Nanobody against BAFF, preparation therefor, and use thereof

Abstract

A nanobody against BAFF or an antigen-binding fragment thereof is provided, the heavy chain variable region of which comprises HCDR2 as set forth in SEQ ID NO: 16 and HCDR3 as set forth in SEQ ID NO: 17 or 19. The nanobody against BAFF or the antigen-binding fragment thereof can effectively block the BAFF signaling pathway, effectively inhibit the biological activity of BAFF, and can be used for preventing or treating diseases caused by B cell activation, proliferation, or functional abnormalities resulting from BAFF.

Description

An anti-BAFF nanobody and its preparation and application Technical Field

[0001] This application relates to the field of antibody drug technology, specifically to an anti-BAFF nanobody that can target BAFF and inhibit its activity, and its preparation and application. Background Technology

[0002] B-cell activation factor (BAFF), also known as TNFSF13B, is a member of the tumor necrosis factor (TNF) ligand family. It is a type II transmembrane protein containing 285 amino acids, existing in both soluble and membrane-bound forms. BAFF is expressed on monocytes, dendritic cells, and bone marrow stromal cells. It has three receptors: B-cell activation factor receptor (BAFFR), transmembrane activator and CAML interactor (TACI), and B-cell maturation protein (BCMA). BAFF binds to different receptors, promoting B-cell survival and proliferation, T-cell-independent heterotrophic transformation, and plasma cell survival, respectively. BAFF is involved in the pathogenesis of many autoimmune diseases. Elevated BAFF concentrations have been detected in the serum of patients with systemic lupus erythematosus (SLE), multiple sclerosis (MS), IgA nephropathy, and rheumatoid arthritis. BAFF transgenic mice also exhibit lupus-like phenotypes, characterized by elevated serum immunoglobulin levels, elevated autoantibody levels, and symptoms such as immune complex-mediated glomerulonephritis. SLE is a chronic systemic autoimmune disease clinically characterized by multi-system involvement. Its pathogenesis is complex, including the proliferation and activation of autoreactive T cells and B cells, the production of various autopathogenic antibodies, and abnormal cytokine secretion and receptor expression. Among these, the loss of B cell tolerance plays a crucial pathological role in the occurrence and maintenance of SLE. Blocking BAFF signaling provides a new approach for immunological intervention in autoimmune diseases. Belimumab is a BAFF monoclonal antibody used to treat SLE. It targets soluble BAFF, preventing BAFF from binding to B cells, promoting B cell apoptosis, and significantly reducing circulating B cell levels. Although BAFF monoclonal antibodies are available on the market, unmet clinical needs remain. Summary of the Invention

[0003] The purpose of this invention is to provide a novel and superior anti-BAFF nanobody or its antigen-binding fragment to address unmet clinical needs. The main strategy involves panning an antibody library using immunized camels and phage display to obtain camel-derived antibodies targeting BAFF. The VHH domain targeting BAFF is then fused with the Fc domain of IgG1 to obtain a camel-derived chimeric anti-BAFF nanobody with in vivo biostability. Through in vitro and in vivo BAFF-targeting activity assays, a camel-derived chimeric anti-BAFF nanobody with superior activity is obtained. This nanobody is then humanized to obtain a humanized anti-BAFF nanobody with superior expression levels and bioactivity, aiming to develop a nanobody drug superior to existing marketed or investigational products.

[0004] In a first aspect, the present invention provides an anti-BAFF nanobody or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR2 as shown in SEQ ID NO:16 and HCDR3 as shown in SEQ ID NO:17 or 19.

[0005] In an alternative example, the heavy chain variable region comprises HCDR1-3 selected from the group consisting of:

[0006] 1) SEQ ID NO: 15, 16, 17;

[0007] 2) SEQ ID NO: 18, 16, 19.

[0008] In one alternative example, the heavy chain variable region is selected from SEQ ID NO:1, 2, 7, 8.

[0009] In an alternative embodiment, the heavy chain variable region is selected from amino acid sequences having at least 99%, 95%, 90%, 85%, or 80% identity with SEQ ID NO: 1, 2, 7, or 8; or, the heavy chain variable region comprises HCDR1-3 of 1) or 2) above. Preferably, the heavy chain variable region has the same function as the heavy chain variable regions selected from SEQ ID NO: 1, 2, 7, or 8.

[0010] In an alternative embodiment, the anti-BAFF nanobody or its antigen-binding fragment further comprises a heavy chain constant region domain.

[0011] In an alternative example, the heavy chain constant region structural domain comprises a heavy chain constant region or a segment thereof.

[0012] In one alternative example, the heavy chain constant region is selected from IgG1, IgG2, IgG3, or IgG4.

[0013] In one alternative, the heavy chain constant region is derived from human or mouse sources.

[0014] In an alternative example, the heavy chain constant region segment comprises CH2-CH3.

[0015] In one alternative, the heavy chain constant region or a segment thereof contains a mutated amino acid. For example, existing Fc mutation methods can improve half-life or stability.

[0016] In an alternative example, the heavy chain constant region structural domain comprises SEQ ID NO:3.

[0017] In an alternative example, the heavy chain constant region structural domain is connected to the N-terminus or C-terminus of the heavy chain variable region.

[0018] In one alternative example, the connection is made via a peptide linker.

[0019] In an alternative example, the peptide linker is an L1 short peptide with the amino acid sequence shown in SEQ ID NO:4.

[0020] In an alternative embodiment, the nanobody comprises an amino acid sequence selected from SEQ ID NO:5, 6, 9, 10.

[0021] In an alternative embodiment, the anti-BAFF nanobody or its antigen-binding fragment has at least one of the following properties:

[0022] 1) It binds to the same BAFF epitope as the antibodies or their antigen-binding fragments described above;

[0023] 2) Competes with the antibodies or their antigen-binding fragments mentioned above to bind to the BAFF epitope.

[0024] In one alternative, the nanobody or its antigen-binding fragment is camel-derived, chimeric, human, or fully human.

[0025] In one alternative example, the nanobody or its antigen-binding fragment is selected from full-length antibodies, scFv, Fv, Fab, F(ab'), F(ab')2, Fv, Fd, single-chain antibodies, and single-domain antibodies.

[0026] In one alternative, the nanobody or its antigen-binding fragment is monovalent, bivalent, or multivalent.

[0027] In one alternative, the nanobody comprises a monomer or a dimer or trimer formed from the monomer, the dimer or trimer being homologous or heterologous.

[0028] In a second aspect, the present invention provides a polynucleotide molecule that encodes the anti-BAFF nanobody or its antigen-binding fragment as described in the first aspect.

[0029] In an alternative example, the polynucleotide molecule is selected from SEQ ID NO:11-14.

[0030] In a third aspect, the present invention provides an expression vector containing the polynucleotide molecule described in the second aspect.

[0031] In one alternative example, the expression vector is a virus or a plasmid.

[0032] 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.

[0033] In a fourth aspect, the present invention provides a host cell containing the expression vector described in the third aspect or integrating the polynucleotide molecule described in the second aspect.

[0034] In another preferred embodiment, the host cell is selected from the group consisting of COS, CHO, NSO, sf9, sf21, DH5α, BL21(DE3), TG1, BL21(DE3), 293F or 293E cells.

[0035] A fifth aspect of the present invention provides a method for preparing the anti-BAFF nanobody or its antigen-binding fragment as described in the first aspect, the method comprising the following steps:

[0036] 1) Culturing the host cells described in the fourth aspect to obtain a culture containing the anti-BAFF nanobody or its antigen-binding fragment;

[0037] 2) Isolate or recover the anti-BAFF nanobody or its antigen-binding fragment from the culture;

[0038] 3) Optionally, purify and / or modify the anti-BAFF nanobody or its antigen-binding fragment obtained in step 2).

[0039] In a sixth aspect, the present invention provides an immunoconjugate comprising:

[0040] 1) The anti-BAFF nanobody or its antigen-binding fragment as described in the first aspect; and

[0041] 2) The coupling part selected from the following group: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes or combinations thereof.

[0042] A seventh aspect of the present invention provides a multispecific antibody comprising the anti-BAFF nanobody or its antigen-binding fragment as described in the first aspect.

[0043] In one alternative example, the multispecific antibody is a bispecific antibody.

[0044] An eighth aspect of the present invention provides a pharmaceutical composition comprising an effective amount of the anti-BAFF nanobody or its antigen-binding fragment as described in the first aspect, or the immunoconjugate as described in the sixth aspect, or the multispecific antibody as described in the seventh aspect; and a pharmaceutically acceptable carrier or excipient.

[0045] In one alternative embodiment, the dosage form of the pharmaceutical composition includes a gastrointestinal dosage form or a parenteral dosage form.

[0046] 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.

[0047] A ninth aspect of the present invention provides the use of the anti-BAFF nanobody or its antigen-binding fragment as described in the first aspect, or the immunoconjugate as described in the sixth aspect, or the multispecific antibody as described in the seventh aspect, or the pharmaceutical composition as described in the eighth aspect, said use including any of the following applications:

[0048] 1) To prepare drugs for the prevention and / or treatment of diseases associated with BAFF;

[0049] 2) To prepare drugs for the prevention and / or treatment of autoimmune diseases, inflammatory diseases or tumors;

[0050] 3) To prepare drugs for the prevention and / or treatment of BAFF-mediated or BAFF-abnormally expressed autoimmune diseases, inflammatory diseases or tumors;

[0051] 4) Prepare drugs that reduce immune response.

[0052] A tenth aspect of the present invention provides a method for preventing / treating a disease, the method comprising administering to a subject in need an anti-BAFF nanobody or an antigen-binding fragment thereof according to a first aspect of the present invention, or an immunoconjugate according to a sixth aspect, or a multispecific antibody according to a seventh aspect, or a pharmaceutical composition according to an eighth aspect, wherein the disease is selected from:

[0053] 1) Diseases related to BAFF;

[0054] 2) Autoimmune diseases, inflammatory diseases, or tumors;

[0055] 3) BAFF-mediated or BAFF-abnormally expressed autoimmune diseases, inflammatory diseases, or tumors;

[0056] 4) Diseases with abnormal immune responses.

[0057] In one alternative example, the aforementioned disease is a B-cell tumor or an autoimmune or inflammatory disease involving B cells.

[0058] In one alternative example, the aforementioned diseases are selected from: lupus nephritis, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, connective tissue diseases, interstitial lung diseases, systemic scleroderma, diffuse scleroderma, immune thrombocytopenic purpura, multiple sclerosis (MS), myositis, polyangiitis, IgA nephropathy, glomerulonephritis, nephrotic syndrome, idiopathic membranous nephropathy, autoimmune hepatitis, chronic graft-versus-host disease, bronchial diseases, rheumatic diseases, B-cell non-Hodgkin's lymphoma, B-cell chronic lymphocytic leukemia, and multiple myeloma; preferably, the systemic lupus erythematosus includes adult-onset and pediatric systemic lupus erythematosus.

[0059] The main advantages of this invention are as follows: the anti-BAFF nanobody of this invention can effectively block the BAFF signaling pathway and effectively inhibit the biological activity of BAFF. It can be used to treat or prevent diseases such as SLE caused by BAFF-induced B cell activation, proliferation, or functional abnormalities, and its activity at multiple levels is superior to the positive control Belimumab. Furthermore, the nanobody of this invention also possesses excellent stability and can be considered a promising clinical drug.

[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 a polynucleotide molecule encoding the aforementioned antibody or its antigen-binding fragment. 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 a plasmid, viral vector (e.g., adenovirus, retrovirus), bacteriophage, yeast plasmid, or other vector. 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 vector preferably includes pDR1, pcDNA3.4, 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] BAFF

[0083] 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 signaling crucial for B cell formation and maintenance. Excessive BAFF levels can lead to abnormally high antibody production, exhibiting autoimmunity. BAFF exists in three forms: membrane-bound (mbBAFF), soluble trimerized BAFF (sBAFF), and a polymeric form composed of 60 BAFF monomers.

[0084] "BAFF-related diseases" refer to diseases or symptoms caused by BAFF activity, BAFF mediation, or abnormal BAFF expression, such as autoimmune diseases, inflammatory diseases, tumors, or cancers. Autoimmune diseases include, but are not limited to, lupus nephritis, systemic lupus erythematosus (SLE), connective tissue diseases, interstitial lung disease, systemic scleroderma, immune thrombocytopenic purpura, multiple sclerosis (MS), myositis, polyangiitis, IgA nephropathy, and glomerulonephritis; tumors or cancers include, but are not limited to, non-Hodgkin's lymphoma, B-cell chronic lymphocytic leukemia, and multiple myeloma.

[0085] 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.

[0086] Pharmaceutical Compositions and Applications

[0087] 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.

[0088] 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).

[0089] 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.

[0090] 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.

[0091] 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

[0092] Figure 1A shows the ELISA binding activity of BAFF-1A2-Fc with human BAFF.

[0093] Figure 1B ELISA binding activity of BAFF-3B2-Fc with human BAFF

[0094] Figure 2A ELISA blocking activity of BAFF-1A2-Fc against the binding of BAFF and its receptor BAFFR.

[0095] Figure 2B ELISA blocking activity of BAFF-3B2-Fc against the binding of BAFF and its receptor BAFFR.

[0096] Figure 3 Camel-derived chimeric anti-BAFF nanobody and 293FT-hBAFF △85-136 The binding activity (the vertical axis represents the ratio of the MFI of the sample at the same concentration to the MFI of the positive control Belimumab, with Beli being 1)

[0097] Figure 4A UPLC-SEC spectrum of humanized anti-BAFF nanobody hBAFF-1A2-Fc

[0098] Figure 4B shows the UPLC-SEC spectrum of the humanized anti-BAFF nanobody hBAFF-3B2-Fc.

[0099] Figure 5A DSC spectrum of humanized anti-BAFF nanobody hBAFF-1A2-Fc

[0100] Figure 5B DSC spectrum of humanized anti-BAFF nanobody hBAFF-3B2-Fc

[0101] Figure 5C DSC spectrum of anti-PD-L1 nanobody KN035

[0102] Figure 6A shows the ELISA binding activity of the humanized anti-BAFF nanobody to human BAFF.

[0103] Figure 6B shows the ELISA binding activity of the humanized anti-BAFF nanobody to cynomolgus monkey BAFF.

[0104] Figure 7. Cell-blocking activity of humanized anti-BAFF nanobody against BAFF.

[0105] Figure 8A. Inhibitory effect of humanized anti-BAFF nanobody on anti-dsDNA in spontaneous SLE mouse model.

[0106] Figure 8B shows the inhibitory effect of humanized anti-BAFF nanobody on IgA in a spontaneous SLE mouse model.

[0107] Figure 8C shows the inhibitory effect of humanized anti-BAFF nanobody on IgG in a spontaneous SLE mouse model.

[0108] Figure 8. Inhibitory effect of humanized anti-BAFF nanobody on splenomegaly in spontaneous SLE mouse model. Detailed Implementation

[0109] The sequence of the present invention is shown in the table below:

[0110] The antibody sequences of this invention all adopt the Kabat system numbering rules.

[0111] Experimental materials

[0112] pcDNA 3.4: Brand Thermo Fisher, product number A14697.

[0113] HEK-293F: Brand: Thermo Fisher, Product No.: A14527.

[0114] 0.45μm filter: brand Millipore, part number SLHV013SL.

[0115] Hitrap Mabselect Sure affinity chromatography column: Brand: Sitopan, Product No. 11003493.

[0116] Waters MassPREP™ Micro Desalting Column: Brand: Waters, Model: 186004032.

[0117] E. coli Top 10 competent cells: Brand: Shenggong, Product No.: B528412.

[0118] Endotoxin-free plasmid extraction kit: Brand: Tiangen, Product No.: DP117.

[0119] DNA Purification and Recovery Kit: Brand: Tiangen, Product No.: DP214.

[0120] ClonExpress™ II One Step Cloning Kit: Brand: Novizan, Product No.: C112.

[0121] HS DNA Polymerase: Brand: Takara, Product No.: R010B.

[0122] Coating solution: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L with double distilled water.

[0123] PBST: PBS + 0.05% Tween 20.

[0124] Tween 20: Brand Aladdin, item number T104863.

[0125] ELISA blocking solution: PBST + 1% BSA.

[0126] BSA: Brand Shenggong, Item No. A60332.

[0127] TMB: Brand BD Biosciences, catalog number 555214.

[0128] Termination solution: 2M sulfuric acid solution.

[0129] Recombinant human BAFF protein: Brand: Kaika Biotechnology, Product No.: BAF-HM112.

[0130] Goat anti-human IgG (Fc specific)-HRP antibody: brand Merck, catalog number A0170.

[0131] Streptavidin HRP: Brand BD, Product No. 554066.

[0132] Lipofectamine 3000 transfection reagent: Brand: Thermo Fisher, Product No.: L3000015.

[0133] 0.25% Pancreatic Enzyme-EDTA: Brand GIBCO, Product No. 25200-072.

[0134] FBS: Brand GIBCO, Item No. 10099-141.

[0135] Pen Strep: Brand GIBCO, Product No. 1514022.

[0136] Sodium pyruvate: Brand GIBCO, Product No. 11360-070.

[0137] GlutaMAX: Brand GIBCO, Product No. 35050-061.

[0138] Goat anti-human FITC secondary antibody: Brand Thermo Fisher, Product No. MA1-10379.

[0139] Goat anti-human PE secondary antibody: Brand: Jackson ImmunoResearch, Product No. 109-115-098.

[0140] MTS / PMS: Brand Promega, Product No. G5421.

[0141] Example 1. Construction of Camel-Derived Chimeric Anti-BAFF Nanobody

[0142] 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: 1) and BAFF-3B2 (SEQ ID NO: 2). BAFF-1A2 was linked to the human Fc domain (SEQ ID NO: 3) via adapter L1 (SEQ ID NO: 4) to obtain the camel-derived chimeric anti-BAFF nanobody BAFF-1A2-Fc (SEQ ID NO: 5); BAFF-3B2 was linked to the human Fc domain via adapter L1 to obtain the camel-derived chimeric anti-BAFF nanobody BAFF-3B2-Fc (SEQ ID NO: 6).

[0143] Example 2. Purification of Camel-Derived Chimeric Anti-BAFF Nanobodies

[0144] The nucleotide sequence of the camel-derived chimeric 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. 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. The purity of the purified protein was determined by UPLC-SEC.

[0145] Example 3. Binding activity of camel-derived chimeric anti-BAFF nanobody to BAFF

[0146] The binding activity of camel-derived chimeric anti-BAFF nanobody to human BAFF was determined by ELISA, with the anti-BAFF monoclonal antibody Belimumab used as a positive control (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). doi:10.3389 / fimmu.2018.02698). The specific detection method is as follows:

[0147] Dilute recombinant human BAFF with coating buffer to 100 ng / mL, and add 100 μL / well to each well of 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 each well of the microplate and incubate at room temperature for 2 hours. Remove the blocking buffer (remove any residual droplets with absorbent paper). Dilute the camel-derived chimeric anti-BAFF nanobody and the positive control Belimumab with blocking buffer to 10 μg / mL, then perform four-fold dilutions to create 12 concentration gradients. Add 100 μL / well to each well of the diluted solution 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 with blocking buffer at a ratio of 1:3000, and add 100 μL / well to each well of the microplate. 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 .

[0148] The experimental results are shown in Figures 1A-1B. Both camel-derived chimeric anti-BAFF nanobodies BAFF-1A2-Fc and BAFF-3B2-Fc can bind to human BAFF, EC... 50 The concentrations were 0.039 nM and 0.031 nM, respectively, for the positive control EC. 50 The binding activities of BAFF-1A2-Fc and BAFF-3B2-Fc were 0.117 nM and 0.133 nM, respectively, which were superior to those of the positive control.

[0149] Example 4. Blocking activity of camel-derived chimeric anti-BAFF nanobody against the binding of BAFF and its receptor BAFFR

[0150] The blocking activity of camel-derived chimeric anti-BAFF nanobody against human BAFF binding to the BAFFR receptor was determined by ELISA. The specific detection method is as follows: Dilute recombinant human BAFFR with coating buffer to 500 ng / mL, and add 100 μL / well to each well of the ELISA plate, incubating overnight at 4°C. Remove the coating buffer (remove any residual droplets with absorbent paper), add 200 μL / well of blocking buffer to the ELISA plate, and incubate at room temperature for 2 hours. Remove the blocking buffer (remove any residual droplets with absorbent paper), dilute biotin-labeled recombinant human BAFF with the blocking buffer to 1 μg / mL, and then dilute the camel-derived chimeric anti-BAFF nanobody and the positive control Belimumab with this solution to 500 nM, creating 12 concentration gradients through two-fold dilutions. Add 100 μL / well sequentially to each well of the blocked ELISA plate, and incubate at 37°C for 1 hour. Wash the plate three times with PBST (remove any residual droplets with absorbent paper). Dilute 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 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 .

[0151] The experimental results are shown in Figures 2A-2B. Both the camel-derived chimeric anti-BAFF nanobody BAFF-1A2-Fc and the positive control Belimumab can block the binding of human BAFF to BAFFR, with IC50 values ​​of 0.031 nM and 0.035 nM, respectively. Similarly, both the camel-derived chimeric anti-BAFF nanobody BAFF-3B2-Fc and the positive control Belimumab can block the binding of human BAFF to BAFFR, with IC50 values ​​of 0.031 nM and 0.035 nM, respectively. 50 The concentrations were 0.029 nM and 0.031 nM, respectively. The blocking activities of BAFF-1A2-Fc and BAFF-3B2-Fc were superior to those of the positive control.

[0152] Example 5. Camel-derived chimeric anti-BAFF nanobody and 293FT-hBAFF △85-136 Binding activity

[0153] Constructing 293FT-hBAFF △85-136 Cell line: The packaged viral vector pLVX-hBAFF was transfected using Lipofectamine 3000 transfection reagent. △85-136 (The human BAFF gene [gene number Q9Y275-1] was inserted into the vector, with amino acid deletions at positions 85-136) 293FT cells were transfected and cultured in puromycin-containing medium until the cells reached stable growth to obtain 293FT-hBAFF. △85-136 Cell populations were further diluted to 2-3 cells / mL and added to 96-well plates for single-clone sorting to obtain 293FT-hBAFF cells. △85-136 Cell lines. Flow cytometry was used to detect camel-derived chimeric anti-BAFF nanobodies BAFF-1A2-Fc and BAFF-3B2-Fc, as well as the positive control Belimumab and 293FT-hBAFF. △85-136 The binding activity of cell lines.

[0154] The experimental results are shown in Figure 3. The vertical axis represents the ratio of the mean fluorescence intensity (MFI) of the camel-derived chimeric anti-BAFF nanobody to the MFI of the positive control Belimumab. The camel-derived chimeric anti-BAFF nanobodies BAFF-1A2-Fc and BAFF-3B2-Fc are compared with 293FT-hBAFF. △85-136 The binding activity of the cell line was higher than that of the positive control Belimumab.

[0155] Example 6. Humanization of Camel-Derived Anti-BAFF Nanobody

[0156] The camel-derived chimeric anti-BAFF nanobody was humanized. The CDR loop structure of the VHH domain was determined. The closest homologous sequence for each V / J region of the VHH domain was found in the human ancestral sequence database. The human ancestry that best matched the VHH domain and the lowest amount of reversion mutation 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. Finally, the humanized anti-BAFF nanobodies hBAFF-1A2-Fc (SEQ ID NO.9) and hBAFF-3B2-Fc (SEQ ID NO.10) were obtained.

[0157] Example 7. Purification of humanized anti-BAFF nanobody

[0158] The nucleotide sequence of the humanized 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. 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. The purity of the purified protein was determined by UPLC-SEC.

[0159] The UPLC-SEC test results are shown in Figures 4A-4B. The protein purity of the humanized anti-BAFF nanobody hBAFF-1A2-Fc is 96.02%, and the protein purity of the humanized anti-BAFF nanobody hBAFF-1A2-Fc is 98.16%.

[0160] Example 8. Determination of the thermal stability of humanized anti-BAFF nanobody

[0161] Differential scanning calorimetry (DSC) was used to determine the change in molar heat capacity of the anti-humanized anti-BAFF nanobody as a function of temperature to assess its thermal stability. Solutions of the humanized anti-BAFF nanobody hBAFF-1A2-Fc and hBAFF-3B2-Fc were diluted to 0.5 mg / mL with 1×PBS (pH 7.4) buffer. Anti-PD-L1 nanobody KN035 (IMGT / 2D structure-DB card for INN 10930) was treated in the same manner as a control. 500 μL of each was analyzed using an automated capillary differential scanning calorimeter.

[0162] The DSC spectra are shown in Figures 5A-5C. With increasing temperature, the Tm1 and Tm2 of the humanized anti-BAFF nanobody hBAFF-1A2-Fc were 69.63℃ and 84.83℃, respectively, while those of the humanized anti-BAFF nanobody hBAFF-3B2-Fc were 71.03℃ and 84.84℃, respectively. The Tm onsets of hBAFF-1A2-Fc and hBAFF-3B2-Fc were 58.58℃ and 61.35℃, respectively. In contrast, the Tm1 and Tm2 of the control nanobody KN035 were 66.17℃ and 86.89℃, respectively, with a Tm onset of 57.87℃. These results indicate that the thermal stability of hBAFF-3B2-Fc and hBAFF-1A2-Fc is superior to that of KN035.

[0163] Example 9. ELISA binding activity of humanized anti-BAFF nanobody to BAFF

[0164] The binding activity of humanized anti-BAFF nanobody to human BAFF (gene ID Q9Y275-1, Thr141-Leu285) and cynomolgus monkey BAFF (gene ID A0A2K5V2X4, Thr141-Leu285) 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 200 ng / mL and recombinant cynomolgus monkey BAFF to 1 μg / mL with coating buffer, and add 100 μL / well to each well of the plate. 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). Dilute the humanized anti-BAFF nanobody and the positive control Belimumab to 10 μg / mL with the blocking buffer, then perform a four-fold dilution to create 12 concentration gradients. Add 100 μL / well to each well of the diluted solution 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 buffer and add 100 μL / well 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 / well of TMB and incubate at room temperature in the dark for 5 minutes. 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 .

[0165] The binding of human BAFF to human BAFF is shown in Figure 6A. Humanized anti-BAFF nanobodies hBAFF-1A2-Fc and hBAFF-3B2-Fc, as well as the positive antibody Belimumab, can all bind to human BAFF. EC 50 The binding activity of the humanized anti-BAFF nanobody was superior to that of the positive control at concentrations of 0.035 nM, 0.038 nM, and 0.080 nM, respectively. The binding to cynomolgus monkey BAFF is shown in Figure 6B. The humanized anti-BAFF nanobody hBAFF-1A2-Fc, hBAFF-3B2-Fc, and the positive antibody Belimumab all bound to cynomolgus monkey BAFF, EC50, 0.035 nM, 0.038 nM, and 0.080 nM. 50 The binding activity of the humanized anti-BAFF nanobody was comparable to that of the positive control at concentrations of 0.018 nM, 0.021 nM, and 0.020 nM, respectively.

[0166] Example 10. Cell-blocking activity of humanized anti-BAFF nanobody against BAFF and BAFFR binding.

[0167] Construction of Jurkat BAFFR:Fas reporter cell line: Jurkat cells were transfected with the packaged viral vector pLVX-BAFFR:Fas (the vector contains human BAFFR and Fas genes) using Lipofectamine 3000 transfection reagent. The cells were cultured and passaged in puromycin-containing medium until they reached stable growth to obtain Jurkat BAFFR:Fas reporter 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 Jurkat BAFFR:Fas reporter cell line. Jurkat BAFFR:Fas reporter cells were diluted with culture medium at a concentration of 100,000 cells / 100 μL / well and added to 96-well plates. Simultaneously, human BAFF was diluted with culture medium to 500 ng / mL. Humanized anti-BAFF nanobodies hBAFF-1A2-Fc and hBAFF-3B2-Fc, as well as the positive control Belimumab, were then diluted with this solution to 500 nM, creating nine concentration gradients through three-fold dilutions. The plates were incubated at 37°C for 1 hour, and then 100 μL / well was added to each well of the cell-containing 96-well plate. After incubation at 37°C for 24 hours, 40 μL of MTS / PMS was added, and the reaction was continued at 37°C for 3 hours. The OD value at 490 nm was read using a microplate reader, and the IC50 was calculated. 50 .

[0168] The experimental results are shown in Figure 7. Humanized anti-BAFF nanobodies hBAFF-1A2-Fc, hBAFF-3B2-Fc, and the positive antibody Belimumab can all block the binding of BAFF to its receptor BAFFR, with an IC50 concentration of [missing value]. 50 The concentrations were 2.644 nM, 2.306 nM, and 3.609 nM, respectively. Compared with the positive control, the humanized anti-BAFF nanobodies hBAFF-1A2-Fc and hBAFF-3B2-Fc showed superior blocking activity.

[0169] Example 11. Inhibitory effect of humanized anti-BAFF nanobody on spontaneous SLE symptoms in C57BL / 6JGpt-Tg(hBAFF) mice

[0170] C57BL / 6JGpt-Tg(hBAFF) mice (humanized BAFF mice, background C57BL / 6) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. This mouse model can exhibit typical SLE symptoms, such as elevated SLE serological markers and autoantibodies, including anti-double-stranded DNA antibody (anti-dsDNA), immunoglobulin A (IgA) and immunoglobulin G (IgG), and can also cause symptoms such as visceral enlargement.

[0171] Female C57BL / 6JGpt-Tg (hBAFF) mice, aged 8-10 weeks, were acclimatized for 3-7 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: a model control group (G2, n=6), an hBAFF-Fc-1A2 test product group (G3, n=8), an hBAFF-Fc-3B2 test product group (G4, n=8), and a Belimumab positive control group (G5, n=8). A healthy C57BL / 6JGpt mouse model group (G1, n=5) was also established. Grouping types and administration methods are shown in Table 1. Administration was once weekly for 22 weeks. Mice were weighed weekly. Serum was collected at week 22 for ELISA detection of anti-dsDNA, IgG, and IgA. Mice were euthanized at week 22, and the spleen was collected and weighed.

[0172] Table 1. Grouping and administration methods of C57BL / 6JGpt-Tg(hBAFF) mouse models

[0173] The results of SLE serological markers and autoantibody levels are shown in Figures 8A-8C. Compared with the G1 (healthy control) group, the levels of anti-dsDNA, IgA, and IgG in G2 (hBAFF+Vehicle) mice were significantly increased at week 22 of drug administration; the anti-dsDNA results are shown in Figure 8A. Compared with the G2 (hBAFF+Vehicle) group, the anti-dsDNA levels in G3 (hBAFF+hBAFF-Fc-1A2) and G4 (hBAFF+hBAFF-Fc-3B2) mice were significantly decreased at week 22 of drug administration, while the anti-dsDNA level in G5 (hBAFF+Belimumab) mice showed no significant change; the IgA results are shown in Figure 8B. Compared with the G2 (hBAFF+Vehicle) group, the levels of anti-dsDNA in G3 (hBAFF+hBAFF-Fc-1A2) and G4 (hBAFF+hBAFF-Fc-3B2) mice were significantly decreased at week 22 of drug administration, while the levels of anti-dsDNA in G5 (hBAFF+Belimumab) mice showed no significant change; the IgA results are shown in Figure 8B. In the G2 (hBAFF+Vehicle) group, the IgA levels of G3 (hBAFF+hBAFF-Fc-1A2) and G4 (hBAFF+hBAFF-Fc-3B2) mice were significantly reduced at week 22 of drug administration, while the IgA levels of G5 (hBAFF+Belimumab) mice showed no significant change. The IgG results are shown in Figure 8C. Compared to the G2 (hBAFF+Vehicle) group, the IgG levels of G3 (hBAFF+hBAFF-Fc-1A2), G4 (hBAFF+hBAFF-Fc-3B2), and G5 (hBAFF+Belimumab) mice were significantly reduced at week 22 of drug administration. Compared to the positive control group, the test group showed significant inhibitory effects on anti-dsDNA, IgA, and IgG levels at week 22 of drug administration, all of which were superior to the control group. Compared with hBAFF-Fc-3B2, hBAFF-Fc-1A2 showed better inhibitory activity at week 22 of administration.

[0174] The spleen weighing results are shown in Figure 8D. Compared with the G1 (healthy control) group, the spleen weight of G2 (hBAFF+Vehicle) mice was significantly increased. Compared with the G2 (hBAFF+Vehicle) group, the spleen weight of G3 (hBAFF+hBAFF-Fc-1A2) and G4 (hBAFF+hBAFF-Fc-3B2) mice was significantly decreased, and the spleen weight of G5 (hBAFF+Belimumab) mice was also significantly decreased. Compared with the positive control group, the test sample group showed a better inhibitory effect on splenomegaly. Among hBAFF-Fc-1A2 and hBAFF-Fc-3B2 in the test sample group, hBAFF-Fc-3B2 showed a better inhibitory effect on splenomegaly.

[0175] in conclusion

[0176] The above experiments show that the anti-BAFF nanobodies hBAFF-1A2-Fc and hBAFF-3B2-Fc provided by this invention can bind to the ligand BAFF and effectively block the binding of the ligand BAFF to its receptor BAFFR. In a spontaneous mouse SLE model, the anti-BAFF nanobodies hBAFF-1A2-Fc and hBAFF-3B2-Fc can effectively inhibit the increase of SLE serological markers and autoantibodies, as well as typical SLE symptoms such as splenomegaly, with significant effects. Compared with Belimumab, the anti-BAFF nanobodies outperformed Belimumab in vitro, at the cellular level, and in a spontaneous mouse SLE model, especially in the mouse model. This indicates that nanobodies can exhibit better efficacy than monoclonal antibodies in treating diseases caused by abnormal B cell survival and proliferation due to BAFF.

Claims

1. An anti-BAFF nanobody or its antigen-binding fragment, characterized in that, Its heavy chain variable region includes HCDR2 as shown in SEQ ID NO:16 and HCDR3 as shown in SEQ ID NO:17 or 19.

2. The nanobody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain variable region contains HCDR1-3 selected from the following groups: 1) SEQ ID NO: 15, 16, 17; 2) SEQ ID NO: 18, 16, 19.

3. The nanobody 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, 2, 7, 8; or 2) The heavy chain variable region is selected from amino acid sequences having at least 99%, 95%, 90%, 85%, or 80% identity with SEQ ID NO: 1, 2, 7, or 8; or 3) The heavy chain variable region includes HCDR1-3 of 1) or 2).

4. The nanobody or its antigen-binding fragment as described in claim 1, characterized in that, It also includes a heavy chain constant region structural domain; preferably, the heavy chain constant region structural domain is selected from human IgG1, human IgG2, human IgG3, human IgG4 or fragments thereof.

5. The nanobody as described in claim 4, characterized in that, It contains amino acid sequences selected from SEQ ID NO:5, 6, 9, and 10.

6. An anti-BAFF nanobody or its antigen-binding fragment, characterized in that, It has at least one of the following properties: 1) The antibody or its antigen-binding fragment according to any one of claims 1-5 binds to the same BAFF epitope; 2) Competes with the antibody or its antigen-binding fragment according to any one of claims 1-5 for binding to the BAFF epitope.

7. A polynucleotide molecule, characterized in that, The polynucleotide molecule encodes the anti-BAFF nanobody or its antigen-binding fragment as described in any one of claims 1-6; preferably, the polynucleotide molecule is selected from SEQ ID NO:11-14.

8. An expression carrier, characterized in that, The expression vector contains the polynucleotide molecule as described in claim 7.

9. A host cell, characterized in that, The host cell contains the expression vector as described in claim 8 or its genome is integrated with the polynucleotide as described in claim 7.

10. A method for preparing anti-BAFF nanobodies or antigen-binding fragments thereof, characterized in that, Including the following steps: 1) Culturing the host cells of claim 9 to obtain a culture containing the anti-BAFF nanobody or its antigen-binding fragment; 2) Isolate or recover the anti-BAFF nanobody or its antigen-binding fragment from the culture; 3) Optionally, purify and / or modify the anti-BAFF nanobody or its antigen-binding fragment obtained in step 2).

11. An immunoconjugate, characterized in that, The immunoconjugate contains: 1) The anti-BAFF nanobody or its antigen-binding fragment as described in any one of claims 1-6; and 2) The coupling part selected from the following group: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes or combinations thereof.

12. A multispecific antibody, characterized in that, It contains the anti-BAFF nanobody or its antigen-binding fragment as described in any one of claims 1-6.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: an anti-BAFF nanobody or its antigen-binding fragment as described in any one of claims 1-6, an immunoconjugate as described in claim 11, or a multispecific antibody as described in claim 12; and a pharmaceutically acceptable carrier or excipient.

14. Use of an anti-BAFF nanobody or its antigen-binding fragment as described in any one of claims 1-6, or an immunoconjugate as described in claim 11, or a multispecific antibody as described in claim 12, or a pharmaceutical composition as described in claim 13, characterized in that, The uses include any of the following applications: 1) To prepare drugs for the prevention and / or treatment of diseases associated with BAFF; 2) To prepare drugs for the prevention and / or treatment of autoimmune diseases, inflammatory diseases or tumors, kidney diseases, and graft rejection; 3) Prepare drugs for the prevention and / or treatment of BAFF-mediated or BAFF-abnormally expressed autoimmune diseases, inflammatory diseases or tumors; preferably B-cell tumors or autoimmune diseases or inflammatory diseases involving B cells; 4) Prepare drugs that reduce immune response.

15. The use as described in claim 14, characterized in that, The diseases are selected from: lupus nephritis, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, connective tissue diseases, interstitial lung diseases, systemic scleroderma, diffuse scleroderma, immune thrombocytopenic purpura, multiple sclerosis (MS), myositis, polyangiitis, IgA nephropathy, glomerulonephritis, nephrotic syndrome, idiopathic membranous nephropathy, autoimmune hepatitis, chronic graft-versus-host disease, bronchial diseases, rheumatic diseases, B-cell non-Hodgkin's lymphoma, B-cell chronic lymphocytic leukemia, and multiple myeloma; preferably, the systemic lupus erythematosus includes adult-onset and pediatric systemic lupus erythematosus.

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