Antigen-binding polypeptide against human b lymphocyte stimulator factor receptor baffr and use thereof

Abstract

The present invention relates to an antigen-binding polypeptide against human B lymphocyte stimulator factor receptor BAFFR and the use thereof. Specifically, the present invention relates to an anti-BAFFR nanobody. Also provided in the present invention are a nucleotide sequence encoding the antigen-binding polypeptide, an expression vector or a host cell containing the nucleotide sequence, and a method for preparing the nanobody. Further provided are a pharmaceutical composition containing the nanobody of the present invention and the use thereof.

Description

Antigen binding polypeptides against human B lymphocyte stimulator receptor BAFFR and uses thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent document claims priority to and the benefit of Chinese patent application No. 202311250418.3, filed on September 26, 2023, entitled “Antigen-binding polypeptides against human B lymphocyte stimulator receptor BAFFR and uses thereof.” The entire contents of the aforementioned patent application are incorporated by reference into this patent document. Technical Field

[0003] The present invention relates to the field of molecular biology and biomedicine technology, and in particular to a nanobody (VHH antibody) that specifically binds to a B lymphocyte stimulator receptor BAFFR, a preparation method thereof, and related uses. Background Art

[0004] B lymphocyte stimulator factor (BAFF; also known as TNFSF13B) is a cytokine belonging to the tumor necrosis factor family and a key factor in B cell survival and maturation. BAFF can promote B cell survival by promoting B cell proliferation and / or differentiation, and plays a vital role in the generation and maintenance of mature B cells. Dysregulation of B cell development can lead to immunodeficiency, escape of autoimmune B cells, and the production of autoimmune antibodies, and is a key factor in the initiation and progression of autoimmune diseases and B cell-related tumors. As an important immunoregulatory molecule, BAFF has strong B cell chemotaxis and plays a key role in maintaining B cell homeostasis. Abnormally high expression of BAFF can inhibit the death of autoreactive B cells and induce the production of autoimmune antibodies, thereby disrupting the balance of autoimmune tolerance and leading to the development of autoimmune diseases or tumors.

[0005] Research has shown that BAFF is involved in the progression of various autoimmune diseases. BAFF expression levels are significantly elevated in many autoimmune diseases. For example, in patients with systemic lupus erythematosus, BAFF expression is consistently high, and serum BAFF concentrations are positively correlated with anti-dsDNA antibody titers. Furthermore, BAFF expression levels are significantly elevated in the serum of patients with Sjögren's syndrome, rheumatoid arthritis, and multiple sclerosis.

[0006] BAFF can bind to three membrane receptors of the TNFR family on the surface of B cells, namely BAFFR (BAFF receptor, B lymphocyte stimulator receptor), TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor), and BCMA (B cell maturation antigen, B cell maturation antigen). Among them, the binding of BAFF and BAFFR plays the most important role in the maturation of peripheral B cells.

[0007] Currently, B cell deletion using CD19 or CD20 antibodies is the main treatment for autoimmune diseases, but the treatment effect is not ideal. On the one hand, CD19 or CD20 antibodies target all immature and mature B cells, which can easily cause immunodeficiency and even infection in patients. On the other hand, CD19 or CD20 antibodies may reduce patients' responsiveness to vaccines, such as the COVID-19 vaccine. In addition, autoantibodies exist in patients with autoimmune diseases, such as dsDNA antibodies in patients with systemic lupus erythematosus, MOG antibodies in patients with multiple sclerosis, and AQP4 antibodies in patients with neuromyelitis optica, and CD20 monoclonal antibodies cannot effectively reduce the concentration of these pathogenic autoantibodies in patients (30-70%).

[0008] Currently, therapeutic antibodies targeting BAFFR, such as lanalumab, have been developed. Lanalumab is a human-mouse chimeric antibody. These antibodies are typically produced by obtaining the target antibody's V region (variable region) gene from hybridoma cells producing a mouse monoclonal antibody. This gene is then recombined with the human C region (constant region) gene and cloned into a suitable vector for expression in recipient cells. Consequently, these antibodies have a relatively low humanization ratio, typically 60-70%, potentially leading to stronger immunogenicity and lower safety.

[0009] Given the limitations and complexity of current treatments, there is still a need in this field to find new treatments and / or products that can eliminate specific pathogenic B cell subsets or regulate B cell activation and function in a more precise manner, thereby treating B cell-related diseases such as tumors or autoimmune diseases.

[0010] Summary of the Invention

[0011] To address the above issues, the inventors have screened nanobodies targeting BAFFR using phage display technology combined with a high-throughput expression platform. These nanobodies offer advantages such as simple humanization, high affinity, high stability, microbial expression, low immunogenicity, good solubility, strong penetration, and the ability to recognize hidden epitopes, thus completing the present invention. Furthermore, the present invention provides methods for preparing these nanobodies and their use in the treatment or diagnosis of autoimmune diseases and tumors.

[0012] Therefore, in a first aspect, the present invention provides an antigen-binding polypeptide specific for human BAFFR, wherein the antigen-binding polypeptide comprises a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2), and a complementarity determining region 3 (CDR3) selected from the following:

[0013] The amino acid sequence of the CDR1 is shown in SEQ ID NO: 8 or 9,

[0014] The amino acid sequence of the CDR2 is shown in any one of SEQ ID NOs: 14-17,

[0015] The amino acid sequence of the CDR3 is shown in any one of SEQ ID NOs: 22-25.

[0016] In a specific embodiment, the CDR1, CDR2 and CDR3 are selected from the following groups:

[0017] (a) the amino acid sequence of the CDR1 is shown in SEQ ID NO: 8;

[0018] The amino acid sequence of the CDR2 is shown in SEQ ID NO: 14; and

[0019] The amino acid sequence of the CDR3 is shown in SEQ ID NO: 22;

[0020] (b) the amino acid sequence of the CDR1 is shown in SEQ ID NO: 8;

[0021] The amino acid sequence of the CDR2 is shown in SEQ ID NO: 15; and

[0022] The amino acid sequence of the CDR3 is shown in SEQ ID NO: 23;

[0023] (c) the amino acid sequence of the CDR1 is shown in SEQ ID NO: 9;

[0024] The amino acid sequence of the CDR2 is shown in SEQ ID NO: 16; and

[0025] The amino acid sequence of the CDR3 is shown in SEQ ID NO: 24; or

[0026] (d) the amino acid sequence of the CDR1 is shown in SEQ ID NO: 9;

[0027] The amino acid sequence of the CDR2 is shown in SEQ ID NO: 17; and

[0028] The amino acid sequence of the CDR3 is shown in SEQ ID NO: 25.

[0029] Preferably, the antigen-binding polypeptide is an anti-BAFFR antibody. Preferably, the antibody comprises FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, for example as heavy chain variable regions.

[0030] More preferably, the anti-BAFFR antibody is an anti-BAFFR Nanobody, which consists of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0031] More preferably, the amino acid sequence of FR1 is selected from the sequence shown in any one of SEQ ID NOs: 5-7, the amino acid sequence of FR2 is selected from the sequence shown in any one of SEQ ID NOs: 10-13, the amino acid sequence of FR3 is selected from the sequence shown in any one of SEQ ID NOs: 18-21, and / or the amino acid sequence of FR4 is selected from the sequence shown in any one of SEQ ID NOs: 26-27.

[0032] More specifically, the anti-BAFFR Nanobody has an amino acid sequence selected from any one of the following:

[0033] (a) the amino acid sequence shown in SEQ ID NO: 1 (Nanobody NB467-55);

[0034] (b) the amino acid sequence shown in SEQ ID NO: 2 (Nanobody NB467-46);

[0035] (c) the amino acid sequence shown in SEQ ID NO: 3 (Nanobody NB467-35);

[0036] (d) The amino acid sequence shown in SEQ ID NO: 4 (Nanobody NB467-8).

[0037] In a second aspect, the present invention further provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the antigen-binding polypeptide. Preferably, the nucleic acid molecule comprises or consists of a nucleotide sequence selected from any one of SEQ ID NOs: 28-31.

[0038] In a third aspect, the present invention further provides a recombinant expression vector comprising the isolated nucleic acid molecule of the second aspect.

[0039] In a fourth aspect, the present invention further provides a host cell comprising the nucleic acid molecule of the second aspect or the recombinant expression vector of the third aspect. Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0040] In a fifth aspect, the present invention further provides a pharmaceutical composition comprising the antigen-binding polypeptide of the first aspect and a pharmaceutically acceptable carrier.

[0041] In a sixth aspect, the present invention further provides a fusion protein comprising the antigen-binding polypeptide of the first aspect. For example, the fusion protein is a chimeric antigen receptor, wherein the extracellular domain of the chimeric antigen receptor comprises the antigen-binding polypeptide of the first aspect, and the chimeric antigen receptor further comprises a transmembrane domain and an intracellular domain.

[0042] In a seventh aspect, the present invention further provides a method for preparing an anti-human BAFFR nanobody, comprising expressing the isolated nucleic acid molecule of the second aspect, or expressing the recombinant vector of the third aspect, or culturing the host cell of the fourth aspect, and optionally purifying.

[0043] In an eighth aspect, the present invention further provides use of the antigen-binding polypeptide of the first aspect, the isolated nucleic acid molecule of the second aspect, the recombinant expression vector of the third aspect, or the host cell of the fourth aspect in the preparation of a drug for treating or preventing autoimmune diseases or tumors.

[0044] Preferably, the autoimmune disease is selected from the following group: patients with nervous system immune diseases such as multiple sclerosis, neuromyelitis optica, myasthenia gravis, etc., and other autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, etc.

[0045] Preferably, the tumor is a hematological tumor, such as B-cell lymphoma, such as Hodgkin's lymphoma and nodular lymphocyte-predominant Hodgkin's lymphoma, and non-Hodgkin's lymphoma, such as diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma / chronic lymphocytic leukemia, mantle cell lymphoma (MCL).

[0046] In the ninth aspect, the present invention further provides a method for treating or preventing an autoimmune disease or tumor in a subject, comprising administering to the subject a therapeutically effective amount of the antigen-binding polypeptide of the first aspect, the isolated nucleic acid molecule of the second aspect, the recombinant expression vector of the third aspect, the host cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect.

[0047] The Nanobodies of the present invention have the superior properties of Nanobodies, such as small molecular weight and high degree of humanization, while also having excellent antigen binding specificity, the ability to competitively block the BAFF-BAFFR interaction and ADCC activity, and therefore have at least the following advantages:

[0048] (1) The anti-human BAFFR nanobody of the present invention has a high affinity for human BAFFR and has a stronger ADCC phagocytic effect than the existing anti-human BAFFR antibodies, and can induce macrophages to phagocytose tumor cells. 50 The values ​​were lower than those of the reference antibody.

[0049] (2) The anti-human BAFFR nanobody of the present invention has a stronger competitive blocking ability on BAFF-BAFFR interaction.

[0050] (3) Compared with monoclonal antibodies, especially chimeric antibodies, the anti-human BAFFR nanobody of the present invention has higher safety and lower immunogenicity.

[0051] (4) The nanoantibodies provided by the present invention have a small molecular weight and therefore have extremely strong tissue penetration, which can directly penetrate certain body barriers, facilitating the diagnosis and targeted treatment of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG1 is a graph showing the affinity of nanobodies and control antibodies to 293T cells overexpressing BAFFR on their surface as detected by flow cytometry. MFI refers to median fluorescence intensity.

[0053] FIG2 is a graph showing the half-inhibitory concentrations (IC50 values) of Nanobodies and control antibodies on BAFF-BAFFR interaction as determined by ELISA.

[0054] FIG3 is a graph showing the effective concentrations (EC50 values) of Nanobodies and control antibodies for inducing T cell cytotoxicity against BAFFR-overexpressing 293T cells as determined by ELISA. DETAILED DESCRIPTION

[0055] I. Terminology

[0056] In the context of the present invention, unless otherwise indicated, BAFFR refers to human BAFFR (Gene ID: 115650; nucleotide: NM_052945.4; amino acid: NP_443177.1), the amino acid sequence and nucleotide sequence of which are shown in SEQ ID NO: 39 and SEQ ID NO: 40, respectively.

[0057] "Antigen-binding polypeptide" refers to a polypeptide sequence that specifically binds to an antigen and can be a complete protein, polypeptide, modified or unmodified, or an antigen-binding fragment with immunological function.

[0058] "Binding to BAFFR" or "binding to BAFFR" refers to an interaction that enables specific binding to BAFFR.

[0059] "Nanobodies", also known as VHH antibodies, refer to single-domain antibodies obtained from camelids consisting only of a single variable domain on the heavy chain. Nanobodies or antigen-binding fragments thereof can be produced, for example, by recombinant technology, phage display technology, synthetic technology such as CDR grafting, or a combination of such or other techniques known in the art.

[0060] "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between an antibody and an antigen. Affinity can be measured by common methods known in the art, including those known in the art and described herein.

[0061] "Competition" refers to competition between antigen-binding polypeptides when applied to antigen-binding polypeptides (e.g., neutralizing antigen-binding polypeptides or neutralizing antibodies) that compete for the same epitope. Competition between antigen-binding polypeptides can be determined by an assay in which the antigen-binding polypeptide to be detected (e.g., an antibody or immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen-binding polypeptide (e.g., a ligand or reference antibody) to a common antigen (e.g., BAFFR or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding polypeptide competes with another. Inhibition of competitive binding can be measured by measuring the amount of a marker bound to a solid surface or cell in the presence of the antigen-binding polypeptide being measured. Typically, the antigen-binding polypeptide being measured is present in excess.

[0062] "Treatment" means administering a therapeutic agent, either internally or externally, such as a composition comprising an antigen-binding polypeptide that specifically binds BAFFR, to a patient experiencing one or more symptoms of a disease. Generally, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated, whether by inducing regression of such symptoms or inhibiting progression of such symptoms to any clinically measurable degree.

[0063] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0064] A "pharmaceutical composition" refers to a mixture containing one or more antigen-binding polypeptides described herein and other pharmaceutical ingredients, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient, and thereby exert its biological activity.

[0065] II. Antigen-binding polypeptides and nanobodies

[0066] The present invention provides a variety of antigen-binding polypeptides that specifically bind to BAFFR. In some embodiments, the antigen-binding polypeptide that specifically binds to BAFFR is an antibody, preferably a nanobody.

[0067] In some embodiments, the antibody, preferably a Nanobody, comprises a complementarity determining region 1 (CDR1 ), a complementarity determining region 2 (CDR2) and a complementarity determining region 3 (CDR3).

[0068] In a preferred embodiment, the CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 8 or 9, or comprises or consists of an amino acid sequence having 1 or 2 amino acid differences from the amino acid sequence shown in SEQ ID NO: 8 or 9. In a more preferred embodiment, the amino acid sequence of the CDR1 is shown in SEQ ID NO: 8 or SEQ ID NO: 9.

[0069] In a preferred embodiment, the CDR2 comprises or consists of an amino acid sequence as shown in any one of 14 to 17, or comprises or consists of an amino acid sequence having 1 or 2 amino acid differences from an amino acid sequence as shown in any one of 14 to 17. In a more preferred embodiment, the amino acid sequence of the CDR2 is as shown in any one of 14 to 17.

[0070] In a preferred embodiment, the CDR3 comprises or consists of an amino acid sequence as shown in any one of 22 to 25, or comprises or consists of an amino acid sequence having 1 or 2 amino acid differences from an amino acid sequence as shown in any one of 22 to 25. In a more preferred embodiment, the amino acid sequence of the CDR2 is as shown in any one of 22 to 25.

[0071] In a more preferred embodiment, the CDR1, CDR2 and CDR3 are selected from the following groups:

[0072] (a) the amino acid sequence of the CDR1 is shown in SEQ ID NO: 8;

[0073] The amino acid sequence of the CDR2 is shown in SEQ ID NO: 14; and

[0074] The amino acid sequence of the CDR3 is shown in SEQ ID NO: 22;

[0075] (b) the amino acid sequence of the CDR1 is shown in SEQ ID NO: 8;

[0076] The amino acid sequence of the CDR2 is shown in SEQ ID NO: 15; and

[0077] The amino acid sequence of the CDR3 is shown in SEQ ID NO: 23;

[0078] (c) the amino acid sequence of the CDR1 is shown in SEQ ID NO: 9;

[0079] The amino acid sequence of the CDR2 is shown in SEQ ID NO: 16; and

[0080] The amino acid sequence of the CDR3 is shown in SEQ ID NO: 24; or

[0081] (d) the amino acid sequence of the CDR1 is shown in SEQ ID NO: 9;

[0082] The amino acid sequence of the CDR2 is shown in SEQ ID NO: 17; and

[0083] The amino acid sequence of the CDR3 is shown in SEQ ID NO: 25.

[0084] In a specific embodiment, the antibody, preferably a nanobody, comprises four framework sequences (FR) spaced apart from the three CDRs. Specifically, the antibody, preferably a nanobody, comprises FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 in that order.

[0085] In a specific embodiment, the antibody, preferably a Nanobody, comprises or consists of the amino acid sequence shown below:

[0086] (a) the amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto;

[0087] (b) the amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto;

[0088] (c) the amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto;

[0089] (d) the amino acid sequence as shown in SEQ ID NO: 4, or an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0090] The present invention also provides a nucleotide sequence encoding the antigen-binding polypeptide. In a preferred embodiment, the nucleotide sequence is selected from the group consisting of:

[0091] (a) the nucleotide sequence shown in SEQ ID NO: 28, or a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto and encoding the amino acid sequence shown in SEQ ID NO: 1;

[0092] (b) the nucleotide sequence as shown in SEQ ID NO: 29, or a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto and encoding the amino acid sequence as shown in SEQ ID NO: 2;

[0093] (c) the nucleotide sequence as shown in SEQ ID NO: 30, or a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto and encoding the amino acid sequence as shown in SEQ ID NO: 3;

[0094] (d) the nucleotide sequence shown in SEQ ID NO: 31, or a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto and encoding the amino acid sequence shown in SEQ ID NO: 4.

[0095] The present invention also provides an expression vector comprising a nucleotide molecule encoding an antigen-binding polypeptide of the present invention. The expression vector may contain a promoter, a ribosome binding site for translation initiation, a transcription terminator, and other regulatory elements. The promoter may be a promoter suitable for expression in a production cell.

[0096] The present invention also provides a host cell comprising a nucleotide molecule encoding an antigen-binding polypeptide of the present invention. The host cell can be used in the

[0097] Production of antigen-binding polypeptides. Host cells can be transiently or non-transiently transfected with the expression vectors of the present invention. In preferred embodiments, the cells are selected from CHO or a cell line derived therefrom, 293T or a cell line derived therefrom.

[0098] In some embodiments, the antigen-binding polypeptide of the present invention is a single domain antibody (VHH) or a heavy chain antibody. In some embodiments, the antigen-binding polypeptide is a humanized antibody and / or a fully human antibody. In some embodiments, the antigen-binding polypeptide is an antibody fragment, such as Fv, Fab, Fab', scFv, diabody or F(ab')2. In some embodiments, the antigen-binding polypeptide is a multispecific antibody, such as a bispecific antibody or a trispecific antibody. In some embodiments, the antigen-binding polypeptide of the present invention is a VHH antibody. In some embodiments, the antigen-binding polypeptide of the present invention is a VHH-Fc antibody fused to the Fc region of a human immunoglobulin, wherein the human immunoglobulin is preferably IgG, more preferably IgG1.

[0099] The present invention also provides a fusion protein comprising an antigen-binding polypeptide of the present invention. In one embodiment, the fusion protein is a chimeric antigen receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an internal signaling domain, wherein the extracellular domain comprises an antigen-binding polypeptide of the present invention, such as a nanobody of the present invention.

[0100] In some embodiments, the transmembrane domain of the CAR can be derived from a transmembrane domain commonly used in the art, such as a transmembrane domain from a T cell co-stimulatory molecule.

[0101] In some embodiments, the intracellular signaling domain of the CAR may include an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain may include an immunoreceptor tyrosine-based inhibition motif (ITIM). In some embodiments, the intracellular signaling domain can comprise an intracellular domain of a molecule selected from the group consisting of: Fcγ receptor (FcγR), Fcε receptor (FcεR), Fcα receptor (FcαR), neonatal Fc receptor (FcRn), CD3, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247ζ, CD247η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, and Zap70.

[0102] In some embodiments, the CAR may further include a costimulatory domain. In some embodiments, the costimulatory domain may include the following signal transduction domains: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors or Toll ligand receptors.

[0103] III. Application

[0104] The antigen-binding polypeptides or antibodies of the present invention, such as nanobodies, can specifically bind to BAFFR with high affinity, thereby inhibiting BAFF-BAFFR interaction, and are therefore particularly suitable for treating autoimmune diseases and tumors.

[0105] In one embodiment, the antigen-binding polypeptide or antibody, such as a Nanobody, of the invention is used to treat a subject suffering from an autoimmune disease. For example, the subject suffering from the autoimmune disease has elevated BAFF expression levels compared to a healthy subject.

[0106] In one embodiment, the antigen-binding polypeptides or antibodies such as Nanobodies of the invention are used to treat a subject suffering from a tumor, particularly a malignant tumor.

[0107] In the context of the present invention, the subject is a vertebrate, preferably a mammal, such as a human. Such mammals include, but are not limited to, rodents, monkeys, humans, farm animals, sports animals, and pets. The term "subject" is broadly understood to also encompass tissues, cells, and progeny of biological entities obtained in vivo or cultured in vitro.

[0108] The antigen-binding polypeptides, antibodies, nanobodies or fusion proteins of the present invention may be contained in pharmaceutical compositions. Said pharmaceutical compositions may be used for preventive and / or therapeutic treatments.

[0109] In therapeutic applications, the pharmaceutical compositions of the invention can be administered to a subject already suffering from a disease in an amount sufficient to cure or at least partially arrest the progression of the symptoms of the disease. The therapeutically effective amount can vary depending on the severity of the disease, the course of the disease, previous therapy, the subject's health, body weight, response to the drug, and the judgment of the treating physician. The antigen-binding polypeptides, antibodies, Nanobodies or fusion proteins of the invention can be used in combination with other therapeutic agents, for example, administered sequentially or simultaneously.

[0110] In preventive applications, the pharmaceutical compositions of the invention can be administered before, during, or after the onset of a disease. For example, the pharmaceutical compositions of the invention can be used as prophylactics to prevent the onset of a disease.

[0111] The antigen-binding polypeptides, antibodies, Nanobodies, fusion proteins or pharmaceutical compositions of the invention can be administered by injection, for example, direct injection, stereotactic injection, injection via a micropump infusion system. The injection can be performed by intravenous, parenteral, intraperitoneal and / or subcutaneous routes, thereby delivering the antigen-binding polypeptides, antibodies, Nanobodies, fusion proteins or pharmaceutical compositions to cells, tissues or organs of the subject.

[0112] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions or according to the conditions recommended by the manufacturer. Reagents for which specific sources are not specified are conventional reagents purchased from the market.

[0113] Example

[0114] The present invention will be further explained below in conjunction with specific examples. However, it should be noted that the following examples are only used to explain the present invention and are not intended to limit the present invention. All technical solutions that are the same or similar to the present invention are within the scope of protection of the present invention. Where specific techniques or conditions are not specified in the present examples, the operations were carried out in accordance with conventional technical methods and instrument specifications in the art; where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0115] Example 1. Obtaining anti-BAFFR nanobodies by phage display

[0116] In this example, alpacas were immunized with human BAFFR as an antigen to obtain a variety of antibody encoding fragments, and candidate antibody clones were subsequently screened using a phage library and a mammalian expression system. Specifically, the specific steps of the anti-BAFFR nanobody screening process are as follows.

[0117] a. Preparation of Antigen

[0118] Based on the amino acid and nucleotide sequences of human BAFFR, an antigen that can effectively induce alpacas to produce specific antibodies against human BAFFR was analyzed and designed. Human IgG1 Fc was linked to its C-terminus to obtain a modified antigen, designated "human BAFFR hFC antigen", whose amino acid sequence is shown in SEQ ID NO:33 and the coding sequence is shown in SEQ ID NO:32.

[0119] b. Immunize alpacas

[0120] The human BAFFR hFC antigen obtained in step a was thoroughly mixed with an equal volume of Freund's adjuvant and used for subcutaneous injection into alpacas.

[0121] Specifically, on day 1, alpacas were primed with an emulsified mixture of 500 μg of human BAFFR hFC antigen and an equal volume of Freund's complete adjuvant. Four booster immunizations were performed on days 21, 42, 63, and 84, using an emulsified mixture of 250 μg of human BAFFR hFC antigen and an equal volume of Freund's complete adjuvant. Seven days after the first four immunizations, 10 ml of peripheral blood was collected from the alpacas to measure the anti-BAFFR serum titer using ELISA.

[0122] The specific steps for ELISA testing are as follows. Human BAFFR his antigen was diluted to 2 μg / mL in 0.05 M carbonate buffer (pH 9.6) and added to the culture plate at 100 μL / well. The culture plate was coated overnight at 4°C. The coating solution was discarded and the plate was washed three times with PBST. 300 μL of 5% skim milk was added to each well and blocked at 37°C for 1 hour. The plate was washed three times with PBST buffer. 100 μL / well of serum dilutions (starting from 1:2000 dilutions) was added and incubated at 37°C for 45 minutes. The plate was washed five times with PBST. 100 μL of goat anti-Alpaca IgG (H+L) HRP (Chengdu Apac Biotechnology Co., Ltd., Cat#: S001H, Chengdu Apac Biotechnology Co., Ltd., diluted 1:1 in PBS) was added to each well and incubated at 37°C for 45 minutes. The plate was washed five times with PBST. TMB color development solution was added (100 μL / well) and incubated at 37°C for 5 min. Stop solution was added to terminate the reaction (50 μL / well) and the optical density was measured at 450 nm.

[0123] One week after the fifth immunization, 50 mL of peripheral blood was collected from the alpacas and mononuclear cells were isolated.

[0124] c. Library construction

[0125] Extract RNA from the PBMCs obtained in step b, and after reverse transcription, perform nested PCR to obtain the target gene fragment. Clone the target gene fragment into a eukaryotic expression vector, and transform the resulting expression vector into competent cells to construct a BAFFR-VHH phage display library. The specific steps are as follows.

[0126] Using the PBMC cells screened in step b as a template, total cell RNA was extracted using RNAiso Plus, and PrimeScript TM RNA was reverse transcribed into cDNA using the II 1st Strand cDNA Synthesis Kit. The target fragment was obtained by nested PCR. The amplification system for the target fragment is shown in Table 1, and the amplification procedure is shown in Table 2.

[0127] Table 1 Nested PCR one-round reaction system

[0128] Table 2 Nested PCR second round reaction system

[0129] The amplified nucleotide fragment encoding the alpaca VHH was cloned into the eukaryotic expression vector pComb3XSS (Chengdu Apak Biotechnology Co., Ltd.). The resulting recombinant vector was transformed into TG1 competent cells by electroporation to generate a VHH phage display library. To further verify the successful construction of the BAFFR-VHH phage display library, the library was cultured on 2-YT-A plates, and 48 clones from the resulting colonies were selected for sequencing.

[0130] The sequencing results showed that the successful insertion rate of the clones was 100%. Based on the number of transformants, library insertion rate and diversity sequencing analysis results, the library capacity was calculated to be 1.92×10 9 , indicating that the obtained BAFFR-VHH phage display library has good library capacity and diversity.

[0131] d. Acquisition of Nanobodies

[0132] The phage display library obtained in step c was used for panning and positive clones were selected. The coding sequence of the selected VHH antibody was fused with the coding sequence of human IgG1 Fc and constructed into the pTT5 expression vector (Chengdu Apec Biotechnology Co., Ltd.) for transfection into eukaryotic cells for expression and purification. Ultimately, an anti-human BAFFR nanobody was obtained. The specific method is as follows.

[0133] The SA-magnetic beads (MPHTSA-300, Suzhou Nawei Technology Co., Ltd.) were removed and washed twice with PBS for later use; Bio-Human BAFFR-his (Acro, BAR-H82E3) antigen was diluted with PBS to a final concentration of 5 μg / ml, and 2×10 11 The phage library was incubated at 37°C for 1 hour. The incubated mixture was added to a magnetic bead tube and shaken at 4°C for 45 minutes. The supernatant was aspirated under the action of a magnetic stand and washed three times with PBST and twice with PBS. 800 μL of Gly-HCl eluent was added and incubated at 37°C for 8 minutes to elute the specifically bound phage. The eluate was transferred to a 1.5 mL sterile centrifuge tube and quickly neutralized with 160 μL of Tris-HCl neutralization buffer. 10 μL was taken for serial dilution, the titer was determined, and the panning recovery rate was calculated. The remaining eluate was mixed, amplified and purified, and used for the next round of affinity panning.

[0134] After two rounds of screening, monoclonal phage supernatant was identified by ELISA after the second round of screening. Bio-Human BAFFR-his antigen was immobilized in a 96-well microtiter plate at a concentration of 2 μg / ml per 100 μl and incubated overnight at 4°C. The coating solution was discarded and the plates were washed three times with PBST. 300 μl of 5% skim milk was added to each well and blocked at 37°C for 1 hour. The plates were washed three times with PBST buffer, and 100 μl of two-fold diluted phage supernatant was added to each well and incubated at 37°C for 45 minutes. The plates were washed five times with PBST, and 100 μl of mouse anti-M13 antibody HRP (Chengdu Apoc Biotechnology Co., Ltd.) was added to each well and incubated at 37°C for 45 minutes. The plates were washed five times with PBST. TMB color development solution was added at 100 μl / well and incubated at 37°C for 5 minutes.

[0135] The reaction was terminated by adding 50 μL / well of 1M HCl stop solution, and the optical density was measured at 450 nm. Positive clones were screened based on the results (OD450 > 1). Human IgG1 Fc fusion constructs were constructed into the pTT5 plasmid and transfected into HEK293T mammalian cells. The cells were expressed for 7 days in a shaker at 37°C and 5% CO2. After culture, the cell supernatant was collected and purified using Protein A affinity media (Suzhou Nawei Technology Co., Ltd., 17010-050100) to obtain four target antibodies, designated NB467-55, NB467-46, NB467-35, and NB467-8.

[0136] The VHH amino acid sequences and nucleotide sequences of Nanobodies NB467-55, NB467-46, NB467-35, and NB467-8 are shown below.

[0137] Table 3. Sequence information of NB467-55, NB467-46, NB467-35, and NB467-8

[0138] Example 2. Binding of anti-BAFFR nanobodies to 293T cells overexpressing BAFFR

[0139] The four anti-BAFFR nanobodies NB467-55, NB467-46, NB467-35, and NB467-8 obtained in Example 1 were subjected to flow cytometry for characterization of binding affinity kinetics. Meanwhile, the anti-BAFFR antibody Lanalumab (Chengdu Apoc Biotechnology Co., Ltd.) was used as a positive control antibody.

[0140] Specifically, HEK-293T-BAFFR cells were collected from cell culture flasks, washed twice with FACS buffer, and centrifuged. The cells were resuspended in an appropriate volume of flow cytometry staining solution to a final concentration of 2 × 10 cells per tube. 5 cells / mL.

[0141] Antibodies NB467-55, NB467-46, NB467-35, and NB467-8, as well as lanalumab, generated in Example 1, were diluted to 200 nM. Add 50 μl of the antibody solution to the tube containing HEK-293T-BAFFR cells. Mix thoroughly and incubate at 4°C for 60 minutes. After incubation, wash the cells once with FACS buffer.

[0142] Anti-human FC-647 secondary antibody (Jackson, 109-605-003) was diluted according to the manufacturer's instructions, and 100 μl of the secondary antibody dilution was added to the sample tube. After mixing, the mixture was incubated at 4°C for 60 minutes. After incubation, the cells were washed three times with FACS buffer, and the cell pellet obtained by centrifugation was resuspended in 200 μl of PBS and analyzed using a flow cytometer (Sony, SA3800). The results are shown in Table 4 and Figure 1.

[0143] Table 4. Binding of Nanobodies to 293T cells overexpressing BAFF (EC 50 )

[0144] Figure 1 shows the mean fluorescence intensity (MFI) values ​​of each antibody, which confirms that the nanobody of the present invention has a stronger ability to bind to 293T cells overexpressing human BAFFR than Lanalumab. Table 4 shows that the nanobody EC 50 The values ​​were better than or close to the control antibody Lanalumab, among which NB467-46, NB467-55, and NB467-8 were stronger than NB467-35.

[0145] Example 3. Competitive blocking ability of human anti-BAFFR nanobodies on BAFF-BAFFR interaction

[0146] This example measured the competitive functional blocking ability of the four anti-BAFFR nanobodies obtained in Example 1 on the BAFF-BAFFR interaction. Lanalumab (same as above) was also used as a positive control antibody.

[0147] Specifically, human BAFFR-his protein (Acro, BAR-HP2H6) was added to a 96-well microplate at 200 ng / well and incubated overnight at 4°C. The next day, the plates were washed three times with PBST and blocked with PBST containing 5% w / v skim milk powder at 37°C for 1 hour. The plates were then washed again with PBS.

[0148] The test antibodies were diluted in a 1:3 series starting at 800 nM (using 5% skim milk). Biotin Human BAFF his protein (Acro, BAF-H82Q2) was also diluted to 0.2 μg / mL. Control group: 50 μL of diluted Biotin Human BAFF his protein mixed with 50 μL of 5% milk; experimental group: 50 μL of diluted Biotin Human BAFF his protein mixed with 50 μL of the serially diluted antibodies.

[0149] Then, the antibody / Biotin Human BAFF his mixture was added to the Human BAFFR-his protein-coated plate. After incubation at 37°C for 1 hour, it was washed 5 times with PBST. The secondary antibody (Chengdu Apak Biotechnology Co., Ltd.) diluted with blocking solution 1:10K was added and incubated at 37°C for 1 hour. The ELISA plate incubated with the secondary antibody was washed 5 times with PBST, 100 μl of TMB single-component colorimetric solution was added to each well, and incubated at 37°C for 7 minutes. The reaction was terminated with 50 μL / well 1M HCL, and the absorbance at 450 nm was measured. The data were analyzed using Graphpad Prism software to obtain the half inhibitory concentration (IC 50 The specific results are shown in Table 5 and Figure 2.

[0150] Table 5. Competitive blocking of BAFF-BAFFR interaction by various nanobodies

[0151] The results showed that compared with Lanalumab, the IC 50 Lower value, IC 50 The absorption rate can reach about one-tenth of that of Lanalumab or even less, indicating that the competitive blocking ability of the Nanobody of the present invention on the BAFF-BAFFR interaction is significantly stronger than that of Lanalumab. Figure 2 shows the absorbance value at 450nm, indicating that the antibody of the present invention can block the human BAFF-BAFFR interaction and has better BAFF-BAFFR blocking activity than the reference antibody.

[0152] Example 4. ADCC activity of anti-human BAFFR nanobody

[0153] This example measured the ADCC activities of the four anti-BAFFR nanobodies obtained in Example 1. Lanalumab (same as above) was also used as a positive control antibody.

[0154] Specifically, HEK293T-BAFFR cells as described in Example 2 were digested, harvested by centrifugation, resuspended in DEME + 1% FBS, and plated at 35,000 cells per well in a 96-well assay plate in a volume of 50 μl. Jurkat-NFAT-CD16a cells (purchased from Sichuan Sibowo Biotechnology Co., Ltd.) were harvested by centrifugation, resuspended in RPMI 1640 + 1% FBS, and plated at 100,000 cells per well in a volume of 50 μl.

[0155] Dilute the antibody to be tested in RPMI1640 + 1% FBS. Starting at 50 μg / mL, dilute the antibody 5-fold for a total of 11 concentration points. Add 100 μl / well of the diluted antibody to the plate, and include a 50 μg / mL isotype control (Isotype Control) as a negative control. Incubate in a 37°C CO2 incubator for 5 hours. After incubation, add 30 μl of Bio-Lite Luciferase Assay System luciferase substrate to each well, incubate with shaking for 3 minutes, and detect on the SpectraMax i3x.

[0156] The data were analyzed using Graphpad Prism software to obtain EC 50 The measurement results are shown in Table 6.

[0157] Table 6. EC values ​​of four antibodies and control antibody 50 value

[0158] The results in Table 6 show that the Nanobodies of the present invention can induce ADCC effects, and their EC 50 The values ​​were significantly lower than those of the reference antibody, demonstrating a stronger ADCC activity than the reference antibody, Lanalumab. The fluorescence value-concentration curve in Figure 3 shows that the antibodies of the present invention exhibited stronger fluorescence intensity than the reference antibody, Lanalumab, indicating a stronger ADCC activity. Isotype indicates the isotype control antibody.

[0159] The foregoing description is intended only to provide specific embodiments of the present disclosure, intended to enable those skilled in the art to understand and implement the present disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the scope of the present disclosure. Therefore, the present disclosure is not intended to be limited to the embodiments described herein, but rather to be construed in the broadest manner consistent with the principles and novel features disclosed herein.

[0160] Table 7. Sequence information

Claims

1. An antigen-binding polypeptide that specifically binds to human B lymphocyte stimulator receptor BAFFR, characterized in that: A group comprising CDR1, CDR2 and CDR3 selected from the following groups: (a) the amino acid sequence of CDR1 is shown in SEQ ID NO: 8; The amino acid sequence of the CDR2 is shown in SEQ ID NO: 14; and The amino acid sequence of the CDR3 is shown in SEQ ID NO: 22; (b) the amino acid sequence of the CDR1 is shown in SEQ ID NO: 8; The amino acid sequence of the CDR2 is shown in SEQ ID NO: 15; and The amino acid sequence of the CDR3 is shown in SEQ ID NO: 23; (c) the amino acid sequence of the CDR1 is shown in SEQ ID NO:9; The amino acid sequence of the CDR2 is shown in SEQ ID NO: 16; and The amino acid sequence of the CDR3 is shown in SEQ ID NO: 24; or (d) the amino acid sequence of the CDR1 is shown in SEQ ID NO:9; The amino acid sequence of the CDR2 is shown in SEQ ID NO: 17; and The amino acid sequence of the CDR3 is shown in SEQ ID NO:

25.

2. The antigen-binding polypeptide according to claim 1, characterized in that The antigen-binding polypeptide is a VHH single domain antibody, or a VHH-Fc antibody in which VHH is fused to an Fc region from a human immunoglobulin.

3. The antigen-binding polypeptide according to claim 2, characterized in that Comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-4, or wherein the VHH consists of an amino acid sequence as shown in any one of SEQ ID NOs: 1-4.

4. An isolated nucleic acid molecule, characterized in that The method comprises a nucleotide sequence encoding the antigen-binding polypeptide according to any one of claims 1 to 3.

5. The isolated nucleic acid molecule according to claim 4, characterized in that The nucleotide sequence comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 28-31 or consists of the nucleotide sequence.

6. A recombinant expression vector, characterized in that: Comprising the isolated nucleic acid molecule according to claim 4 or 5.

7. A host cell, characterized in that Comprising the recombinant expression vector according to claim 6.

8. A pharmaceutical composition, characterized in that Comprising the antigen-binding polypeptide according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

9. Use of the antigen-binding polypeptide according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4 or 5, the recombinant expression vector according to claim 6, the host cell according to claim 7 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating a tumor or an autoimmune disease.

10. A fusion protein, characterized in that Comprising the antigen-binding polypeptide according to any one of claims 1 to 3.

Images