Anti-baffr antibodies and uses thereof

Abstract

Described herein are anti-BAFF-R antibodies and antigen-binding fragments thereof having potential high stability and low immunogenicity. Also described are nucleic acids encoding the antibodies, compositions containing the antibodies, methods of making the antibodies, and methods of using the antibodies to treat or prevent diseases, such as cancer.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to International Application No. PCT / CN2020 / 076131, filed on February 21, 2020, pursuant to 35 U.SC §119(c), the disclosure of which is incorporated herein by reference in its entirety. Invention Field

[0003] This invention relates to monoclonal anti-B cell activating factor (BAFF) receptor antibodies, nucleic acids encoding said antibodies and expression vectors, recombinant cells comprising said vectors, and compositions containing said antibodies. Methods for preparing said antibodies and methods for treating diseases (including cancer) using said antibodies are also provided.

[0004] References to sequence lists submitted electronically

[0005] This application contains a sequence list, which was submitted electronically via EFS-Web in ASCII format, named "065815.1WO2 Sequence List", created on February 16, 2021, and is 10kb in size. The sequence list submitted via EFS-Web is part of this specification and is incorporated herein by reference in its entirety. Background of the Invention

[0007] Monoclonal antibody immunotherapy has been very successful in treating cancer, especially hematologic malignancies. However, monoclonal antibody therapy alone cannot cure the disease, and drug resistance can be a problem. One target for treating hematologic malignancies is CD20, which rituximab can target. Resistance to rituximab has been observed and is believed to be caused by multiple mechanisms, including the downregulation of CD20 expression.

[0008] Another target for B-cell tumors is the B-cell activating factor (BAFF) receptor, a member of the TNF receptor superfamily specifically involved in B lymphocyte development and survival. The BAFF receptor is expressed almost exclusively on B cells, and its surface expression has been found in various human B-cell lymphomas. Given that much of the signaling pathways of BAFF and its receptor are known, it remains an attractive target for B-cell lymphomas; therefore, anti-BAFF receptor monoclonal antibodies (mAbs) could be used as potential anticancer therapies. Invention Summary

[0009] This invention provides isolated monoclonal antibodies or antigen-binding fragments thereof that specifically bind to the B-cell activating factor (BAFF) receptor. Compared to previously disclosed anti-BAFF receptor antibodies or antigen-binding fragments thereof, the isolated monoclonal antibodies or antigen-binding fragments thereof exhibit higher stability and / or reduced immunogenicity.

[0010] An isolated monoclonal antibody or its antigen-binding fragment is provided, wherein the isolated monoclonal antibody or its antigen-binding fragment comprises heavy chain complementarity-determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3, each having the polypeptide sequences shown in SEQ ID NOs: 3, 4, 5, 6, 7, and 8, wherein the antibody or its antigen-binding fragment specifically binds to a BAFF receptor, preferably a human BAFF receptor.

[0011] In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region having a polypeptide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1, and a light chain variable region having a polypeptide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2.

[0012] In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 2.

[0013] In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment is human or humanized.

[0014] In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment binds to the BAFF receptor and is able to induce effector-mediated tumor cell lysis.

[0015] Also provided is an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0016] A vector comprising isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention is also provided.

[0017] A host cell comprising a vector containing isolated nucleic acid encoding a monoclonal antibody of the present invention or an antigen-binding fragment thereof is also provided.

[0018] In some embodiments, a pharmaceutical composition is provided comprising the isolated monoclonal antibody or its antigen-binding fragment as described in this invention, and a pharmaceutically acceptable carrier.

[0019] Methods for treating cancer in subjects in need are also provided, including administering the pharmaceutical composition of the invention to the subject. In some embodiments, the cancer is lymphoma, leukemia, or myeloma. The cancer can be, but is not limited to, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other fluid-filled tumors. For example, non-Hodgkin lymphoma (NHL) can be mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, or Burkitt lymphoma.

[0020] A method for generating the monoclonal antibody or antigen-binding fragment thereof of the present invention is also provided. The method includes culturing cells containing nucleic acids encoding the monoclonal antibody or antigen-binding fragment thereof under conditions for generating the monoclonal antibody or antigen-binding fragment thereof, and recovering the monoclonal antibody or antigen-binding fragment thereof from the cells or culture.

[0021] A method for preparing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment of the present invention is also provided. The method includes conjugating the monoclonal antibody or antigen-binding fragment to a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0022] A method for determining BAFF receptor levels in a subject is also provided. The method includes: (a) obtaining a sample from the subject; (b) contacting the isolated monoclonal antibody or its antigen-binding fragment of the present invention with the sample; and (c) determining the subject's BAFF receptor levels. For example, the sample may be a tissue sample or a blood sample. For example, the tissue sample may be a cancer tissue sample.

[0023] Brief description of the attached figures

[0024] The foregoing summary and the following detailed description of preferred embodiments of this application will be better understood when read in conjunction with the accompanying drawings. However, it should be understood that this application is not limited to the explicit embodiments shown in the figures.

[0025] Figure 1A-1B The human germline light chain Ig sequence (IGKV4-1*01 (SEQ ID NO:17); IGKV3-11*01 (SEQ ID NO:19)) and H90-11 monoclonal antibody were displayed. Figure 1A (SEQ ID NO:2) and H90-5 monoclonal antibody ( Figure 1B Alignment of the light chain sequence of (SEQ ID NO:10) )(SEQ ID NO:10).

[0026] Figures 2A-2D This demonstrates the SMAC-HPLC determination of rituximab ( Figure 2A C90 Figure 2B H90-5 Figure 2C ) and H90-11 ( Figure 2D The experimental results of the residence time (RT) of the ) are shown in the figure.

[0027] Figure 3 The immunogenicity results of the H90-5, H90-11 and C90 monoclonal antibodies, as determined by an indirect ELISA method, are shown. Invention Details

[0029] Various publications, articles, and patents are referenced or described in the background and throughout this specification; each of these references is incorporated herein by reference in its entirety. Discussions of documents, actions, materials, devices, articles, or similar matters contained in this specification are intended to provide background to the invention. Such discussion does not, with respect to any disclosed or claimed invention, acknowledge that any or all of these matters constitute part of the prior art.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings specified in this specification.

[0031] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise.

[0032] Unless otherwise stated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine, through conventional experimentation, equivalent substitutions for many of the specific embodiments of the invention described herein. This invention is intended to include these equivalent substitutions.

[0033] As described herein, the terms “comprising,” “including,” “containing,” “having,” “possessing,” “having,” “included,” or “owning,” or any other variation thereof, will be understood to implicitly include a specified integer or group of integers, but do not exclude any other integer or group of integers, and are non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent in those compositions, mixtures, processes, methods, articles, or apparatus. Furthermore, unless expressly stated to the contrary, “or” refers to inclusive or, not exclusive or. For example, any of the following satisfy conditions A or B: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); A and B are both true (or exist).

[0034] As used herein, the term “composed of” or variations thereof, such as “constituting of” or “made of”, used throughout the specification and claims, indicates the inclusion of any of the stated integers or groups of integers, but no additional integers or groups of integers may be added to the specified method, structure, and combination.

[0035] As used herein, “subject” means any animal, preferably a mammal, and most preferably a human. The term “mammal” as used herein includes any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans, with humans being more preferred.

[0036] In the context of two or more nucleic acid or polypeptide sequences (such as anti-BAFF receptor antibodies and the polynucleotides encoding them, BAF receptor polypeptides and the BAFF receptor polynucleotides encoding them), the term "identical" or percentage "identity" means that the two or more sequences or subsequences are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned using one of the following sequence comparison algorithms or visual inspection to obtain the maximum correspondence.

[0037] For sequence comparisons, a reference sequence is typically used to compare with the test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, with subsequence coordinates specified if necessary, along with the sequence algorithm program parameters. Then, based on the specified program parameters, the sequence comparison algorithm calculates the percentage of sequence identity between the test sequence and the reference sequence.

[0038] Further evidence that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid undergoes an immune cross-reaction with the polypeptide encoded by the second nucleic acid, as described below. Therefore, for example, when the difference between one polypeptide and another is only in conserved substitutions, the two polypeptides are usually substantially identical. Another evidence that two nucleic acid sequences are substantially identical is that the two molecules hybridize under stringent conditions.

[0039] As used herein, the term "polynucleotide," synonymous with "nucleic acid molecule," "nucleotide," or "nucleic acid," refers to any polynucleotide or polydeoxynucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA composed of a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA composed of a mixture of single-stranded and double-stranded regions, and mixed molecules composed of DNA and RNA (which may be single-stranded, more typically double-stranded, or a mixture of single-stranded or double-stranded regions). Furthermore, "polynucleotide" refers to a triple-stranded region containing RNA or DNA, or RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, as well as DNA or RNA containing a backbone modified for stability or other reasons. For example, "modified" bases include trimethylated bases and less common bases such as inosine. A wide variety of modifications can be made to DNA and RNA; therefore, "polynucleotide" includes naturally occurring chemically, enzymatically, or metabolically modified polynucleotide forms, as well as chemical forms of DNA and RNA specific to viruses and cells. "Polynucleotides" also include relatively short nucleic acid chains, often called oligonucleotides.

[0040] As used herein, the term "expression" refers to the biosynthesis of a gene product. The term includes the process of gene transcription into RNA. The term also includes the translation of RNA into one or more polypeptides, further including all naturally occurring post-transcriptional and post-translational modifications. The expressed polypeptide may be located in the cytoplasm of a host cell, enter an extracellular environment such as a cell culture medium, or be anchored to the cell membrane.

[0041] As used herein, the terms “peptide,” “polypeptide,” or “protein” can refer to a molecule composed of amino acids and can be recognized by those skilled in the art as a protein. Conventional single-letter or three-letter codes for amino acid residues are used herein. The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to an amino acid polymer of any length. The polymer can be linear or branched, can comprise modified amino acids, and can be broken down by non-amino acid components. The term also includes amino acid polymers that have undergone natural or artificial modifications, such as disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification, such as coupling with a labeling component. This definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids), and other modifications known in the art.

[0042] The peptide sequences described herein are written according to common convention, with the N-terminal region on the left and the C-terminal region on the right. Although the isoforms of amino acids are known, the L-form of the amino acid is represented unless otherwise specified.

[0043] The term "isolated" can refer to nucleic acids or polypeptides that are substantially free of cellular material, bacterial material, viral material, or culture medium (when produced via recombinant DNA technology), or chemical precursors or other chemical substances (when chemically synthesized). Furthermore, isolated polypeptides refer to polypeptides that can be administered to subjects as isolated polypeptides. In other words, if a polypeptide is attached to a column or in a gel, it cannot simply be considered "isolated." Additionally, "isolated nucleic acid fragments" or "isolated peptides" refer to nucleic acid or protein fragments that do not occur naturally as fragments and / or are not normally in a functional state.

[0044] Antibody

[0045] This application generally relates to isolated anti-BAFF receptor antibodies, nucleic acids encoding said antibodies and expression vectors, recombinant cells comprising said vectors, and compositions containing said antibodies. Methods for preparing said antibodies and methods for using said antibodies to treat diseases, including cancer, are also provided. The antibodies of the present invention possess one or more desired functional properties, including but not limited to high affinity binding to the BAFF receptor, high specificity for the BAFF receptor, increased stability, and reduced immunogenicity.

[0046] The anti-BAFF receptor antibodies described herein aim to achieve optimal developability for clinical trials and approval as a treatment or preventative medicine for cancer in subjects of need. A key characteristic of anti-BAFF receptor antibodies is targeted binding with minimal or no cross-reactivity with non-target molecules. However, once an antibody is found to achieve its intended potency by binding to a suitable target (such as the BAFF receptor), its developability is crucial. Antibody developability can be determined by analyzing various properties of the described anti-BAFF receptor antibodies, including but not limited to antibody expression, antibody solubility, antibody covalent integrity, antibody conformation and colloidal stability, antibody multispecificity, and antibody immunogenicity. Anti-BAFF receptor antibodies with optimal developability exhibit high expression, high solubility, high covalent integrity, high conformation and colloidal stability, low multispecificity, and low immunogenicity. For example, the developability and biophysical properties of antibodies are discussed in Jain et al., PNAS 114(5):944-949 (2017).

[0047] In general, the present invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof that specifically bind to BAFF receptors and possess characteristics desired for improved stability and reduced potential immunogenicity. Such antibodies exhibit greater manufacturability and can induce less anti-drug immune responses in subjects (e.g., human subjects).

[0048] As used herein, the term "antibody" is broad and includes monoclonal or polyclonal human, human-derived, complex, and chimeric immunoglobulin or antibody molecules, as well as antibody fragments. Generally, an antibody is a protein or peptide chain that exhibits binding specificity to a particular antigen. The structure of antibodies is well known. Immunoglobulins can be classified into five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) based on the amino acid sequence of their heavy chain constant domain. IgA and IgG are further subdivided into IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4 isotypes. Therefore, the antibody described in this invention can be any of the five major classes or a corresponding subclass. Preferably, the antibody described in this invention is IgG1, IgG2, IgG3, or IgG4. Vertebrate antibody light chains can be divided into one of two distinct types based on the amino acid sequence of their constant domain, namely κ and λ. Therefore, the antibody described in this invention can comprise κ and λ light chain constant domains. According to a particular embodiment, the antibody described in this invention comprises heavy chain and / or light chain constant regions derived from rat or human antibodies. In addition to the constant heavy and light chain domains, antibodies also contain an antigen-binding region composed of a light chain variable region and a heavy chain variable region. Each heavy chain variable region contains three domains (i.e., complementarity-determining regions 1-3: CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0049] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of antibodies specific to other distinct antigens (e.g., isolated antibodies that specifically bind to the BAFF receptor or antibodies that are substantially free of antibodies that do not bind to the BAFF receptor). Furthermore, isolated antibodies are substantially free of other cellular material and / or chemicals.

[0050] As used herein, the term "monoclonal antibody" refers to an antibody obtained from essentially homogeneous antibodies, meaning that, apart from a small number of naturally occurring mutations, the individual antibodies comprising a population are identical. The monoclonal antibodies described in this invention can be prepared using hybridoma methods, phage display technology, single-lymphocyte gene cloning technology, or DNA recombination methods. For example, the monoclonal antibody can be produced by a hybridoma comprising B cells obtained from transgenic non-human animals (such as transgenic mice or rats) whose genome includes human heavy-chain and light-chain transgenes.

[0051] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, such as a biantibody, Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized biantibody (ds biantibody), single-chain antibody molecule (scFv), single-domain antibody (sdab), scFv dimer (bivalent biantibody), multispecific antibody formed from a portion of an antibody containing one or more CDRs, camel-derived single-domain antibody, nanobody, domain antibody, bivalent domain antibody, or other antibody fragment that binds to an antigen but does not contain the complete antibody structure. The antigen-binding fragment is capable of binding to the same antigen that binds to a parent antibody or a fragment of a parent antibody. According to a specific embodiment, the antigen-binding fragment includes a light chain variable region, a light chain constant region, and a heavy chain Fd segment. According to other specific embodiments, the antigen-binding fragment includes Fab and F(ab').

[0052] As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the art that comprises a heavy chain variable region and a light chain variable region linked by a short peptide consisting of about 15 to 20 amino acids. As used herein, the term "single-domain antibody" refers to a conventional single-domain antibody in the art that comprises a heavy chain variable region and a heavy chain constant region or comprises only a heavy chain variable region.

[0053] As used herein, the term "human antibody" refers to an antibody produced by a human being, or an antibody produced using any technique known in the art that has a corresponding amino acid sequence to a human-produced antibody. The definition of a human antibody includes complete or full-length antibodies, fragments thereof, and / or antibodies containing at least one human heavy chain and / or light chain polypeptide.

[0054] As used in this article, the term "humanized antibody" refers to a non-human antibody that has been modified to increase sequence homology with human antibodies, thereby retaining the antigen-binding properties of the antibody, but with reduced immunogenicity in the human body.

[0055] As used herein, the term "chimeric antibody" refers to an antibody whose amino acid sequence of an immunoglobulin molecule is derived from two or more species. The light and heavy chain variable regions typically correspond to variable regions from one mammal (such as mice, rats, rabbits, etc.) that have the desired specificity, affinity, and performance, while the constant regions correspond to antibody sequences from another mammal (such as humans) to avoid evoking an immune response in that species.

[0056] As used herein, the term "multispecific antibody" refers to an antibody comprising a plurality of immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope, and the second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In one embodiment, the first and second epitopes are located on the same antigen, for example, on the same protein (or a subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes do not overlap or substantially do not overlap. In one embodiment, the first and second epitopes are located on different antigens, for example, on different proteins (or different subunits of a multimeric protein). In one embodiment, the multispecific antibody includes a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

[0057] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds to no more than two epitopes or two antigens. A bispecific antibody is characterized by having a first immunoglobulin variable domain sequence having binding specificity to a first epitope and a second immunoglobulin variable domain sequence having binding specificity to a second epitope. In one embodiment, the first and second epitopes are located on the same antigen, for example, on the same protein (or multimeric protein subunit). In one embodiment, the first and second epitopes overlap and substantially overlap. In one embodiment, the first and second epitopes are located on different antigens, for example, on different proteins (or different multimeric protein subunits). In one embodiment, the bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity to a second epitope. In one embodiment, the bispecific antibody comprises a hapten or fragment thereof having binding specificity to a first epitope, and a hapten or fragment thereof having binding specificity to a second epitope. In one embodiment, the bispecific antibody comprises an scFv or a fragment thereof that binds specifically to a first epitope and an scFv or a fragment thereof that binds specifically to a second epitope. In one embodiment, the first epitope is located on the BAFF receptor, and the second epitope is located on PD-1, PD-L1, CTLA-4, EGFR, HER-2, CD19, CD20, CD33, CD47, CD73, CD3, and / or other tumor-associated immunosuppressants or epitope antigens.

[0058] As used herein, the term "BAFF receptor" refers to the B cell activating factor (BAFF) receptor, also known as tumor necrosis factor receptor superfamily member 13C (TNFRSF 13C and BLyS receptor 3 (BR3), a membrane protein of the TNF receptor superfamily that recognizes BAFF and is essential for B cell maturation and survival. The BAFF receptor is an atypical representative of the TNF receptor superfamily because members of the TNF receptor superfamily typically possess multiple extracellular cysteine-rich domains (CRDs) that can bind to ligands and can also be used for ligand-independent assembly of receptor monomers, dimers, trimers, or multimers. Unlike most TNF-R family members, the BAFF receptor contains only a portion of the CRDs, used for... Ligand binding and self-assembly. BAFF receptors begin to be expressed as immature B cells develop into transitional B cells. Subsequently, these transitional B cells receive BAFF receptor-dependent pro-survival signals, rescuing them from premature cell death. BAFF receptors are expressed at various stages of B cell maturation and in various B-cell lymphomas, and have been identified as potential targets for cancer therapy; that is, BAFF receptors can be used as therapeutic molecules to specifically target cancer cells. An exemplary amino acid sequence of the human BAFF receptor is represented by GenBank accession number NP_443177 (SEQ ID NO:18).

[0059] As used in this article, "antibodies that specifically bind to the BAFF receptor" refer to antibodies that bind to the BAFF receptor, preferably human BAFF receptor, with a KD value of 1×10⁻⁶. -7 M or lower, preferably 1×10 -8 M or lower, more preferably 5×10 -9 M or lower, 1×10 -9 M or lower, 5×10 -10 M or lower or 1×10 -10 M or lower. The term "KD" refers to the dissociation constant, obtained from the ratio of Kd to Ka (i.e., Kd / Ka), and expressed as molar concentration (M). The KD value of an antibody can be determined according to the present invention using methods in the art. For example, the KD of an antibody can be determined by surface plasmon resonance, such as when using a biosensor system, such as... The system, or the use of biological layer interferometry techniques, such as the Octet RED96 system.

[0060] The smaller the KD value of an antibody, the higher its affinity for binding to the target antigen.

[0061] According to one particular aspect, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, said isolated monoclonal antibody or antigen-binding fragment thereof comprising heavy chain complementarity-determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3, having polypeptide sequences of SEQ ID NOs:3, 4, 5, 6, 7, and 8, respectively; wherein said antibody or antigen-binding fragment thereof specifically binds to a BAFF receptor, preferably a human BAFF receptor.

[0062] According to another specific aspect, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, said isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region having at least 85%, preferably 90%, more preferably 95% or higher (e.g., 95%, 96%, 97%, 98% or 99%) identity with the polypeptide sequence of SEQ ID NO:1, or a light chain variable region having at least 85%, preferably 90%, more preferably 95% or higher (e.g., 95%, 96%, 97%, 98% or 99%) identity with the polypeptide sequence of SEQ ID NO:2.

[0063] According to a preferred embodiment, the isolated monoclonal antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region having at least 85%, preferably 90%, more preferably 95% or higher (e.g., 95%, 96%, 97%, 98% or 99%) identity with the polypeptide sequence of SEQ ID NO:1, and a light chain variable region having at least 85%, preferably 90%, more preferably 95% or higher (e.g., 95%, 96%, 97%, 98% or 99%) identity with the polypeptide sequence of SEQ ID NO:2.

[0064] According to another specific aspect, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof comprising a heavy chain variable region having a polypeptide sequence of SEQ ID NO:1 and a light chain variable region having a polypeptide sequence of SEQ ID NO:2.

[0065] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, said isolated monoclonal antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each having a polypeptide sequence of SEQ ID NOs:3, 4, 5, 6, 7, and 8, respectively. In another embodiment, said isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having at least 85%, preferably 90%, more preferably 95% or higher (e.g., 95%, 96%, 97%, 98%, or 99%) the same polypeptide sequence as SEQ ID NO:1, and a light chain variable region having at least 85%, preferably 90%, more preferably 95% or higher (e.g., 95%, 96%, 97%, 98%, or 99%) the same polypeptide sequence as SEQ ID NO:2. Preferably, said isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:1 and a light chain variable region having the polypeptide sequence of SEQ ID NO:2.

[0066] According to another specific aspect, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof exhibits higher stability and / or lower immunogenicity in a subject compared to a control antibody. For example, the control antibody may be another isolated monoclonal antibody that specifically binds to the BAFF receptor, preferably the human BAFF receptor. For example, the control antibody may comprise heavy chain complementarity-determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3, with polypeptide sequences SEQ ID NOs: 11, 12, 13, 14, 15, and 16, respectively.

[0067] According to another particular aspect, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is human or humanized.

[0068] In another general aspect, the present invention relates to isolated nucleic acids encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention. Those skilled in the art will understand that the coding sequence of a protein can be altered (e.g., substitution, deletion, insertion, etc.) without changing the amino acid sequence of the protein. Accordingly, those skilled in the art will understand that the nucleic acid sequence encoding the monoclonal antibody or antigen-binding fragment thereof can be altered without changing the amino acid sequence of the protein.

[0069] In another general aspect, the present invention relates to a vector comprising an isolated nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof of the present invention. In view of this disclosure, any vector known to those skilled in the art, such as plasmids, granules, phage vectors, or viral vectors, may be used. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any elements that establish the conventional function of the expression vector, such as a promoter, ribosome-binding element, terminator, enhancer, selection marker, and origin of replication. The promoter may be a constitutive, inducible, or repressive promoter. Several expression vectors capable of delivering nucleic acids into cells are known in the art and can be used in the present invention to generate antibodies or antigen-binding fragments thereof in cells. According to embodiments of the present invention, recombinant expression vectors can be generated using conventional cloning techniques or artificial gene synthesis. These techniques are well known to those skilled in the art in light of the present disclosure.

[0070] In another general aspect, the present invention relates to host cells comprising isolated nucleic acids encoding the monoclonal antibody or its antigen-binding fragment of the present invention. According to the present invention, any host cell known to those skilled in the art can be used for recombinant expression of the antibody or its antigen-binding fragment of the present invention. In some embodiments, the host cells are *E. coli* TG1 or BL21 cells (for expression of antibodies such as scFv or Fab), CHO-DG44 cells, CHO-K1 cells, or HEK293 cells (for expression of antibodies such as full-length IgG). According to specific embodiments, the recombinant expression vector is transformed into host cells by conventional methods such as chemical transfection, heat shock, or electroporation, stably integrating into the host cell genome, thereby enabling efficient expression of the recombinant nucleic acid.

[0071] In another general aspect, the present invention relates to a method for producing the monoclonal antibody or antigen-binding fragment thereof of the present invention. The method includes culturing cells containing nucleic acids encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention under conditions for producing the monoclonal antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cells or cell culture (e.g., supernatant). The expressed antibody or antigen-binding fragment thereof can be obtained from the cells and purified according to conventional techniques described herein and known in the art.

[0072] Pharmaceutical Composition

[0073] In another general aspect, the present invention relates to a pharmaceutical composition comprising a monoclonal antibody of the present invention or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier thereof. As used herein, the term "pharmaceutical composition" refers to a product comprising the antibody of the present invention and a pharmaceutically acceptable carrier. The antibodies of the present invention and compositions thereof may also be used in the manufacture of medicaments for therapeutic purposes as described herein.

[0074] As used herein, the term "carrier" means any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulating agent, or other material known in the art for pharmaceutical formulation. It should be understood that the properties of the carrier, excipient, or diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceuticalally acceptable carrier" means a non-toxic material that does not interfere with the effectiveness of the compositions of the invention or the bioactivity of the compounds according to the invention. Depending on the specific embodiments, and in view of this disclosure, any pharmaceutically acceptable carrier suitable for antibody pharmaceutical compositions may be used in the present invention.

[0075] Formulation of pharmaceutically active ingredients using pharmaceutically acceptable carriers is known in the art, as described in Remington: Science and Pharmaceutical Practice (e.g., 21st edition (2005) and any subsequent editions). Additional non-limiting examples include buffers, diluents, solvents, tension modifiers, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers may be used to formulate the pharmaceutical compositions of the present invention.

[0076] In one embodiment of the invention, the pharmaceutical composition is a liquid formulation. Preferred examples of liquid formulations are water-soluble formulations, i.e., formulations containing water. The liquid formulation may include solutions, suspensions, emulsions, microemulsions, gels, etc. Water-soluble formulations typically contain at least 50% w / w water, or at least 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% w / w water.

[0077] In one embodiment, the pharmaceutical composition may be formulated as an injectable preparation for administration via an injection device (e.g., a syringe or infusion pump). The injectable preparation may be delivered via subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, or other methods.

[0078] In another embodiment, the pharmaceutical composition is a solid dosage form, such as a lyophilized or spray-dried composition, which may be used as is or have solvents and / or diluents added by a physician or patient prior to use. Solid dosage forms may include tablets (such as compressed tablets and / or coated tablets) and capsules (such as hard or soft gelatin capsules). For example, the pharmaceutical composition may also be in the form of sachets, sugar coatings, powders, granules, tablets, or powders for reconstitution.

[0079] The dosage forms can be immediate release, in which case they may contain a water-soluble or dispersing carrier, or they can be delayed release, sustained release, or modified release, in which case they may include water-insoluble polymers, and the dissolution rate of the dosage form in the gastrointestinal tract or subcutaneous tissue can be modulated.

[0080] In other embodiments, the pharmaceutical composition may be delivered intranasally, orally, or sublingually.

[0081] The pH of the aqueous formulation can be between pH 3 and pH 10. In one embodiment of the invention, the pH of the formulation is about 7.0 to about 9.5. In another embodiment of the invention, the pH of the formulation is about 3.0 to about 7.0.

[0082] In another general aspect, the present invention relates to a method for preparing a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof of the present invention, the method comprising conjugating the monoclonal antibody or an antigen-binding fragment thereof to a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

[0083] How to use

[0084] In another general aspect, the present invention relates to a method for targeting BAFF receptors on the surface of cancer cells in a subject, the method comprising administering to a subject an isolated monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the BAFF receptor, or a pharmaceutical composition of the present invention. The binding of the monoclonal antibody or its antigen-binding fragment to the BAFF receptor can mediate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and / or antibody-dependent cytotoxicity (ADCC) or other effects leading to the death of target cancer cells. For example, the monoclonal antibody or its antigen-binding fragment may be used to recruit conjugated drugs, and / or may form bispecific antibodies with another monoclonal antibody to mediate the death of target cancer cells.

[0085] The functional activity of the antibodies binding to the BAFF receptor and their antigen-binding fragments can be characterized by methods known in the art and those described herein. Methods for identifying antibodies binding to the BAFF receptor and their antigen-binding fragments include, but are not limited to, affinity and specificity analyses, including Biacore, ELISA, and OctetRed analyses; and binding assays using flow cytometry to detect the binding of the BAFF receptor to the antibody on tumor cells or cells recombinantly expressing the BAFF receptor. Depending on the specific implementation, methods for characterizing antibodies binding to the BAFF receptor and their antigen-binding fragments include the methods described below.

[0086] In another general aspect, the present invention relates to a method of treating a subject with cancer, the method comprising administering to the subject an isolated monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the BAFF receptor, or a pharmaceutical composition of the present invention. In some embodiments, the cancer is lymphoma, leukemia, or myeloma. The cancer may be selected from any liquid or solid cancer, for example, it may be selected from, but is not limited to, lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors, non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors. For example, non-Hodgkin lymphoma (NHL) can be mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, or Burkitt lymphoma.

[0087] According to embodiments of the present invention, the pharmaceutical composition comprises a therapeutically effective amount of an anti-BAFF receptor antibody or an antigen-binding fragment thereof. As used herein, the term "therapeutically effective amount" refers to the amount of active ingredient or component that elicits a desired biological or pharmaceutical response in a subject. For the purposes stated, the therapeutically effective amount can be determined empirically and conventionally.

[0088] As used in this article regarding anti-BAFF receptors or their antigen-binding fragments, the therapeutically effective dose refers to the amount of anti-BAFF receptor antibodies or their antigen-binding fragments that modulate the immune response in the required subject.

[0089] According to the specific implementation plan, a therapeutically effective dose refers to a dose sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or improving the severity of the disease, disorder, or condition to be treated, or related symptoms; (ii) shortening the duration of the disease, disorder, or condition to be treated, or related symptoms; (iii) preventing the development of the disease, disorder, or condition to be treated, or related symptoms; (iv) causing the resolution of the disease, disorder, or condition to be treated, or related symptoms; (v) preventing the development or onset of the disease, disorder, or condition to be treated, or related symptoms; (vi) preventing the disease, disorder, or condition to be treated... (vii) To reduce the number of people hospitalized for a disease, disorder, or condition to be treated, or for symptoms related to such disease, disorder, or condition; (viii) To shorten the length of hospital stay for people with a disease, disorder, or condition to be treated, or for symptoms related to such disease, disorder, or condition; (ix) To increase the survival time of subjects with a disease, disorder, or condition to be treated, or for symptoms related to such disease, disorder, or condition; (xi) To suppress or alleviate the disease, disorder, or condition to be treated, or for symptoms related to such disease, disorder, or condition in subjects; and / or (xii) To enhance or improve the preventive or therapeutic effect of another treatment.

[0090] The effective therapeutic dose or dosage may vary due to various factors, such as the disease, disorder, or condition to be treated, the method of administration, the target site, the subject's physiological state (including, for example, age, weight, and health status), whether the subject is human or animal, other medications administered, and whether the treatment is preventative or therapeutic. Optimal titration of the therapeutic dose is performed to optimize safety and efficacy.

[0091] Depending on the specific implementation, the compositions described herein are formulated to suit the intended route of administration to a subject. For example, the compositions described herein may be formulated to suit intravenous, subcutaneous, or intramuscular administration.

[0092] As used herein, the terms “treatment,” “management,” and “method of treatment” are intended to refer to an improvement or reversal of at least one measurable physical parameter associated with cancer, which may or may not be identifiable in the subject. The terms “treatment,” “management,” and “method of treatment” may also refer to causing the remission of a disease, disorder, or condition, preventing its progression, or at least slowing its progression. In specific embodiments, “treatment,” “management,” and “method of treatment” refer to the relief, prevention of the development or onset of one or more symptoms associated with a disease, disorder, or condition (such as a tumor or, more preferably, cancer), or a reduction in the duration of such symptoms. In specific embodiments, “treatment,” “management,” and “method of treatment” refer to the prevention of recurrence of a disease, disorder, or condition. In specific embodiments, “treatment,” “management,” and “method of treatment” refer to an increase in the survival rate of a subject suffering from the disease, disorder, or condition. In specific embodiments, “treatment,” “management,” and “method of treatment” refer to the elimination of a disease, disorder, or condition in a subject.

[0093] According to specific embodiments, compositions for treating cancer are provided. For cancer treatment, said compositions can be used in combination with another treatment method, including but not limited to chemotherapy, anti-CD20 monoclonal antibodies, anti-CD47 monoclonal antibodies, anti-CD73 monoclonal antibodies, anti-apelin monoclonal antibodies, anti-CTLA-4 monoclonal antibodies, anti-PD-L1 monoclonal antibodies, anti-PD-1 monoclonal antibodies, PD-1 / PD-L1 therapy, other immuno-oncology drugs, anti-angiogenic agents, radiotherapy, antibody-drug conjugates (ADCs), targeted therapy, or other anticancer drugs. Anti-BAFF receptor antibodies can be constructed into bispecific antibodies with chaperone monoclonal antibodies against PD-1, PD-L1, CTLA-4, EGFR, HER-2, CD19, CD20, CD33, CD73, CD47, CD3, and / or other tumor surface antigens for the treatment of cancers / tumors that express both BAFF receptors and specific tumor-associated antigens.

[0094] As used herein, the term “combination” refers to the use of one or more therapies when a subject is given two or more treatments. The use of the term “combination” does not restrict the order in which the therapies are given to the subject. For example, the first dose of treatment (as described herein) may be given to the subject before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior), concurrently, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the second dose of treatment.

[0095] In another aspect, the present invention relates to a method for determining BAFF receptor levels in a subject. The method includes: (a) obtaining a sample from the subject; (b) contacting the sample with a monoclonal antibody of the present invention or an antigen-binding fragment thereof; and (c) determining the subject's BAFF receptor levels.

[0096] As used herein, “sample” means a biological sample isolated from a subject, which may include, but is not limited to, whole blood, serum, plasma, blood cells, endothelial cells, biopsy tissue (such as cancerous tissue), lymph, ascites, interstitial fluid, bone marrow, cerebrospinal fluid, saliva, mucus, sputum, sweat, urine or other secretions, excretions or other bodily fluids. “Blood sample” means whole blood or any component thereof, including blood cells, serum and plasma.

[0097] In some embodiments, detection methods can be used to determine BAFF receptor levels in subjects, and these methods are selected from, but not limited to, Western blot assays, ELISA assays, and / or immunohistochemistry (IHC). Relative protein levels can be determined by Western blot analysis and immunohistochemistry (IHC), while absolute protein levels can be determined by ELISA assays. When determining relative BAFF receptor levels, the BAFF receptor levels between at least two samples can be determined, for example, between samples from different time points from the same subject, between samples from different tissues of the same subject, and / or between samples from different subjects. Additionally, when determining absolute BAFF receptor levels, such as by ELISA assays, the absolute level of BAFF receptors in a sample can be determined by establishing standards for the ELISA assay prior to testing the sample. Those skilled in the art will understand which analytical techniques can be used to determine BAFF receptor levels in subject samples using the antibodies or antigen-binding fragments of the present invention.

[0098] Methods for determining BAFF receptor levels from subject samples can diagnose abnormalities (elevated, decreased, or insufficient) in BAFF receptor levels within a disease and allow for appropriate treatment decisions. For example, such diseases may include cancer. Furthermore, by monitoring a subject's BAFF receptor levels, the risk of developing such diseases can be determined based on knowledge of BAFF receptor levels during the progression of a specific disease.

[0099] Implementation Plan

[0100] This application provides the following non-restrictive implementation schemes.

[0101] Implementation scheme 1 is an isolated monoclonal antibody or its antigen-binding fragment, wherein the isolated monoclonal antibody or its antigen-binding fragment includes heavy chain complementarity-determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity-determining region 1 (LCDR1), LCDR2 and LCDR3, which respectively have polypeptide sequences of SEQ ID NOs: 3, 4, 5, 6, 7 and 8, wherein the antibody or its antigen-binding fragment specifically binds to the BAFF receptor, preferably the human BAFF receptor.

[0102] Implementation Scheme 2 is the isolated monoclonal antibody or its antigen-binding fragment described in Implementation Scheme 1, wherein the isolated antibody or its antigen-binding fragment comprises a heavy chain variable region having a polypeptide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1, and a light chain variable region having a polypeptide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2.

[0103] Implementation scheme 3 is the isolated monoclonal antibody or its antigen-binding fragment described in implementation scheme 1 or 2, wherein the isolated monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 2.

[0104] Implementation scheme 4 is the isolated monoclonal antibody or its antigen-binding fragment described in any of implementation schemes 1-3, wherein the antibody or its antigen-binding fragment has increased stability and / or lower immunogenicity compared to the control antibody.

[0105] Implementation scheme 5 is the isolated monoclonal antibody or its antigen-binding fragment described in any of implementation schemes 1-4, wherein the antibody or its antigen-binding fragment is human or humanized.

[0106] Implementation scheme 6 is any of the isolated monoclonal antibodies or antigen-binding fragments thereof described in implementation schemes 1-5, wherein the antibody or antigen-binding fragment thereof is capable of binding to the BAFF receptor and inducing effector-mediated tumor cell lysis.

[0107] Implementation scheme 7 is an isolated nucleic acid encoding any of the monoclonal antibodies or antigen-binding fragments described in implementation schemes 1-6.

[0108] Implementation scheme 8 is a vector containing the isolated nucleic acid described in implementation scheme 7.

[0109] Implementation scheme 9 is a host cell containing the vector described in implementation scheme 8.

[0110] Embodiment 10 is a pharmaceutical composition comprising any of the isolated monoclonal antibodies or antigen-binding fragments thereof described in Embodiments 1-6 and their pharmaceutically acceptable carriers.

[0111] Implementation Scheme 11 is a method for treating cancer in a subject in need, the method comprising administering the pharmaceutical composition of Implementation Scheme 10 to the subject.

[0112] Implementation Scheme 12 is a method for targeting BAFF receptors on the surface of cancer cells in a subject in need, the method comprising administering the pharmaceutical composition described in Implementation Scheme 10 to the subject.

[0113] Implementation Scheme 13 is a method for preparing a monoclonal antibody or antigen-binding fragment thereof as described in any of Implementation Schemes 1-6, the method comprising: culturing cells containing nucleic acids encoding the monoclonal antibody or antigen-binding fragment thereof under conditions for preparing the monoclonal antibody or antigen-binding fragment thereof, and recovering the monoclonal antibody or antigen-binding fragment thereof from the cells or culture.

[0114] Embodiment 14 is a method for preparing a pharmaceutical composition comprising any of the monoclonal antibodies or antigen-binding fragments thereof described in Embodiments 1-6, the method comprising: conjugating the monoclonal antibody or antigen-binding fragment thereof to a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0115] Implementation scheme 15 is a method for determining BAFF receptor levels in a subject, the method comprising:

[0116] a. Obtaining samples from subjects;

[0117] b. Contact the sample with the isolated monoclonal antibody or its antigen-binding fragment as described in any of embodiments 1-6; and

[0118] c. Determine the BAFF receptor level in the subjects.

[0119] Implementation scheme 16 is the method described in implementation scheme 15, wherein the sample is a tissue sample.

[0120] Implementation scheme 17 is the method described in implementation scheme 16, wherein the tissue sample is a cancer tissue sample.

[0121] Implementation scheme 18 is the method described in implementation scheme 15, wherein the sample is a blood sample. Example

[0122] Example 1: Identification and characterization of affinity-matured anti-BAFF receptor monoclonal antibody H90-11

[0123] H90-11 is an anti-BAFF receptor monoclonal antibody identified from affinity maturation screening of the previously identified anti-BAFF receptor monoclonal antibody H90-5 (WO2017 / 214170; Qin et al., Clinical Cancer Research. 24(5):1114-23(2018)). When compared with H90-5, H90-11 did not show a significant increase in affinity for the BAFF receptor (Table 1); however, several characteristics of H90-11 were observed, as follows.

[0124] Table 1: Affinity of anti-BAFF receptor monoclonal antibodies to BAFF receptor

[0125] monoclonal antibody target <![CDATA[K D (M)]]> <![CDATA[K on (1 / Ms)]]> <![CDATA[K dis (1 / s)]]> Total X^2 Full R^2 H90-5 3.4E-09 9.9E+05 3.4E-03 0.0106 0.9816 H90-11 9.5E-10 8.6E+05 8.2E-04 0.0073 0.9927

[0126] Note that H90-11 and H90-5 contain the same heavy chain variable regions (SEQ ID NO:1 and SEQ ID NO:9, respectively). However, H90-11 and H90-5 contain different light chain variable regions (SEQ ID NO:2 and SEQ ID NO:10, respectively).

[0127] Next, the study showed that when using Ig Blast, H90-5 has 12 different amino acid residues compared to the human germline Ig sequence. Figure 1B ), while compared to the same human germline Ig sequence, H90-11 has only 6 different amino acid residues ( Figure 1A Given that the H90-11 monoclonal antibody is more similar to the human germline sequence, it is believed that the H90-11 monoclonal antibody is less likely to induce an immune response in humans. Furthermore, it is believed that the more similar an antibody is to the human germline sequence, the more stable the antibody is.

[0128] Therefore, the stability and immunogenicity of the H90-5 and H90-11 antibodies were evaluated. H90-5 and H90-11 were prepared as full-length human IgG1, and the antibody drug rituximab and the pre-humanized monoclonal antibody C90 of H90-5 were prepared as antibody controls. The antibodies used for stability and immunogenicity studies exhibit the following characteristics, as shown in Table 2.

[0129] Table 2: Antibody Characteristics

[0130] Sample ID <![CDATA[EC280(mL.mg -1 .cm -1 )]]> Provide concentration (mg / mL) pI Rituxan 1.643 0.28 8.66 C90 1.495 2.6 7.91 H90-5 1.500 1.9 8.24 H90-11 1.624 1.4 8.34

[0131] The stability of H90-11, H90-5, C90, and rituximab antibodies was evaluated using vertical monolayer adsorption chromatography (SMAC) high-performance liquid chromatography (HPLC) as described by Kohli et al. (Kohli et al., Monoclonal Antibodies 7(4):752-8(2015)). Antibodies with long residence times on Zenix columns (e.g., >20 min) precipitated over time, while antibodies with short residence times on Zenix columns (e.g., <10 min) did not precipitate. Antibodies with residence times of 10–20 min required further evaluation using different methods.

[0132] In short, the SMAC-HPLC method uses the following HPLC and SMAC columns: Agilent 1100 equipped with DAD for HPLC, Zenix SEC-300, 4.6 x 300 mm. A 3 μM column was used for SMAC. The column flow rate was 0.35 mL / min, and the mobile phase contained 150 mM sodium phosphate at pH 7.0. The column was run for 20 minutes. Samples were analyzed at 5 °C and wavelengths of 214 nm and 280 nm, while the column was stored at room temperature. The sample buffer was Tris-glycine buffer (100 mM Tris / 90 mM glycine, pH 7.5). 3 μg of sample was loaded onto each column.

[0133] SMAC residence time (RT) is negatively correlated with colloidal stability. Proteins with longer residence times on the column exhibit broader peak widths and are more prone to aggregation or precipitation. Similar to rituximab, H90-11 has a low aggregation tendency (RT < 10 min), while C90 and its humanized version, H90-5, have a moderate aggregation tendency (RT > 10 min). Therefore, H90-11 is considered less prone to aggregation than H90-5. SMAC-HPLC results are as follows... Figures 2A-2D As shown in Table 3 below.

[0134] Table 3 Summary of SMAC Results

[0135]

[0136] The immunogenicity of H90-11, H90-5, and C90 antibodies was assessed using an AI-driven immunogenicity prediction and assessment system developed by Sun et al. (Sun et al., Journal of Immunological Methods, 452:6-11 (2018)). Simply, the anti-drug antibody titers of different antibodies were evaluated using Balb / c mice. Female Balb / c mice, approximately 4–6 weeks old, were first immunized with a complete Freund's adjuvant antibody, followed by a booster immunization with an incomplete Freund's adjuvant antibody. Four weeks after the first immunization, blood was collected from the tails of the immunized mice, and the titers of the immunoantibodies were detected using an indirect enzyme-linked immunosorbent assay (ELISA) as described by Sun et al., to assess the potential immunogenicity of H90-11 and H90-5. It was observed that the anti-drug antibody titers of H90-11 in mice were lower than those of H90-5. Figure 3 Therefore, it is believed that H90-11 has lower immunogenicity in the human body than H90-5.

[0137] Those skilled in the art will understand that modifications can be made to the above embodiments without departing from the broad inventive concept. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined in this specification.

Claims

1. An isolated monoclonal antibody or antigen binding fragment thereof, characterized in that, The isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain complementarity determining region 1 (HCDR1), a HCDR2, a HCDR3, a light chain complementarity determining region 1 (LCDR1), a LCDR2, and a LCDR3, each having the polypeptide sequence of: a. SEQ ID NOs: 3, 4, 5, 6, 7, and 8; The antibody or antigen-binding fragment thereof specifically binds to a B-cell activating factor receptor.

2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof specifically binds to a human B-cell activating factor receptor.

3. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof specifically binds to a human B-cell activating factor receptor.

4. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof specifically binds to a human B-cell activating factor receptor.

5. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody or antigen-binding fragment thereof has increased stability and / or lower immunogenicity in a subject compared to a control antibody.

6. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein, The antibody or antigen-binding fragment thereof is humanized.

7. An isolated nucleic acid, comprising, The monoclonal antibody or antigen-binding fragment thereof is capable of binding to a BAFF receptor and inducing effector-mediated tumor cell lysis.

8. A vector, characterized in that, The isolated nucleic acid encodes the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6.

9. A host cell, characterized in that, The vector contains the isolated nucleic acid of claim 7.

10. A pharmaceutical composition, characterized by, The host cell contains the vector of claim 8.

11. A method of producing a monoclonal antibody or antigen binding fragment thereof according to any one of claims 1 to 6, characterized in that, The pharmaceutical composition contains the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6 and a pharmaceutically acceptable carrier.

12. A method of preparing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment of any one of claims 1-6, wherein, The method comprises culturing a cell containing a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof under conditions wherein the monoclonal antibody or antigen-binding fragment thereof is produced, and recovering the antibody or antigen-binding fragment from the cell or culture. The method comprises combining the monoclonal antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

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