modify the anti-APRIL antibody or its antigen-binding fragment or anti-APRIL / BAFF bispecific antibody obtained in step 2).

14. An immunoconjugate, characterized in that, The immunoconjugate contains: 1) The anti-APRIL antibody or its antigen-binding fragment as described in any one of claims 1-3, or the anti-APRIL / BAFF bispecific antibody as described in any one of claims 4-9; and 2) Selected coupling motifs from the following group: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

15. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: an anti-APRIL antibody or its antigen-binding fragment as described in any one of claims 1-3, or an anti-APRIL / BAFF bispecific antibody as described in any one of claims 4-9, or an immunoconjugate as described in claim 14; and a pharmaceutically acceptable carrier or excipient.

16. Use of an anti-APRIL antibody or its antigen-binding fragment as described in any one of claims 1-3, or an anti-APRIL / BAFF bispecific antibody as described in any one of claims 4-9, or an immunoconjugate as described in claim 14, or a pharmaceutical composition as described in claim 15, characterized in that, The uses include any of the following applications: 1) To prepare medicines for the prevention and / or treatment of diseases associated with BAFF and / or APRIL; 2) To prepare drugs for the prevention and / or treatment of autoimmune diseases, inflammatory diseases or tumors; 3) To prepare drugs for the prevention and / or treatment of BAFF and / or APRIL-mediated autoimmune diseases, inflammatory diseases or tumors; 4) Prepare drugs that reduce immune response.

17. The use as described in claim 16, characterized in that, The diseases are selected from inflammatory arthritis (such as rheumatoid arthritis), systemic lupus erythematosus, myasthenia gravis, primary Sjögren's syndrome, kidney diseases, neuromyelitis optica, optic neuritis, multiple sclerosis, B-cell tumors, connective tissue diseases, interstitial lung diseases, systemic scleroderma, diffuse scleroderma, immune thrombocytopenic purpura, myositis, polyangiitis, autoimmune hepatitis, chronic graft-versus-host disease, bronchial diseases, and rheumatic diseases; preferably, the kidney diseases include IgAN, lupus nephritis, and membranous glomerulonephritis.

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