Anti-her4 antibodies and methods of making and using the same

Abstract

The present application provides an anti-HER4 antibody or antigen-binding fragment thereof. The present application further provides a bispecific molecule or a multispecific molecule comprising the antibody or antigen-binding fragment thereof, an immunoconjugate, and a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, a vector and host cell comprising the nucleic acid molecule. The present application also provides a method of preparing the antibody or antigen-binding fragment thereof, a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, and a method and use of the antibody or antigen-binding fragment thereof, or a pharmaceutical composition thereof in treating a disease, which provides a candidate specific antibody drug for the treatment and / or prevention of diseases or disorders related to HER4.

Description

Technical Field

[0001] This invention relates to anti-HER4 antibodies or antigen-binding fragments thereof, polynucleotides encoding the antibody or antigen-binding fragments thereof, expression vectors and host cells, pharmaceutical compositions comprising the antibody or antigen-binding fragments thereof, and their use or methods for treating subjects. Background Technology

[0002] The ErbB / HER family of receptor tyrosine kinases includes ErbB1 (also known as EGFR or HER1), ErbB2 (HER2, c-Neu), ErbB3 (HER3), and ErbB4 (HER4). ErbB / HER family receptors activate downstream signal transduction pathways mediated by their dimerization, including the RAS-RAF-MEK-ERK, PI3K-AKT, and JAK-STAT pathways, thereby participating in the regulation of cell growth, differentiation, and survival. HER4, encoded by the ErbB4 / HER4 gene, is a transmembrane glycoprotein with a molecular weight of approximately 180 kDa. It consists of a ligand-binding extracellular domain (ECD), a transmembrane domain, and an intracellular tyrosine kinase domain. HER4 is composed of ligands including neuregulin-1–4 (NRG-1–4) (J.R. Toggweiler, Nature 1993, 366: 473-475; K.L. Carraway et al., Nature 1997, 387: 512-516; D. Zhang et al., Proc Natl Acad Sci USA, 1997, 94: 9562-9567; D. Harari et al., Oncogene 1999, 18: 2681-2689), heparin-binding EGF (HB-EGF) (K. Elenius et al., EMBO J 1997, 16: 1268-1278), and betacellulin (BTC) (D.J. Riese et al., Oncogene). HER4 is activated by epiregulin (T. Komurasaki et al., Oncogene 1997, 15: 2841-2848) and forms homodimers or heterodimers with other ErbB / HER family members. Although HER4 is expressed in a variety of tumors (e.g., blastoma, breast cancer, lung cancer, melanoma, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, and bladder cancer) (Hollmén M et al., Future Oncol 2010, 6: 37-53), multiple reports show that HER4 expression is downregulated in tumors, or that its expression is associated with a good prognosis of tumors (Srinivasan R et al., Clin Cancer Res 1998, 5: 2877-2883; Witton CJ et al., J Pathol 2003, 200: 290-297).Furthermore, HER4 is considered a tumor suppressor gene in breast cancer (Wang J et al., Oncotarget 2016, 7:76693-76703). Therefore, the role of HER4 in tumorigenesis and progression remains to be further clarified. Currently, HER4 has been shown to play a crucial role in the development and maintenance of the heart and nervous system (FEJones et al., J Cell Biol 1999, 147:77-87; H. Tidcombe et al., Proc Natl Acad Sci USA 2003, 100:8281-8286; T. Junttila et al., Trends Cardiovasc Med 2000, 10:304-310; A. Buonanno et al., Curr Opin Neurobiol 2001, 11:287-296).

[0003] Therefore, there is a desire in the art to develop a neutralizing antibody that can specifically bind to HER4 for more precise treatment of HER4-related diseases or conditions. Summary of the Invention

[0004] This invention provides an anti-HER4 antibody, its preparation method, and its application in HER4-related diseases or conditions.

[0005] On the one hand, the present invention provides an antibody or antigen-binding fragment that can specifically bind to HER4.

[0006] In some embodiments, the anti-HER4 antibody or its antigen-binding fragment specifically binds to human HER4 extracellular domain III and blocks ligand-induced activation of the HER4 receptor. In some embodiments, the activity of the antibody or its antigen-binding fragment specifically binding to human HER4-expressing cells is detected by flow cytometry.

[0007] In some embodiments, the anti-HER4 antibody or its antigen-binding fragment has an inhibitory effect on the HER4 receptor and its downstream signal transduction pathways. In one embodiment, the anti-HER4 antibody or its antigen-binding fragment is capable of inhibiting ligand-induced HER4 receptor dimerization and its downstream signal transduction pathways, the ligands including NRG-1 to 4, preferably NRG-1. In some embodiments, the IC50 value of the antibody or its antigen-binding fragment against NRG-1-induced HER2 / HER4 receptor dimerization, as detected by live-cell bioluminescence, is no more than 1 μg / mL, preferably no more than 0.5 μg / mL.

[0008] In some embodiments, the anti-HER4 antibody or its antigen-binding fragment does not have cross-reactivity with other members of the human ErbB / HER family, such as EGFR, HER2 and / or HER3.

[0009] In some embodiments, the HER4 antibody or its antigen-binding fragment is capable of cross-reacting with HER4 from other species (e.g., mice, rats, dogs).

[0010] On the other hand, the present invention provides a method for screening to obtain the anti-HER4 antibody of the present invention or its antigen-binding fragment, the method including but not limited to hybridoma technology, phage display technology, and single lymphocyte gene cloning technology.

[0011] On the other hand, the present invention provides isolated nucleic acid molecules (also referred to as "polynucleotides") encoding the anti-HER4 antibody of the present invention or an antigen-binding fragment thereof, as well as expression vectors containing said nucleic acids and host cells containing the nucleic acids or expression vectors. The present invention also relates to a method for preparing the anti-HER4 antibody of the present invention or an antigen-binding fragment thereof using said host cells, said method comprising culturing said host cells and recovering said antibody or antigen-binding fragment thereof from the culture medium.

[0012] On the other hand, the present invention provides bispecific or multispecific molecules, immune conjugates, chimeric antigen receptors, engineered T-cell receptors, or oncolytic viruses comprising the anti-HER4 antibody of the present invention or its antigen-binding fragment thereof.

[0013] On the other hand, the present invention provides a pharmaceutical composition comprising the anti-HER4 antibody of the present invention or its antigen-binding fragment, a bispecific antibody or a multispecific antibody, an immune conjugate, a chimeric antigen receptor, an engineered T-cell receptor, or an oncolytic virus, and a pharmaceutically acceptable carrier.

[0014] On the other hand, the present invention provides a kit containing an effective amount of the anti-HER4 antibody of the present invention or its antigen-binding fragment, or the pharmaceutical composition herein, and optionally at least one other therapeutic agent.

[0015] On the other hand, the present invention provides the use of the anti-HER4 antibody of the present invention or its antigen-binding fragment in the preparation of pharmaceutical compositions or formulations for the treatment and / or prevention of HER4-related diseases or conditions, and in the preparation of diagnostic reagents for the diagnosis of HER4-related diseases or conditions.

[0016] On the other hand, the present invention provides a method for treating and / or preventing HER4-related diseases or conditions, the method comprising administering to a subject an effective amount of the anti-HER4 antibody of the present invention or its antigen-binding fragment, pharmaceutical composition, or kit.

[0017] On the other hand, the present invention provides a method for inhibiting the HER4 receptor and its downstream signal transduction pathways, comprising using the anti-HER4 antibody of the present invention or its antigen-binding fragment, pharmaceutical composition or kit to inhibit ligand-induced HER4 dimerization in vitro or in vivo, wherein the ligands include NRG-1 to 4, preferably NRG-1.

[0018] On the other hand, the present invention provides a method for detecting the content or expression level of HER4 in a test sample (e.g., a biological sample, such as serum or tissue), comprising: (a) contacting the test sample with an antibody or antigen-binding fragment of the present invention, and (b) detecting the binding of the antibody or antigen-binding fragment of the present invention to HER4 in the test sample.

[0019] This application is further illustrated by the following drawings and specific embodiments. Other features and advantages disclosed in this application will be apparent, and these drawings and specific embodiments should not be considered as limiting the scope of this application. Modifications readily conceived by those skilled in the art are included within the spirit of this application and the scope of protection of the appended claims. All references cited in this application, including publications, patents, and patent applications, are incorporated herein by reference in their entirety. Attached Figure Description

[0020] Figure 1 The binding activity of the anti-HER4 antibody Ab4261 to the extracellular domain proteins of human ErbB / HER family proteins (including EGFR, HER3, and HER4) and the chimeric HER4 extracellular domain III recombinant protein (HER4_DⅢ) was detected by ELISA.

[0021] Figure 2 Flow cytometry was used to detect the binding activity of the optimized molecule of antibody Ab4261 containing a combination mutation and the control antibody (maternal antibody Ab4261) to HER4-expressing 293T cells.

[0022] Figure 3 The binding activity of the optimized molecule of antibody Ab4261 (G202-K201) to human ErbB / HER family protein members (including EGFR, HER2, HER3 and HER4) was detected by ELISA.

[0023] Figure 4 The binding activity of the optimized molecule (G202-K201) of antibody Ab4261 to HER4 in different species (including humans, mice, rats, and dogs) was detected by ELISA.

[0024] Figure 5The binding activity of optimized molecules of antibody Ab4261 (G201-K203 and G202-K201) and control antibody (maternal antibody Ab4261) to HER4-expressing 293T cells was detected by flow cytometry.

[0025] Figure 6 The effect of the optimized molecule of antibody Ab4261 (G202-K201) on NRG-1-induced HER2 / HER4 receptor dimerization was studied. The control antibodies included the parental antibody Ab4261 and Pertuzumab. Invention Details

[0026] definition

[0027] 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. For the purposes of this invention, the following terms are defined below.

[0028] In this article, the terms “HER4,” “HER4 receptor,” and “HER4 protein” are used interchangeably; the protein is also known as ErbB4, c-ErbB-4, or p180erbB4. Unless otherwise specified as originating from a non-human species, “HER4” as used herein refers to human HER4.

[0029] Any natural form of HER4 can be the amino acid sequence shown in SEQ ID NO:86 and / or the full-length HER4 amino acid sequence shown in UniProtKB registry number Q15303.1, and can be naturally expressed by cells (including cardiomyocytes, breast cells, and tumor cells) or expressed by cells transfected with the HER4 gene or cDNA. This term includes naturally occurring HER4 allelic variants and splice variants, isotypes, homologs, and species homologs. HER4 can be isolated from the human body or produced through recombinant or synthetic methods.

[0030] The full-length HER4 protein consists of 1308 amino acids. Residues 1-25 form the signal peptide, residues 26-651 form the extracellular domain (ECD), residues 652-675 form the transmembrane domain, and residues 676-7308 form the intracellular domain. The extracellular domain comprises four subdomains, namely subdomain 1...

[0031] The subdomains consist of subdomain 1 (D1, approximately amino acid residues 26-210), subdomain 2 (D2, approximately amino acid residues 211-332), subdomain 3 (D3, approximately amino acid residues 333-501), and subdomain 4 (D4, approximately amino acid residues 502-651). D1 and D3 are involved in ligand binding, while cysteine-rich D2 and D4 are responsible for receptor dimerization.

[0032] In this article, "cells expressing HER4" can be naturally occurring cells or cell lines (e.g., cardiomyocytes, breast cells, tumor cells) or cells generated by recombinantly introducing nucleic acids encoding HER4 into host cells.

[0033] In this document, the terms "antigen-binding domain," "antigen-binding region," or "epitaxy-binding domain" are used interchangeably to refer to a specific region on an antibody, antigen-binding fragment, or its derivative that is directly involved in the specific interaction with the target antigen. This interaction occurs, for example, through binding, steric hindrance, stabilization / instability, or spatial distribution, to achieve a dynamic equilibrium with the target antigen. In this invention, "antigen-binding domain" also refers to a specific region on the antibody, antigen-binding fragment, or its derivative that interacts with a specific epitope on the HER4 receptor, achieving a dynamic equilibrium through binding, steric hindrance, stabilization / instability, or spatial distribution.

[0034] An antibody is a polypeptide or protein that is encoded by one or more immunoglobulin genes, or fragments thereof, and is capable of specifically recognizing and binding to an antigen. Recognized immunoglobulin genes include constant regions of κ, λ, α, γ, δ, ε, and μ, as well as numerous variable regions. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which respectively define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE. Several of these classes can be further divided into subclasses / isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. A typical immunoglobulin (e.g., antibody) structural unit consists of a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The N-terminal domain of each chain defines a variable (V) region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The antibody heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH), where the heavy chain constant region typically includes three domains: CH1, CH2, and CH3. The light chain consists of a light chain variable region (VL) and a light chain constant region (CL), where the light chain constant region typically contains one domain, CL. H and V L The paired molecules together form a single antigen-binding site. Endogenous V L It is encoded by gene segments V (variable) and J (conjugating), and endogenous V H Encoded by V, D (diversity), and J. V L or V H Both include a hypervariable region, also known as a complementarity-determining region (CDR), and a framework region (FR). The terms "variable region" or "V region" are used interchangeably and refer to a heavy chain variable region or light chain variable region arranged in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the amino terminus to the carboxyl terminus. The term "J region" refers to the subsequence encoding the C-terminal portion of the variable region containing CDR3 and FR4. V or J regions can be naturally occurring, recombinant, or synthetic. In this document, the antibody light chain variable region and / or antibody heavy chain variable region are sometimes collectively referred to as the "antibody variable region," and the antibody light chain and / or antibody heavy chain are collectively referred to as the "antibody chain." In some embodiments, the FR of the antibody or its antigen-binding fragment provided herein may be identical to a human germline sequence or may be naturally occurring or artificially modified.

[0035] The locations of CDR and FR can be determined using a variety of well-known definitions in the art, such as Kabat, Chothia, IMGT, and Contact (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, 1991, Department of Health and Human Services, NIH Publication No. 91-3242; Johnson et al., Nucleic Acids Res 2001, 29:205-206; Chothia & Lesk, J Mol Biol 1987, 196:901-917; Chothia et al., Nature 1989, 342:877-883; Chothia et al., J Mol Biol 1992, 227:799-817; Al-Lazikani et al., J Mol Biol 1997, 273:927-748; Lefranc MP et al., Nucleic Acids Research). 1999,27:209–212; MacCallum RM et al., J Mol Biol 1996,262:732-745). The definition of antigen-binding sites is also described in the following literature: Ruiz et al., Nucleic Acids Res 2000, 28:219-221; Lefran MP, Nucleic Acids Res 2001, 29:207-209; Lefranc MP, The Immunologist 1999, 7:132-136; Lefranc MP et al., Dev Comp Immunol 2003, 27:55-77; MacCallum et al., J Mol Biol 1996, 262:732-745; Martin et al., Proc Natl Acad Sci USA 1989, 86:9268-9272; Martin et al., Methods Enzymol 1991, 203:121-153; Sternberg M JE (ed.), Protein Structure Prediction, Oxford University Press, Oxford 1996, 141-172. This invention encompasses any method of definition for determining the CDR in the anti-HER4 antibody or its antigen-binding fragment of this application. Table 1 shows the position numbers of the amino acid sequences of the antibody CDRs determined using different methods of definition. The exact number of amino acid residues covering a particular CDR varies with the sequence of the CDR.Once the amino acid sequence of the antibody's variable region is known, those skilled in the art can determine the antibody's CDR using conventional methods, including but not limited to those defined above.

[0036] Table 1. CDRs determined by different definition methods 1

[0037]

[0038]

[0039] 1 All CDR definitions in Table 1 are numbered according to the numbering system proposed by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, 1991, Department of Health and Human Services).

[0040] Furthermore, Kabat et al. defined a numbering system for the variable region sequence, which can be applied to any antibody. Those skilled in the art can readily apply this "Kabat numbering" system to the variable region sequence of any antibody without relying on any experimental data other than the antibody sequence itself to determine the variable region sequence. Unless otherwise stated, the numbering of specific amino acid residue positions in the variable region of the antigen-binding domain of the anti-HER4 antibody of this application is determined according to the Kabat numbering system.

[0041] Antibodies exist either as intact immunoglobulins or as numerous fragments produced by digestion with various peptidases. Although various antibody fragments are defined according to the digestion of intact antibodies, those skilled in the art will understand that such fragments can be synthesized de novo by chemical cleavage methods or by using recombinant DNA methods. In this document, the term "antigen-binding fragment" (or simply "antibody moiety" or "antibody fragment") refers to an antibody moiety containing one or more CDRs or any other antibody fragment capable of binding to an antigen (e.g., HER4 or its extracellular domain) but not possessing an intact antibody structure. Antigen-binding fragments can have the same specific antigen-binding activity as intact antibodies. In some embodiments, an antigen-binding fragment may contain one or more CDRs from a particular human antibody, transposed to a frame region from one or more different human antibodies. Antigen-binding fragments include, but are not limited to: (i) “Fab” fragments, monovalent antibody fragments composed of VH, VL, CL, and CH1 domains; (ii) “F(ab')2” fragments, bivalent fragments containing two Fab fragments linked by disulfide bonds in the hinge region; (iii) “Fv” fragments, composed of the VL and VH domains of the antibody arm, which are the smallest antibody fragments containing complete antigen-binding sites; (iv) “Fd” fragments, composed of VH and CH1 domains; (v) “single-chain Fv antibody (scFv)”, “single-chain antibody”, or “scFv molecule”, which refers to engineered antibodies composed of light chain variable regions directly linked to heavy chain variable regions or linked by a single peptide chain (Huston JS et al., Proc Natl Acad Sci USA).

[0042] 1988, 85:5879-5883; Bird et al., Science 1988, 242:423-426); In addition, single-chain antibodies also include “linear antibodies” containing a pair of tandem Fv segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen-binding regions (Zapata et al., Protein Eng 1995, 8:1057-1062; US Patent 5,641,870); (vi) “dAb” fragments (Ward et al., Nature 1989, 341:544-546; PCT Publication No. WO 90 / 05144A1) contain a single variable domain, such as a VH domain. Single-domain antibodies (sdAbs) are independent immunoglobulin domains; (vii) "Diabody" is a bivalent bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, but the peptide linker used is too short to allow the two domains on the same chain to pair, thus forcing these two domains to pair with the complementary domain of another chain to form two antigen-binding sites (Holliger P et al., Proc Natl Acad Sci USA 1993, 90:6444-6448; Poljak et al., Structure 1994, 2:1121-1123; Patent EP404097; PCT Publication No. WO93 / 11161).

[0043] In this document, the term "Fc region" or "Fc domain" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region, such as the immunoglobulin heavy chain constant region other than the first constant region (CH1). For IgG, the Fc region may comprise immunoglobulin domains CH2 and CH3, as well as the hinge region between CH1 and CH2. The Fc region as used herein includes native sequence Fc regions and / or Fc region variants, and may be part of the anti-HER4 antibody of this application. It should be understood in the art that the boundaries of the Fc region can vary; however, the human IgG heavy chain Fc region is generally defined as containing a cysteine ​​residue at position 226 or a proline residue at position 230 at its amino terminus, according to the EU numbering system / scheme, as seen in Kabat et al., NIHP Publication 1991, 91-3242, National Technical Information Service.

[0044] The term "anti-HER4 antibody" or "HER4-specific antibody" refers to any form of antibody or fragment thereof that specifically binds to HER4, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that the fragment specifically binds to HER4. The anti-HER4 antibody of the present invention is preferably an antibody or antibody fragment that specifically binds to the extracellular domain of HER4.

[0045] In this document, the terms "specific binding," "binding specificity," "specific to," or "binding" refer to a binding reaction that determines the presence of a target molecule (e.g., an antigen) in a heterogeneous population of proteins and other biological products (e.g., biological samples such as blood, serum, plasma, or tissue samples). In other words, this binding is selective for the target molecule and distinguishes between undesirable or nonspecific interactions. For example, an antibody that specifically binds to a target molecule (which may be an antigen) has greater affinity, stronger binding activity, easier binding, and / or longer binding duration when binding to the target molecule compared to binding to other non-target molecules. Various immunoassays can be used to select antibodies that specifically bind to a particular protein, such as solid-phase ELISA, flow cytometry (FACS), cellular fluorescence assays, or surface plasmon resonance (SPR) techniques to determine the binding reaction of the antibody or its antigen-binding fragment to the target antigen / protein. Typically, under the established assay conditions, the specific or selective binding reaction of an antibody or conjugate to an antigen produces a signal at least twice the background signal, more generally at least 10-100 times the background signal, and substantially does not bind to other antigens / proteins present in the sample in significant quantities.

[0046] In this document, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical except for the possibility of small amounts of naturally occurring mutations. Monoclonal antibodies exhibit specificity and affinity for binding to specific epitopes. Monoclonal antibodies can be prepared by the hybridoma method first described by Kohler et al., Nature 1975, 256:495, or by recombinant DNA methods (see US Patent 4,816,567), or isolated from phage antibody libraries, for example, by techniques described in Clackson et al., Nature 1991, 352:624-628; Marks et al., J Mol Biol 1991, 222:581-597.

[0047] As used in this article, "epitope" refers to a protein determinant, an antigenic moiety that can be recognized and specifically bound by antibodies. Epitopes are typically composed of surface groups of a molecule, such as amino acids or sugar side chains, and usually have specific three-dimensional structural features and specific charge characteristics.

[0048] The term "affinity" or "binding affinity" refers to the intrinsic binding strength of an interaction between a molecule (e.g., a receptor or antigen) and its pair (e.g., a ligand or antibody), i.e., the strength of the sum of all non-covalent interactions. Unless otherwise stated, "binding affinity" as used herein is used to reflect the intrinsic binding affinity between members of a binding pair (e.g., receptor and ligand or antigen and antibody) in a one-to-one interaction. The affinity of molecule X for its pair Y can generally be expressed by the equilibrium dissociation constant (K0). D This indicates that the equilibrium dissociation constant is the dissociation rate constant (K). dis or K off Affinity is the ratio of the binding rate constant (Ka or Kon) to the binding rate constant. Affinity can be measured by common methods known in the art, including the methods used in this invention.

[0049] In this article, the terms "cross-reactivity," "cross-reactivity," or "cross-reactive activity" are used interchangeably to refer to the ability of an antibody to be specific to one antigen and also react with a second antigen; it is a measure of the affinity between two different antigens. Therefore, if an antibody binds to an antigen other than its target antigen, it is cross-reactive. Cross-reactive epitopes typically contain many amino acid sequences homologous to or similar structural features to the target antigen epitope, and in some cases may bind to antibodies more precisely than the target antigen epitope. If an antibody does not bind to any sequence or structure other than the target antigen epitope or cross-reactive epitope, the antibody can be considered to have "high specificity" for that antigen epitope.

[0050] In this paper, the terms "fusion" or "fusion-related," when used for amino acid sequences (e.g., peptides, polypeptides, or proteins), refer to the combination of two or more amino acid sequences into a single, non-naturally occurring amino acid sequence through chemical bonding or recombination. Fusion amino acid sequences can be generated by recombination of two genes encoding polynucleotide sequences and can be expressed by introducing a construct containing the recombinant polynucleotide into a host cell.

[0051] In this document, the term "antibody variant" or "antibody variant" refers to an antibody polypeptide sequence containing at least one amino acid mutation in the variable region of a reference antibody. Variants may be substantially homologous to or substantially identical to the unmodified antibody. In some embodiments, one, two, three, four, five, and / or six CDRs of the inventive anti-HER4 antibody contain amino acid mutations to improve and optimize the performance of the antibody or antigen-binding moiety, including but not limited to increased humanization, enhanced specificity to the target protein / antigen (e.g., HER4), binding activity, increased yield, and / or improved stability (e.g., reducing or eliminating the risk of aspartic acid isomerization and / or asparagine deacylation). In some embodiments, one, two, three, and / or four FRs of the inventive anti-HER4 antibody contain amino acid mutations to improve and optimize the performance of the antibody or antigen-binding moiety, including but not limited to increased humanization, enhanced specificity to the target molecule (e.g., HER4), binding activity, increased yield, and / or improved stability (e.g., reducing or eliminating the risk of aspartic acid isomerization and / or asparagine deacylation). In some embodiments, one or more amino acid mutations are performed on the CDRs and FRs of the anti-HER4 antibody of this application to improve the humanization of the antibody or antigen-binding moiety, enhance specificity for the target molecule (e.g., HER4), improve binding activity, increase yield, and / or improve stability (e.g., reduce or eliminate the risk of aspartic acid isomerization and / or asparagine deacylation). In some embodiments, the amino acid mutations include amino acid substitutions, deletions, insertions, or any combination thereof.

[0052] Amino acid substitution includes conserved and non-conserved amino acid substitution. Conserved amino acid substitution involves replacing the amino acid with another amino acid of the same class (e.g., with similar chemical properties or functions), while non-conserved substitution involves replacing the amino acid with a different class (with dissimilar chemical properties or functions). Those skilled in the art can perform conserved or non-conserved amino acid substitution based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the residues involved. For example, (i) nonpolar (hydrophobic) amino acids include alanine (Ala, A), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), proline (Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W), and methionine (Met, M); (ii) polar neutral amino acids include glycine (Gly, G), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N), and glutamine (Gln, Q); (iii) positively charged (basic) amino acids include arginine (Arg, R), lysine (Lys, K), and histidine (His, H); and (iv) negatively charged (acidic) amino acids include aspartic acid (Asp, D) and glutamic acid (Glu, E). Generally, conserved amino acid substitutions do not substantially alter the functional properties of proteins, while non-conserved amino acid substitutions can lead to significant changes in protein properties or functions. Although the sites or regions where amino acid sequence mutations are introduced can be predetermined, the potential changes in protein properties or functions resulting from non-conserved substitutions are unpredictable. In some embodiments, the anti-HER4 antibody of the present invention unexpectedly achieves significant changes in function and performance through non-conserved amino acid substitutions, such as binding activity and molecular stability.

[0053] In this document, for two or more polypeptide sequences, the terms "identical," "identity," "percentage of identity," or "percentage sequence identity" are used interchangeably. They refer to the percentage of amino acid residues in a candidate sequence that are identical to a reference sequence, relative to the total number of amino acid residues in the candidate sequence, after introducing necessary intervals to maximize the number of identical amino acids during amino acid sequence alignment. Conservative substitutions of these amino acid residues may or may not be considered identical residues. Sequence alignment can be performed using tools disclosed in the art, such as BLASTp, ClustalW2 (see also Higgins DG et al., Methods Enzymol 1996, 266:383-402; Larkin MA et al., Bioinformatics 2007, 23:2947-2948), and ALIGN or Megalign (DNASTAR) software to determine the percentage sequence identity of the amino acid sequences. Those skilled in the art can use the default parameters of these tools or adjust the parameters appropriately according to the needs of the alignment, for example, by selecting a suitable algorithm for sequence alignment.

[0054] The amino acid sequences mentioned in this article that "reduce or eliminate the risk of deamidation" refer to sequences in which amino acid residues that are easily deamidged are replaced by amino acid residues that are less easily or reluctantly deamidged. Deamidation is a chemical reaction in which the amide functional group in the side chain of asparagine or glutamine is removed or converted into another functional group. Typically, asparagine (Asn) can be converted into aspartic acid or isoaspartic acid, and glutamine (Gln) can be converted into glutamic acid or pyroglutamic acid. Amino acid sequences containing Asn-Gly, Asn-Ser, or Asn-Thr sites are prone to deamidation, and asparagine is more easily deamidged than glutamine. Deamidation of asparagine and / or glutamine can alter the structure and stability and / or function of antibodies (i.e., antibody-antigen binding), therefore, it is necessary to reduce or eliminate the risk of deamidation. The risk of deamidation at these sites can be reduced or eliminated by predicting amino acid residues in the antibody variable region that are prone to deamidation (Jasmin F Sydow et al., PLoS ONE 2014, 9(6):e100736) and by substituting them with amino acid residues that are less or not prone to deamidation.

[0055] In this document, the term "aspartic acid isomerization" refers to an amino acid sequence containing aspartic acid residues that are prone to isomerization. Since isomerization causes conformational changes in antibodies, thereby altering the antibody surface charge and leading to antibody charge heterogeneity, the antibody of this application preferably does not contain aspartic acid isomer sites. Aspartic acid isomerization readily occurs at the DG sequence, and it has also been reported that isomerization can occur at the DH or DS sequences, resulting in isoaspartic acid residues. Because these amino acid residues introduce a linker bond into the polypeptide chain, the stability of the polypeptide chain is reduced (also known as the isoaspartic effect), and it may also lead to a decrease in antibody-antigen binding affinity.

[0056] In this document, when referring to proteins, the term "isolated" means that the protein is substantially free of other cellular components bound to it in its native state, preferably in a homogeneous state; for example, the isolated protein may be removed from its native or natural environment. The isolated protein may be lyophilized or aqueous. Typically, its purity and homogeneity can be determined using analytical chemistry techniques (e.g., polyacrylamide gel electrophoresis or high-performance liquid chromatography). In the isolated preparation, the protein is substantially purified. The term "purified" means that the protein produces substantially only one band in a non-reducing electrophoresis gel. Specifically, this means that the protein has a purity of at least 85%, more preferably at least 95%, and most preferably at least 99%. For the purposes of this application, in some embodiments, recombinant proteins expressed in host cells are considered isolated; the same applies to native or recombinant proteins separated, fractionated, or partially or substantially purified by any technique well known to those skilled in the art. In some embodiments, "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to HER4 is substantially free of antibodies that specifically bind to antigens other than HER4). Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals. In some embodiments, the recombinant polynucleotide encoding the polypeptide or protein of this application (e.g., an anti-HER4 antibody) contained in a vector is considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides contained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.

[0057] Unless the context otherwise indicates, a “derivative” is a polypeptide or fragment thereof having one or more non-conserved or conserved amino acid substitutions relative to the parent antibody or polypeptide; or a polypeptide or fragment thereof modified by covalently linking to a second molecule, for example by linking to a heteropeptide, or by glycosylation, acetylation, phosphorylation, etc. The definition of “derivative” may also include polypeptides of one or more amino acid analogs (e.g., non-natural amino acids, etc.), and other modifications known in the art (natural and non-natural).

[0058] In this paper, the term "expression vector" or "vector" refers to a delivery vehicle into which a polynucleotide or nucleic acid encoding a protein can be operatively inserted to enable the expression of that protein. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic material they carry within the host cells.

[0059] In this document, the term "host cell" refers to a cell into which a foreign polynucleotide, nucleic acid, and / or vector has been introduced. Host cells include "transformers" and "transformed cells," which include the initially transformed cell and its progeny, regardless of the number of passages. Progeny cells may not be identical to parental cells in their nucleic acid content and may contain mutations. This invention includes screening or selecting mutant progeny cells with the same function or biological activity from the initially transformed cells.

[0060] In this document, the terms “subject,” “patient,” or “individual” are used interchangeably and include, but are not limited to: mammals such as humans, non-human primates (e.g., monkeys), mice, pigs, dogs, cats, cattle, goats, rabbits, rats, guinea pigs, hamsters, horses, sheep, or other non-human mammals; non-mammals such as non-mammalian vertebrates, such as birds (e.g., chickens, emus, or ducks) or fish; and non-mammalian invertebrates. In some embodiments, the subject and pharmaceutical composition involved in the use or method of the present invention are used (preventively and / or therapeutically) to treat non-human animals.

[0061] In this article, “treating” or “to treat” refers to alleviating or alleviating a disease or symptom, slowing the onset or development of a disease or symptom, reducing the risk of developing a disease or symptom, or delaying the development of symptoms associated with a disease or symptom, reducing or terminating symptoms associated with a disease or symptom, producing a complete or partial reversal of a disease or symptom, curing a disease or symptom, or a combination of the above.

[0062] As used herein, "HER4-related disease" or "symptom" refers to any disease or symptom caused, exacerbated, or otherwise associated with increased or decreased HER4 expression or activity. In some embodiments, HER4-related disease is cancer, such as glioma. In some embodiments, HER4-related disease is glial proliferative disease.

[0063] The terms "therapeutic effective amount," "effective dose," or "effective amount" refer to the quantity or concentration of an active ingredient or agent that, at the required dose and for the required duration, effectively prevents or improves symptoms associated with a disease or condition, alleviates or reduces the severity of the disease or condition, and / or delays or stops disease progression. The therapeutically effective amount of the formulations, antibodies, or antigen-binding fragments thereof, or compositions of the present invention can vary depending on various factors such as disease state, individual age, sex, and weight, and the ability of the antibody or antigen-binding portion to elicit the desired response in the individual. A therapeutically effective amount can also be considered as the therapeutically beneficial effect of the formulation, antibody, or antigen-binding fragment thereof significantly outweighing any toxic or harmful effects it causes.

[0064] In this document, the terms “pharmaceutical acceptable” or “medicinally acceptable” mean that the carrier, solvent, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients in the formulation and physiologically compatible with the subject.

[0065] The term “about” when used in conjunction with a numerical value means to encompass a range of numerical values ​​having a lower limit 5% smaller than the specified numerical value and an upper limit 5% larger than the specified numerical value, or in one embodiment a lower limit 10% smaller and an upper limit 10% larger, or in another embodiment a lower limit 15% smaller and an upper limit 15% larger, or in yet another embodiment a lower limit 20% smaller and an upper limit 20% larger.

[0066] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0067] As used herein, the terms “comprising,” “including,” “containing,” “having,” or “involving” are used interchangeably to mean including the stated elements, integers, or steps, but not excluding any other elements, integers, or steps. In this document, when the terms “comprising,” “including,” “containing,” “having,” or “involving” are used, unless otherwise specified, they also cover situations consisting of the stated elements, integers, or steps.

[0068] As used herein, the term “optional” indicates whether the object it modifies exists or does not exist; for example, “the pillbox contains at least one optional additional therapeutic agent” means that the pillbox may or may not contain at least one additional therapeutic agent.

[0069] As used herein, "some embodiments," "an embodiment," "a specific embodiment," or "a specific embodiment," or combinations thereof, indicates that a particular feature, structure, or characteristic associated with the described embodiment is included in at least one embodiment of this application. Therefore, the foregoing terms appearing throughout this specification do not necessarily refer to the same embodiment. Furthermore, the particular feature, structure, or characteristic may be combined in any suitable manner in one or more embodiments.

[0070] Unless the context clearly indicates otherwise, singular terms encompass the plural referents, and vice versa.

[0071] For purposes of description and disclosure, all patents, patent applications and other publications are expressly incorporated herein by reference. These publications are provided only because their publication predates the filing date of this application. All statements regarding the dates of these documents or representations of their contents are based on information available to the applicant and do not constitute any admission of the accuracy of the dates or contents of these documents.

[0072] The various aspects of this application will be described in more detail in the following sections.

[0073] 1. Anti-HER4 antibodies and their antigen-binding fragments

[0074] On the one hand, this application provides an isolated anti-HER4 antibody or its antigen-binding fragment, which can specifically recognize and bind to the extracellular domain III of human HER4 and can block ligand-induced HER4 receptor dimerization.

[0075] The anti-HER4 antibody or its antigen-binding fragment of the present invention specifically binds to the extracellular domain of human HER4 and exhibits high binding activity. In some embodiments, flow cytometry was used to detect the binding activity of the antibody or its antigen-binding fragment to HER4-expressing 293T cells, and the mean fluorescence intensity (MFI) value was high.

[0076] The anti-HER4 antibody or its antigen-binding fragment of the present invention has an inhibitory effect on the HER4 receptor and its downstream signal transduction pathways. In some embodiments, the anti-HER4 antibody or its antigen-binding fragment can inhibit ligand-induced HER4 receptor dimerization and its downstream signal transduction pathways, wherein the ligands include NRG-1 to 4, preferably NRG-1. In some embodiments, the inhibitory activity (IC50 value) of the antibody or its antigen-binding fragment against NRG-1-induced HER2 / HER4 receptor dimerization is not more than 1 μg / mL, preferably not more than 0.5 μg / mL, as detected by live-cell bioluminescence.

[0077] The anti-HER4 antibody or its antigen-binding fragment of the present invention has no cross-reactivity with other members of the human ErbB / HER family (including EGFR, HER2, and HER3). The degree of binding of the anti-HER4 antibody or its antigen-binding fragment to non-target proteins (e.g., EGFR, HER2, or HER3) is approximately 10% lower than the binding of the antibody or its antigen-binding fragment to HER4, which can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation (RIA).

[0078] The anti-HER4 antibody or its antigen-binding fragment of the present invention has cross-reactivity with HER4 of other species (e.g., mice, rats, dogs), and its cross-reactivity is comparable to that of human HER4.

[0079] The anti-HER4 antibody of this application may optionally include Fab, Fab', F(ab')2, Fv, scFv, scFv-Fc, single-domain antibody (sdAb), or of IgG type. The anti-HER4 antibody of this application may be a murine antibody, chimeric antibody, humanized antibody, or fully human antibody; it may be a monoclonal antibody, polyclonal antibody, monospecific antibody, bispecific antibody, multispecific antibody, or antibody fragment, as long as the antibody can specifically recognize and bind to the extracellular domain of human HER4 and has no cross-reactivity with other members of the ErbB / HER family. In some embodiments, the anti-HER4 antibody is selected from fully human anti-HER4 antibodies or optimized antibodies or antibody fragments thereof.

[0080] On the other hand, the present invention provides anti-HER4 antibodies or antigen-binding fragments thereof as shown in Table 2, comprising VH and VL. In some embodiments, the anti-HER4 antibody or antigen-binding fragment of the present invention specifically binds to the extracellular domain III of the HER4 receptor, which comprises the VH amino acid sequence as shown in SEQ ID NO:36, and / or the VL amino acid sequence as shown in SEQ ID NO:59.

[0081] In some embodiments, the anti-HER4 antibody of the present invention or its antigen-binding fragment comprises one or more CDRs of the VH amino acid sequence shown in SEQ ID NO:36 or amino acid sequences such as SEQ ID NOs:1, 2 and 21 or variants thereof, said variants including optimized antibodies or any other variants described in the present invention.

[0082] In some embodiments, the anti-HER4 antibody of the present invention or its antigen-binding fragment comprises one or more CDRs of the VL amino acid sequence shown in SEQ ID NO:59 or the amino acid sequence shown in SEQ ID NOs:23, 30 and 34 or its variants, said variants including optimized antibodies or any other variants described in the present invention.

[0083] In some embodiments, the anti-HER4 antibody or its antigen-binding fragment of the present invention comprises: one or more CDRs of the VH amino acid sequence shown in SEQ ID NO:36 or amino acid sequences or variants thereof shown in SEQ ID NOs:1, 2 and 21, and one or more CDRs of the VL amino acid sequence shown in SEQ ID NO:59 or amino acid sequences or variants thereof shown in SEQ ID NOs:23, 30 and 34, said variants including optimized antibodies or any other variants described in the present invention.

[0084] In some specific embodiments, the anti-HER4 antibody or its antigen-binding fragment comprises: the HCDR1 amino acid sequence shown in SEQ ID NO:1, the HCDR2 amino acid sequence shown in SEQ ID NO:2, and the HCDR3 amino acid sequence shown in SEQ ID NO:21, as well as the LCDR1 amino acid sequence shown in SEQ ID NO:23, the LCDR2 amino acid sequence shown in SEQ ID NO:30, and the LCDR3 amino acid sequence shown in SEQ ID NO:34.

[0085] On the other hand, the VH and / or VL of the anti-HER4 antibody (e.g., Ab4261) of the present invention can be used as starting materials for engineering modification to prepare antibodies more suitable for the human body as described herein, namely, optimized antibodies of the parent antibody Ab4261.

[0086] In some embodiments, the anti-HER4 optimized antibody of the present invention comprises an antibody or antigen-binding fragment thereof having a modified VH and / or VL compared to the VH (SEQ ID NO: 36) and / or VL (SEQ ID NO: 59) of the parent antibody Ab4261. Modifications introduced into the parent antibody include introducing amino acid mutations (including amino acid substitution, deletion, insertion, or any combination thereof) into the heavy chain variable regions (CDRs), light chain variable regions (CDRs), and / or FRs of the parent antibody, which can improve the humanization degree, binding activity, and / or reduce or eliminate the risk of aspartic acid isomerization and / or asparagine deacylation of the parent antibody.

[0087] In some specific embodiments, the anti-HER4 optimized antibody of the present invention has one or more amino acid mutations relative to the FR region of the parent antibody Ab4261 to improve the humanization of the parent antibody. For example, one or more amino acid mutations can be made in the FR region (including the heavy chain variable region FR region and / or the light chain variable region FR region) of the VH (SEQ ID NO:36) of Ab4261. Further, amino acid mutations can be introduced into HFR1, HFR3, and / or HFR4, for example, a mutation can be made in the threonine residue at position 7 of HFR1, a mutation can be made in the glycine residue at position 23 of HFR3 (corresponding to position 85 of the VH amino acid sequence shown in SEQ ID NO:36), and / or a mutation can be made in the methionine residue at position 6 of HFR4 (corresponding to position 108 of the VH amino acid sequence shown in SEQ ID NO:36). Amino acid mutations can also be introduced into LFR2 and / or LFR3, for example, a mutation can be made in the methionine residue at position 14 of LFR2 (corresponding to position 48 of the VL amino acid sequence shown in SEQ ID NO:59) and / or the serine residue at position 15 (corresponding to position 49 of the VL amino acid sequence shown in SEQ ID NO:59), and / or the methionine residue at position 29 of LFR3 (corresponding to position 48 of the VL amino acid sequence shown in SEQ ID NO:59). The valine at position 85 of the VL amino acid sequence shown in NO:59 was mutated.

[0088] In one specific embodiment, the anti-HER4 optimized antibody of the present invention has one or more amino acid mutations in the HFR region relative to the VH amino acid sequence (SEQ ID NO:36) of the parent antibody Ab4261, including T7S, G85E and / or M108L, and / or has one or more amino acid mutations in the LFR region relative to the VL amino acid sequence (SEQ ID NO:59) of the parent antibody Ab4261, including M48I, S49Y and / or I85V, which can significantly improve the humanization degree of the parent antibody Ab4261 while retaining its cell-binding activity with the parent antibody Ab4261.

[0089] In some specific embodiments, the anti-HER4 optimized antibody of this application has one or more amino acid mutations in the CDR regions (including heavy chain variable regions CDRs and / or light chain variable regions CDRs) of the parent antibody Ab4261 to enhance the binding activity of the parent antibody to HER4-expressing cells. For example, one or more amino acid mutations are made in the CDRs of VH (SEQ ID NO:36) and / or VL (SEQ ID NO:59) of Ab4261. Further, one or more amino acid mutations may be introduced in HCDR2 and / or HCDR3, for example, alanine at position 1 (corresponding to position 50 of the VH amino acid sequence shown in SEQ ID NO:36), serine at position 8 (corresponding to position 56 of the VH amino acid sequence shown in SEQ ID NO:36) of HCDR2, and / or glycine at position 2 (corresponding to position 96 of the VH amino acid sequence shown in SEQ ID NO:36) and position 3 (corresponding to position 97 of the VH amino acid sequence shown in SEQ ID NO:36) of HCDR3. Mutations in the alanine residue at position 1 of HCDR2 (corresponding to position 50 of the VH amino acid sequence shown in SEQ ID NO:36) include A50E, A50G, A50I, A50R, A50S, A50V, A50W, or A50Y; mutations in the serine residue at position 8 (corresponding to position 56 of the VH amino acid sequence shown in SEQ ID NO:36) include S56D, S56N, S56R, S56V, or S56Y (preferably S56N, S56R, or S56Y); mutations in the serine residue at position 2 of HCDR3 (corresponding to position 50 of SEQ ID NO:36) include A50E, A50G, A50I, A50R, A50S, A50V, A50W, or A50Y. Mutations in the glycine residue at position 96 of the VH amino acid sequence shown in SEQ ID NO:36 include G96A, G96D, G96E, G96F, G96H, G96I, G96K, G96Q, G96R, G96S, G96V, G96W, or G96Y (preferably G96Y). Mutations in the glycine residue at position 3 (corresponding to position 97 of the VH amino acid sequence shown in SEQ ID NO:36) include G97A, G97D, G97E, G97F, G97H, G97K, G97L, G97N, G97Q, G97R, G97T, G97V, G97W, or G97Y.

[0090] One or more amino acid mutations can also be introduced into LCDR1, LCDR2, and / or LCDR3. For example, histidine at position 8 (corresponding to position 27d of the VL amino acid sequence shown in SEQ ID NO:59), tyrosine at position 12 (corresponding to position 30 of the VL amino acid sequence shown in SEQ ID NO:59), histidine at position 13 (corresponding to position 31 of the VL amino acid sequence shown in SEQ ID NO:59), aspartic acid at position 16 (corresponding to position 34 of the VL amino acid sequence shown in SEQ ID NO:59), leucine at position 1 (corresponding to position 50 of the VL amino acid sequence shown in SEQ ID NO:59), asparagine at position 4 (corresponding to position 53 of the VL amino acid sequence shown in SEQ ID NO:59), methionine at position 1 (corresponding to position 89 of the VL amino acid sequence shown in SEQ ID NO:59), and methionine at position 3 (corresponding to position 89 of the VL amino acid sequence shown in SEQ ID NO:59). Amino acid mutations were performed on alanine at position 91 of the VL amino acid sequence shown in SEQ ID NO:59 and glutamine at position 5 (corresponding to position 93 of the VL amino acid sequence shown in SEQ ID NO:59).The histidine residue mutation at position 8 of LCDR1 (corresponding to position 27d of the VL amino acid sequence shown in SEQ ID NO:59) includes H27Y; the tyrosine residue mutation at position 12 (corresponding to position 30 of the VL amino acid sequence shown in SEQ ID NO:59) includes Y30D, Y30G, Y30K, Y30N, or Y30R; the histidine residue mutation at position 13 (corresponding to position 31 of the VL amino acid sequence shown in SEQ ID NO:59) includes H31D, H31K, H31N, H31Q, H31S, H31T, or H31Y (preferably H31K or H31T); the aspartic acid residue mutation at position 16 (corresponding to position 34 of the VL amino acid sequence shown in SEQ ID NO:59) includes D34A, D34G, D34H, D34N, D34Q, D34S, or D34Y; the histidine residue mutation at position 1 of LCDR2 (corresponding to position 27d of the VL amino acid sequence shown in SEQ ID NO:59) includes H27 ... (corresponding to position 27d of the VL amino acid sequence shown in SEQ ID NO:59) includes H27Y; the histidine residue mutation at position 1 (corresponding to position 27d of the VL amino acid sequence shown in SEQ ID NO:59) includes H27Y; the histidine residue mutation at position 1 (corresponding to position 27d of the VL amino acid sequence shown in SEQ ID NO:59) includes H27Y; the histidine residue Mutations in the leucine residue at position 50 of the VL amino acid sequence shown in SEQ ID NO:59 include L50A, L50E, L50G, L50K, L50Q, L50R, L50S, L50V, L50W, or L50Y (preferably L50A, L50W, or L50Y); mutations in the asparagine residue at position 4 (corresponding to position 53 of the VL amino acid sequence shown in SEQ ID NO:59) include N53D, N53E, N53K, N53Q, N53R, N53S, or N53T; mutations in the methionine residue at position 1 of LCDR3 (corresponding to position 89 of the VL amino acid sequence shown in SEQ ID NO:59) include M89Q, M89A, M89H, M89L, or M89S (preferably M89L); mutations in the methionine residue at position 3 (corresponding to position 50 of ... Mutations in the alanine residue at position 91 of the VL amino acid sequence shown in SEQ ID NO:59 include A91D, A91F, A91G, A91H, A91L, A91R, A91S, A91T, A91W, or A91Y; mutations in the glutamine residue at position 5 (corresponding to position 93 of the VL amino acid sequence shown in SEQ ID NO:59) include Q93D, Q93G, Q93H, Q93I, Q93R, Q93S, or Q93T.

[0091] In some specific embodiments, the anti-HER4 optimized antibody of the present invention has one or more amino acid mutations relative to the CDR regions (including heavy chain variable region CDRs and / or light chain variable region CDRs) of the parent antibody Ab4261 to reduce the potential risk of aspartic acid isomerization and / or asparagine deamidation. For example, the heavy chain variable region CDRs or light chain variable region CDRs of the parent antibody Ab4261 are mutated by no more than 1, 2, 3, 4, 5, or 6 amino acid residues, said mutations may be amino acid substitutions, additions, or deletions, preferably amino acid substitutions.

[0092] In one specific embodiment, one or more amino acid mutations are made in the CDR region of VH (SEQ ID NO:36) and / or VL (SEQ ID NO:59) of Ab4261. Further, one or more amino acid mutations can be introduced into HCDR2 and / or LCDR1, such that the mutant of the parent antibody contains amino acid sequences that reduce or eliminate the risk of deamidation. For example, amino acid mutations can be made in the asparagine residues at positions 3 (corresponding to position 52 of the VH amino acid sequence shown in SEQ ID NO:36), 4 (corresponding to position 52a of the VH amino acid sequence shown in SEQ ID NO:36), 5 (corresponding to position 53 of the VH amino acid sequence shown in SEQ ID NO:36), and 6 (corresponding to position 54 of the VH amino acid sequence shown in SEQ ID NO:36). Mutations in the 3rd position (corresponding to the 52nd position of the VH amino acid sequence shown in SEQ ID NO:36) of HCDR2 include N52Y, N52S, N52W, N52S, N52G, or N52H (preferably N52S); mutations in the 4th position (corresponding to the 52a position of the VH amino acid sequence shown in SEQ ID NO:36) of HCDR2 include N52Aa, N52aF, N52aH, N52aP, N52aQ, N52aR, N52aS, N52aT, N52aW, N52aY, or N52aG (preferably N52aF, N52aH, N52aP, N52aS, N52aY, N52aW, or N52aG); mutations in the 5th position (corresponding to the 52a position of SEQ ID NO:36) of HCDR2 include N52Aa, ...). Mutations in the asparagine residue at position 53 of the VH amino acid sequence shown in SEQ ID NO:36 include N53E, N53H, N53I, N53R, N53Y, N53G or N53S (preferably N53I, N53R, N53Y or N53S), and mutations in the glycine residue at position 6 (corresponding to position 54 of the VH amino acid sequence shown in SEQ ID NO:36) include G54A, G54F or G54K (preferably G54A).

[0093] Amino acid mutations can also be made in the asparagine residue at position 10 (corresponding to position 28 of the VL amino acid sequence shown in SEQ ID NO:59) and the glycine residue at position 11 (corresponding to position 29 of the VL amino acid sequence shown in SEQ ID NO:59). Mutations in the asparagine residue at position 10 (corresponding to position 28 of the VL amino acid sequence shown in SEQ ID NO:59) include N28G, N28H, N28I, N28Y, N28L, N28S, N28T, and N28V, and mutations in the glycine residue at position 11 (corresponding to position 29 of the VL amino acid sequence shown in SEQ ID NO:59) include G29A, G29I, G29N, G29P, G29R, G29S, G29T, or G29V (preferably G29R, G29S, or G29T).

[0094] In one specific embodiment, one or more amino acid mutations are made in the CDR region of the VH (SEQ ID NO:36) of the parent antibody Ab4261 to isomerize the parent antibody. In a preferred embodiment, the anti-HER4 optimized antibody of the present invention does not contain an aspartic acid isomer site. One or more amino acid mutations can be introduced into HCDR2 of Ab4261. For example, mutations can be made in the aspartic acid at position 13 (corresponding to position 61 of the VH amino acid sequence shown in SEQ ID NO:36) and the serine at position 14 (corresponding to position 62 of the VH amino acid sequence shown in SEQ ID NO:36) of HCDR2 to reduce or eliminate the aspartic acid isomerization of the amino acid sequence of the parent antibody Ab4261. The mutations in the aspartic acid residue at position 13 of HCDR2 (corresponding to position 61 of the VH amino acid sequence shown in SEQ ID NO:36) include D61Q or D61P (preferably D61P), and the mutations in the serine residue at position 14 (corresponding to position 62 of the VH amino acid sequence shown in SEQ ID NO:36) include S62K.

[0095] By performing the aforementioned amino acid mutations on the CDR and framework regions of the parent antibody Ab4261, the optimized antibody not only achieved a higher level of humanization and a lower risk of aspartic acid isomerization and / or asparagine deamidation, but also improved the binding activity of the parent antibody to human HER4.

[0096] On the other hand, the amino acid sequence numbers of the heavy chain CDR region, light chain CDR region, heavy chain variable region, and light chain variable region of the anti-HER4 antibody (including the parent antibody Ab4261 and its optimized antibody) of this application are summarized in Table 2, wherein the heavy chain variable region CDRs and light chain variable region CDRs are defined by the Kabat numbering system. However, as is known in the art, CDR regions can also be defined based on other numbering systems / methods such as Chothia and IMGT, AbM, or Contact based on the heavy chain / light chain variable region sequence. CDR regions defined by other numbering systems / methods and the CDR regions defined by Kabat adopted in this invention are both within the scope of protection of this invention.

[0097] Table 2. Amino acid sequence ID numbers of the CDR region, heavy chain variable region, and light chain variable region of the anti-HER4 antibody.

[0098]

[0099]

[0100] In some embodiments, the present invention provides an isolated anti-HER4 monoclonal antibody or its antigen-binding fragment comprising (1) the HCDR1 amino acid sequence as shown in SEQ ID NO:1; (2) the amino acid sequence being X1IX2X3X4X5GX6TYYAX7X8VKG (X1 = A, E, G, I, R, S, V, W or Y; X2 = N, Y, S, W, S, G or H; X3 = N, A, F, H, P, Q, R, S, T, W, Y or G; X4 = N, E, H, I, R, Y, G or S; X5 = G, A, F or K; X6 = S, D, N, R, V or Y; X7 = D, Q or P; X8 = S or K; i.e. SEQ ID NO:1) The HCDR2 (shown in NO:81) preferably has the following amino acid sequence: X1 = A, X2 = N or S, X3 = A, F, H, P, S, Y, W or G, X4 = I, R, Y or S, X5 = G or A, X6 = S, N, R or Y, X7 = P, X8 = S; (3) The amino acid sequence is GX9X 10 AFDI(X9=G,A,D,E,F,H,I,K,Q,R,S,V,W or Y; 10 =G,A,D,E,F,H,K,L,N,Q,R,T,V,W orY; i.e., HCDR3 as shown in SEQ ID NO:82), preferably, X9 = G or Y, X 10 =G; (4) The amino acid sequence is KSSQSLLX 11 SX 12 X 13 X 14 X 15 YLX 16 (X 11=H or Y; X 12 =N,G,H,I,Y,L,S,T orV;X 13 =G,A,I,N,P,S,T orV;X 14 =Y,D,G,K,N orR;X 15 =H,D,K,N,Q,S,T orY;X 16 =D,A,G,H,N,Q,S orY; i.e., the LCDR1 shown in SEQ ID NO:83, preferably, X 11 =H,X 12 =N,X 13 =G,R,S or T,X 14 =Y,X 15 =H,K or T,X 16 =D; (5) The amino acid sequence is X 17 GSX 18 RAS(X 17 =L,A,E,G,K,Q,R,S,V,W or Y; 18 =N,D,E,K,Q,R,S orT; i.e., LCDR2 as shown in SEQ ID NO:84, preferably, X 17 =L, A, W or Y, X 18 =N; (6) The amino acid sequence is X 19 QX 20 LX 21 TTRT(X 19 =M,Q,A,H,L or S;X 20 =A,D,F,G,H,L,R,S,T,W or Y; 21 =Q,D,G,H,I,R,S,T; i.e., LCDR3 as shown in SEQ ID NO:85, preferably, X 19 =L,X 20 =A,X 21 =Q, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0101] In some specific embodiments, the above-mentioned anti-HER4 antibody or its antigen-binding fragment comprises: the HCDR1 amino acid sequence shown in SEQ ID NO:1, the HCDR2 amino acid sequence shown in any one of SEQ ID NO:2-22, and the HCDR3 amino acid sequence shown in SEQ ID NO:23 or 24, the LCDR1 amino acid sequence shown in any one of SEQ ID NO:25-31, the LCDR2 amino acid sequence shown in any one of SEQ ID NO:32-35, and the LCDR3 amino acid sequence shown in SEQ ID NO:36 or 37.

[0102] In some specific embodiments, the above-mentioned anti-HER4 antibody or its antigen-binding fragment comprises: the HCDR1 amino acid sequence as shown in SEQ ID NO:1; the HCDR2 amino acid sequence as shown in SEQ ID NO:2, 14, 18, 20, 21 or 22; and the HCDR3 amino acid sequence as shown in SEQ ID NO:23; the LCDR1 amino acid sequence as shown in SEQ ID NO:25, 28, 29 or 31; the LCDR2 amino acid sequence as shown in SEQ ID NO:32 or 34; and the LCDR3 amino acid sequence as shown in SEQ ID NO:36.

[0103] In one specific embodiment, the anti-HER4 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, and HCDR3, as well as LCDR1, LCDR2, and LCDR3, wherein each CDR comprises:

[0104] (1) The HCDR1 amino acid sequence shown in SEQ ID NO:1, the HCDR2 amino acid sequence shown in any one of SEQ ID NO:2-22, the HCDR3 amino acid sequence shown in SEQ ID NO:23; and the LCDR1 amino acid sequence shown in SEQ ID NO:25, the LCDR2 amino acid sequence shown in SEQ ID NO:32, the LCDR3 amino acid sequence shown in SEQ ID NO:36, or amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or

[0105] (2) The HCDR1 amino acid sequence shown in SEQ ID NO:1, the HCDR2 amino acid sequence shown in SEQ ID NO:2, the HCDR3 amino acid sequence shown in SEQ ID NO:24; and the LCDR1 amino acid sequence shown in SEQ ID NO:25, the LCDR2 amino acid sequence shown in SEQ ID NO:32, and the LCDR3 amino acid sequence shown in SEQ ID NO:36, or amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or

[0106] (3) The HCDR1 amino acid sequence shown in SEQ ID NO:1, the HCDR2 amino acid sequence shown in SEQ ID NO:2, and the HCDR3 amino acid sequence shown in SEQ ID NO:23; and the LCDR1 amino acid sequence shown in any one of SEQ ID NO:26-30, the LCDR2 amino acid sequence shown in SEQ ID NO:32, and the LCDR3 amino acid sequence shown in SEQ ID NO:36, or amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or

[0107] (4) The HCDR1 amino acid sequence shown in SEQ ID NO:1, the HCDR2 amino acid sequence shown in SEQ ID NO:2, the HCDR3 amino acid sequence shown in SEQ ID NO:23; and the LCDR1 amino acid sequence shown in SEQ ID NO:25, the LCDR2 amino acid sequence shown in SEQ ID NO:33 or 35, the LCDR3 amino acid sequence shown in SEQ ID NO:36, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or

[0108] (5) The HCDR1 amino acid sequence shown in SEQ ID NO:1, the HCDR2 amino acid sequence shown in SEQ ID NO:2, and the HCDR3 amino acid sequence shown in SEQ ID NO:23; and the LCDR1 amino acid sequence shown in SEQ ID NO:25, 28, or 31, the LCDR2 amino acid sequence shown in SEQ ID NO:34, and the LCDR3 amino acid sequence shown in SEQ ID NO:36, or amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or

[0109] (6) The HCDR1 amino acid sequence shown in SEQ ID NO:1, the HCDR2 amino acid sequence shown in SEQ ID NO:2, and the HCDR3 amino acid sequence shown in SEQ ID NO:23; and the LCDR1 amino acid sequence shown in SEQ ID NO:25, the LCDR2 amino acid sequence shown in SEQ ID NO:32, and the LCDR3 amino acid sequence shown in SEQ ID NO:37, or amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or

[0110] (7) The HCDR1 amino acid sequence shown in SEQ ID NO:1, the HCDR2 amino acid sequence shown in SEQ ID NO:21 or 22, the HCDR3 amino acid sequence shown in SEQ ID NO:23; and the LCDR1 amino acid sequence shown in SEQ ID NO:28 or 31, the LCDR2 amino acid sequence shown in SEQ ID NO:34, the LCDR3 amino acid sequence shown in SEQ ID NO:36, or the amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0111] In one embodiment, the anti-HER4 antibody or its antigen-binding fragment of the present invention comprises HCDR1, HCDR2, and HCDR3, as well as LCDR1, LCDR2, and LCDR3. Each CDR comprises: the amino acid sequence of HCDR1 as shown in SEQ ID NO:1, the amino acid sequence of HCDR2 as shown in SEQ ID NO:21 or 22, the amino acid sequence of HCDR3 as shown in SEQ ID NO:23, the amino acid sequence of LCDR1 as shown in SEQ ID NO:28 or 31, the amino acid sequence of LCDR2 as shown in SEQ ID NO:34, the amino acid sequence of LCDR3 as shown in SEQ ID NO:36, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. Preferably, the anti-HER4 antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence shown in SEQ ID NO:1, the HCDR2 amino acid sequence shown in SEQ ID NO:22, the HCDR3 amino acid sequence shown in SEQ ID NO:23, the LCDR1 amino acid sequence shown in SEQ ID NO:28, the LCDR2 amino acid sequence shown in SEQ ID NO:34, the LCDR3 amino acid sequence shown in SEQ ID NO:34, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0112] In some embodiments, the anti-HER4 antibody or its antigen-binding fragment of the present invention further comprises a heavy chain variable region having said HCDR1, HCDR2 and HCDR3 and a light chain variable region having said LCDR1, LCDR2 and LCDR3.

[0113] In some specific embodiments, the anti-HER4 antibody or its antigen-binding fragment of the present invention comprises: a heavy chain variable region VH having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in any one of SEQ ID NO: 38-62, and a light chain variable region VL having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in any one of SEQ ID NO: 63-80. In some preferred embodiments, the anti-HER4 antibody or its antigen-binding fragment of the present invention comprises: a VH having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 38, 39, 40, 41, 53, 57 or 59, and a VL having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 63, 64, 65, 66, 67, 70, 71 or 74. In some preferred embodiments, the anti-HER4 antibody or its antigen-binding fragment of the present invention comprises: a VH having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 61 or 62, and a VL having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 77, 78, 79 or 80.

[0114] In one specific embodiment, the anti-HER4 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein each of the heavy chain variable region and the light chain variable region comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the VH shown in any one of SEQ ID NO:38-62 and the VL shown in SEQ ID NO:63; or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the VH shown in SEQ ID NO:38 and the VL shown in any one of SEQ ID NO:64-80 ...L shown in SEQ ID NO:64-80; or having at least 85%, 86%, 87%, 88%, 89%, or 100% identity with the VL shown in any one of SEQ ID NO:64-80; or having at least 85 The VH shown in NO:61 or 62 and the VL shown in any one of SEQ ID NO:77-80 have amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. Preferably, the heavy chain variable region and the light chain variable region each comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with VH shown in SEQ ID NO:61 and VL shown in SEQ ID NO:79, respectively; or having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with VH shown in SEQ ID NO:62 and VL shown in SEQ ID NO:77, respectively; more preferably, the heavy chain variable region and the light chain variable region each comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with VH shown in SEQ ID NO:62 and VL shown in SEQ ID NO:77, respectively; The VL shown in NO:77 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0115] In some embodiments, the anti-HER4 antibody or its antigen-binding fragment of the present invention may further comprise a constant region, which includes a heavy chain constant region and a light chain constant region. The heavy chain constant region comprises a CH1, a hinge, and / or a CH2-CH3 region. The heavy chain constant region comprises both native and mutant forms of the human IgG heavy chain constant region Fc region, and also includes a hinge region that promotes dimer formation. In some embodiments, the Fc region comprises antibody CH2 and CH3 domains. Fusion proteins containing the Fc moiety can be purified using protein A or protein G affinity chromatography columns, and their serum half-life can be prolonged. Preferably, the Fc region is derived from human IgG, including IgG1, IgG2, IgG3, and IgG4. In this document, the positions of specific amino acid residues in the Fc region are determined according to the EU numbering system.

[0116] One function of the Fc region of an antibody is to exert "effective functions" with the immune system when the antibody binds to its target, including antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated by the binding of Fc to Fc receptors (FcRs) on the surface of immune cells; CDC is mediated by the binding of Fc to proteins of the complement system, such as C1q. Effector functions can be assessed using various methods, such as Fc receptor binding assays, C1q binding assays, and cell lysis assays.

[0117] The anti-HER4 antibody or its antigen-binding fragment of the present invention can specifically bind to the extracellular domain III of human HER4, has no cross-reactivity with other members of the human HER4 family (including EGFR, HER2 and HER3), and has cross-reactivity with HER4 of other species (e.g., mice, rats, dogs); the anti-HER4 antibody or its antigen-binding fragment has a high degree of humanization, high sequence stability and high binding activity with human HER4, and can effectively inhibit ligand-induced HER4 dimerization and its downstream signal transduction pathways.

[0118] 2. The method for obtaining the anti-HER4 antibody or its antigen-binding fragment of the present invention by screening

[0119] The anti-HER4 antibody provided by this invention can be a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. Methods for generating monoclonal antibodies are known in the art, and any known method (e.g., hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.) can be used in this invention to prepare monoclonal antibodies that specifically bind to the extracellular domain of HER4.

[0120] In some embodiments, the present invention isolates monocytes (PBMCs) from normal human peripheral blood, amplifies antibody variable region genes from PBMCs, and displays the human antibody variable region on the surface of phages using phage display technology to construct a phage display library.

[0121] In some embodiments, a polypeptide (preferably the extracellular domain of the HER4 receptor) of any fragment in the HER4 receptor amino acid sequence can be selected as the target antigen for panning. The interaction between the antibody variable region displayed on the phage surface and the target antigen is detected. An antibody variable region library is screened and amplified by an in vitro selection method, which is similar to natural selection. In some specific embodiments, the present invention uses a variety of methods to detect and screen the antibody variable region displayed on the phage surface that interacts with the target antigen. These methods include, but are not limited to, flow cytometry (e.g., fluorescence-activated cell sorting (FACS) detection), ELISA detection, and cellular fluorescence detection.

[0122] In some embodiments, the present invention screens antibodies that recognize target antigens from an antibody library by constructing a phage display library and panning for the target antigen. Various phage display methods known in the art can be used to generate the antibodies of the present invention; for example, methods for preparing phage libraries are disclosed in U.S. Patent Nos. 5,223,409, 5,622,699, and 6,068,829. Phage display libraries can also be constructed according to the methods described in "Antibody Phage Display: Methods and Protocols (edited by Philippa M. O'Brien, Robert Aitken)". In some embodiments, nucleic acids encoding the antibody variable region can be inserted into the phage coat protein gene, causing the phage to display the antibody variable region on its surface, while the phage contains nucleic acids encoding the antibody variable region internally, thereby achieving a link between the antibody variable region phenotype and genotype.

[0123] Regarding antibody screening, in phage display, a larger library of antibodies with VH and / or VL regions can be expressed on the surface of filamentous phage particles, thereby pairing to form binding domains. Phages can be screened from the library based on their recognition and binding to the target antigen and the binding domains they display.

[0124] In some embodiments, the panning can be achieved by infecting host bacteria with bacteriophages and allowing them to multiply and amplify within the host. Bacteriophages secreted by host bacteria with single-chain antibody fragments on their surface are collected and panned multiple times as needed until phages capable of selectively or specifically binding to target antigens are obtained. Finally, the amino acid sequence of the antibody variable region is obtained by sequencing the antibody gene in the phage genome (Arap et al., Science 1998, 279:377-380; Smith et al., Science 1985, 228:1315-1317).

[0125] In a specific embodiment, this invention utilizes the HER4 receptor extracellular domain as the target antigen, obtains human antibody heavy chain variable region and light chain variable region gene fragments from PBMCs, prepares scFv fragments based on these fragments, and ligates them to phage surface structural protein gene III. Using a co-expression method, these fragments participate in phage assembly and are displayed on the phage surface, thereby constructing a phage display library. Using the phage display library, for specific target antigens (e.g., the HER4 receptor extracellular domain), through multiple rounds of adsorption-elution-amplification (panning), phages that specifically bind to the target antigen can be enriched. Then, gene sequencing technology is used to obtain the corresponding DNA sequence information, thereby inferring the amino acid sequence of the antibody variable region.

[0126] 3. Polynucleotides, vectors and host cells

[0127] On one hand, the present invention provides a nucleic acid encoding an anti-HER4 antibody or an antigen-binding fragment thereof. This application also includes polynucleotide variants encoding the amino acid sequence described herein.

[0128] Those skilled in the art can determine the nucleic acid sequence encoding the antibody of this application based on the genetic code. Polymerase chain reaction (PCR) can be used to isolate and amplify the DNA sequence encoding the anti-HER4 antibody or its antigen-binding fragment of this application. Oligonucleotides may additionally contain recognition sites for restriction endonucleases to facilitate the insertion of the amplified DNA fragment combination into the expression vector. For PCR techniques, see Saiki et al., Science 1988, 239:487-491; Recombinant DNA Methodology, Wu et al., Academic Press, Inc., San Diego (1989), pp. 189-196; PCR Protocols: A Guide to Methods and Applications, Innis et al., Academic Press, Inc. (1990).

[0129] The nucleic acid molecules of the present invention comprise single-stranded and double-stranded DNA and RNA, and corresponding complementary sequences. The nucleic acid molecules of the present invention also include isolated nucleic acid molecules, preferably derived from purified DNA or RNA in quantities or concentrations that can be identified, manipulated, and recovered by standard biochemical methods (e.g., the methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Preferably, such sequences comprise open reading frames provided and / or constructed as internal untranslated sequences or intron breaks not commonly found in eukaryotic genes. The untranslated DNA sequence may be present at the 5' or 3' of the open reading frame, wherein the sequence does not interfere with the manipulation or expression of the coding region.

[0130] The anti-HER4 antibody or its antigen-binding fragment of the present invention is prepared by the following steps: mutagenesis of specific nucleotide sites in the DNA encoding the anti-HER4 antibody or its antigen-binding fragment using PCR mutagenesis or other techniques known to those skilled in the art to produce DNA encoding a variant, followed by expression / amplification of the recombinant DNA in a cell culture as outlined herein. Alternatively, the anti-HER4 antibody or its antigen-binding fragment can also be prepared in vitro using established techniques.

[0131] As is known to those skilled in the art, due to the degeneracy of the genetic code, the anti-HER4 antibody or its antigen-binding fragment of the present invention is encoded by an extremely large number of nucleic acids, each of which is within the scope of this application and can be prepared using standard techniques. Therefore, by identifying specific amino acid sequences, those skilled in the art can easily modify the respective coding sequences of the anti-HER4 antibody or its antigen-binding fragment of the present invention with one or more codons to prepare many different nucleic acids without altering the amino acid sequence of the anti-HER4 antibody or its antigen-binding fragment of the present invention.

[0132] On the other hand, the present invention also provides an expression vector for a nucleic acid encoding the anti-HER4 antibody or its antigen-binding fragment described herein.

[0133] Nucleic acid encoding the anti-HER4 antibody or its antigen-binding fragment of the present invention can be constructed in a suitable vector for introduction into host cells for expression of the target protein. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. The nucleic acid encoding the target protein in the vector is operatively linked to the promoter.

[0134] As used herein, the term “operationally linked” refers to a functional link between a nucleic acid expression control sequence (e.g., an array of promoters, signal sequences, or transcription factor binding sites) and another nucleic acid sequence, and thus the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0135] Suitable vectors include plasmids, phagemids, Cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages (such as λ phage or M13 phage), and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). Vectors can contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain replication initiation sites. Vectors may also include components that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein coats.

[0136] On the other hand, the present invention also provides a host cell comprising a nucleic acid or expression vector encoding the anti-HER4 antibody of the present application or an antigen-binding fragment thereof.

[0137] The cells can be eukaryotic cells, such as mammalian host cells, including but not limited to SV40-transformed monkey kidney cell lines CV1 (COS-7, ATCC, CRL-1651), human embryonic kidney cell lines (293 or 293 cell subclones in suspension culture, Graham et al., J Gen Virol 1977, 36:59-74), juvenile hamster kidney cells (BHK-21, ATCC, CCL-10), Chinese hamster ovary cells (CHO, Urlaub et al., Proc Natl Acad Sci USA 1980, 77:4216-4220), and mouse testicular supporting cells (TM4, Mather, Biol Reprod). 1980, 23:243-251), monkey kidney cells (CV1, ATCC, CCL-70), African green monkey kidney cells (VERO-76, ATCC, CRL-1587), human cervical cancer cells (HELA, ATCC, CCL-2), canine kidney cells (MDCK, ATCC, CCL-34), Buffalo rat hepatocytes (BRL 3A, ATCC, CRL-1442), human lung cells (W138, ATCC, CCL-75), human liver cancer cell line (HepG2, ATCC, HB-8065), mouse mammary tumor (MMT060562, ATCC, CCL-51), TRI cells (Mather et al., Ann NY Acad Sci 1982, 383:44-68), MRC5 cells, or FS4 cells.

[0138] 4. The present invention relates to a method for preparing anti-HER4 antibody or its antigen-binding fragment.

[0139] The present invention provides a method for preparing the anti-HER4 antibody or its antigen-binding fragment using the host cell.

[0140] The method includes transfecting a nucleic acid or expression vector encoding the anti-HER4 antibody of the present invention or its antigen-binding fragment into host cells, and culturing the host cells in a culture medium for a period of time to express the anti-HER4 antibody of the present invention or its antigen-binding fragment. Commercially available culture media can be used without limitation.

[0141] Preferably, the expressed anti-HER4 antibody or its antigen-binding fragment can be secreted into the culture medium for culturing host cells. The antibody is recovered from the culture medium using standard protein purification methods, such as removing impurities by centrifugation or filtration, or purifying the product by affinity chromatography; other purification techniques can also be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography.

[0142] 5. Bispecific molecules / Multispecific molecules

[0143] The present invention also provides a bispecific molecule comprising the anti-HER4 antibody of the present invention or its antigen-binding fragment linked to another functional molecule (e.g., another polypeptide or protein such as a Fab' fragment), thereby forming a bispecific or multispecific molecule that binds to multiple binding sites or binding (target) epitopes. For example, the anti-HER4 antibody of the present invention or its antigen-binding fragment may be functionally linked to one or more other binding molecules, such as another antibody, antigen-binding fragment, polypeptide, or binding mimic, said functional linking including chemical coupling, gene fusion, non-covalent association, or other methods.

[0144] In one embodiment, the other functional molecule may be an Fc receptor (FcR), such as human FcγRIII (CD16), FcδRⅠ (CD64), or FcαR (CD89). Therefore, the bispecific or multispecific molecules of the present invention can simultaneously target effector cells (e.g., monocytes, macrophages, or polymorphonuclear cells [PMN]) expressing FcγR, FcαR, or FcεR and tumor cells expressing HER4.

[0145] In one embodiment, the other functional molecule is selected from antibodies or antigen-binding fragments thereof that are specific to cancer-associated antigens (TAAs) or immune checkpoint protein antigens. The cancer-associated antigens may be selected from HER2, HER3, VEGF, VEGFR, c-Met, TFR (transferrin receptor), IGF-1R (human insulin-like growth factor I receptor), etc.; the immune checkpoint protein antigens may be selected from PD-1, PD-L1, CTLA-4, CD137, CD47, etc.

[0146] In one embodiment, in addition to the binding specificity and anti-HER4 binding specificity of the functional molecules described above, the dual-specificity or multi-specificity molecules of the present invention may further include a third binding specificity. The third specificity may be an anti-enhancing factor (EF) portion; for example, the molecule may bind to a surface protein involved in cytotoxic activity, thereby increasing the immune response against target cells. The anti-enhancing factor portion may be an antibody (including scFv), and may bind to an FcR or target cell antigen. Furthermore, the target cells bound by the anti-enhancing factor portion may be different from the target cells bound by the first and second binding specific molecules; for example, the anti-enhancing factor portion may bind to cytotoxic T cells (e.g., via CD2, CD3, CD8, CD28, CD4, or ICAM-1) or other immune cells that generate an enhanced immune response to the target cells.

[0147] In this document, the term "target cell" refers to any undesirable cell in a subject to which the molecules of this invention (e.g., anti-HER4 antibodies or their antigen-binding fragments, bispecific molecules, or multispecific molecules) are targeted. In some embodiments, the target cell is a cell that overexpresses HER4, including tumor cells (e.g., gliomas) or other non-tumor cells.

[0148] The bispecific molecules of the present invention can have many different forms. For example, a bispecific molecule retains the traditional antibody form, but instead of having two binding arms with the same specificity, it has two binding arms with different specificities. Each binding arm can consist of two single-chain antibody fragments (scFv) linked by a peptide chain to construct a bispecific molecule, namely the so-called Bs(scFv)2 structure. The bispecific molecule can also include two different F(ab) fragments linked by a peptide linker. These and other forms of bispecific molecules can be prepared by genetic engineering, somatic cell hybridization or chemical synthesis. See, for example, Kufer et al., ibid.; Cao and Suresh, Bioconjugate Chemistry, 9(6), 635-644 (1998); and van Spriel et al., Immunology Today, 21(8), 391-397 (2000), and their cited references.

[0149] 6. Immunoconjugates, chimeric antigen receptors, engineered T-cell receptors, or oncolytic viruses

[0150] On one hand, the present invention provides an immunoconjugate comprising the anti-HER4 antibody of the present invention or its antigen-binding fragment.

[0151] The anti-HER4 antibody or its antigen-binding fragment of the present invention can be conjugated with therapeutic agents to form immunoconjugates, such as antibody-drug conjugates (ADCs). Suitable therapeutic agents include cytotoxins (including paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxanthraquinone, miteramycin, actinomycin D, 1-dehydroprotein sterol, glucocorticoids, procaine, tetracaine, lidocaine, propranolol), alkylating agents (e.g., chloroform, chlorobutyrate, melphalan, carmustine [BSNU] and lomustine [CCNU], cyclophosphamide, butyrylimide, dibromonitrobenzene, streptozotocin). Mitomycin C and cis-dichlorodiamineplatin(II) [DDP] cisplatin), puromycin and its analogues, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil norhydrazine), DNA minor groove binders, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, microtubule binders, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics (e.g., actinomycin, bleomycin, mitrazine, and anthramycin [AMC]), and antimitotic agents (e.g., vincristine, vinblastine). In ADCs, the linkers for antibody-therapeutic agent conjugation can be either cleavable linkers, such as peptide linkers, disulfide linkers, or hydrazone linkers, or non-cleavable linkers. ADCs can be prepared using methods described in U.S. Patent Nos. 7,087,600, 6,989,452 and 7,129,261; PCT Publications WO 02 / 096910, WO 07 / 038,658, WO 07 / 051,081, WO 07 / 059,404, WO 08 / 083,312 and WO 08 / 103,693; and U.S. Patent Publications 20060024317, 20060004081 and 20060247295, the contents of which are incorporated herein by reference.

[0152] On the other hand, the present invention also provides a chimeric antigen receptor, engineered T cell receptor, or oncolytic virus comprising the anti-HER4 antibody of the present invention or its antigen-binding fragment thereof.

[0153] 7. Pharmaceutical Composition

[0154] The present invention provides a pharmaceutical composition comprising the anti-HER4 antibody of the present invention or an antigen-binding fragment thereof (or an immunoconjugate, or a bispecific / multispecific molecule, or a chimeric antigen receptor, or an engineered T-cell receptor, or an oncolytic virus), and a pharmaceutically acceptable carrier.

[0155] The pharmaceutical composition may comprise any kind of pharmaceutically acceptable carrier. Carriers that may be used include excipients, surfactants, thickeners or emulsifiers, solid binders, dispersants or suspending agents, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, or combinations thereof. The selection and use of suitable carriers is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th edition (Lippincott Williams & Wilkins 2003), the contents of which are incorporated herein by reference.

[0156] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Given the different routes of administration, the active ingredient may be coated in a material to protect it from acids and other natural conditions that could inactivate it. As used herein, the term "parenteral administration" refers to non-enteral and non-local routes of administration, including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intracapsular, intra-capsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions. Alternatively, the antibody of this application may be administered via non-parenteral routes (e.g., local, epidermal, or mucosal administration), such as intranasal, oral, vaginal, rectal, sublingual, or local administration.

[0157] Pharmaceutical compositions can be sterile aqueous solutions or dispersants. They can also form microemulsions, liposomes, or other ordered structural combinations suitable for high drug concentrations.

[0158] The amount of active ingredient that can be combined with a carrier material to form a single dosage form is determined based on the subject and the specific route of administration, and is generally also the amount of the composition capable of producing a therapeutic effect. Typically, compositions formed with a pharmaceutically acceptable carrier contain about 0.01% to about 99% of the active ingredient, preferably about 0.1% to about 70%, and most preferably about 1% to about 30% of the active ingredient.

[0159] The dosage regimen can be adjusted to provide the optimal expected response (e.g., therapeutic response). For example, it can be administered as a single dose, in multiple doses, or the dose can be reduced or increased proportionally depending on the treatment outcome. Formulating parenteral compositions in unit dosage form is particularly advantageous, as it facilitates administration and ensures uniform dosage. As used herein, unit dosage form refers to physically discrete units suitable as unit doses for treating subjects; each unit dose contains a predetermined amount of the active ingredient, the predetermined amount of which is calculated to produce the expected therapeutic effect when the active ingredient is administered with the desired drug carrier. Additionally, the anti-HER4 antibody or its antigen-binding fragment of the present invention can also be administered as a sustained-release formulation, thereby reducing the frequency of administration.

[0160] The dosage range of a combination of anti-HER4 antibody or its antigen-binding fragment can be from about 0.0001 mg / kg to 100 mg / kg body weight, typically from 0.001 mg / kg to 50 mg / kg body weight.

[0161] The "therapeutic effective dose" of the anti-HER4 antibody or its antigen-binding fragment of the present invention preferably reduces the severity of disease symptoms, increases the frequency and duration of disease progression-free periods, or prevents bodily damage or disability caused by disease. For example, relative to an untreated subject, the "therapeutic effective dose" preferably inhibits disease progression by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%. The therapeutic effective dose of the therapeutic antibody can improve at least one symptom of the subject's condition, typically a human or other mammal. The "therapeutic effective dose" can also be determined differently based on various factors, including but not limited to formulation method, administration method, age, body size, weight, patient's sex or pathological condition, diet, administration time, administration interval, route of administration, excretion rate, and response sensitivity.

[0162] The drug composition may be selected as a controlled-release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, for example, Sustained and Controlled Release Drug Delivery Systems, ed. J.R. Robinson, Marcel Dekker, Inc., New York, 1978.

[0163] Therapeutic pharmaceutical compositions can be delivered via medical devices selected from: (1) needle-free subcutaneous injection devices (e.g., U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 and 4,596,556); (2) microinfusion pumps (U.S. Patent No. 4,487,603); (3) transdermal devices (U.S. Patent No. 4,486,194); (4) infusion devices (U.S. Patent Nos. 4,447,233 and 4,447,224); and (5) permeation devices (U.S. Patent Nos. 4,439,196 and 4,475,196); the disclosures of which are incorporated herein by reference.

[0164] In some embodiments, the anti-HER4 antibody of the present invention or its antigen-binding fragment can be formulated to ensure biodistribution in vivo. For example, to ensure that the therapeutic antibody of the present application crosses the blood-brain barrier, it can be formulated into liposomes, which may additionally contain a targeting portion to enhance selective delivery to specific cells or organs. See, for example, U.S. Patent Nos. 4,522,811, 5,374,548, 5,416,016, and 5,399,331; Ranade VV, J Clin Pharmacol 1989, 29:685-694; Umezawa et al., Biochem Biophys Res Commun 1988, 153:1038-1044; Bloeman et al., FEBS Lett 1995, 357:140-144; Owais M et al., Antimicrob Agents Chemother 1995, 39:180-184; Briscoe et al., Am J Physiol 1995, 268:L374-380; Schreier et al., J Biol Chem 1994, 269:9090-9098; Keinanen and Laukkanen, FEBS Lett 1994, 346: 123-126; and Killion and Fidler, Immunomethods 1994, 4: 273-279.

[0165] 8. medicine box

[0166] On one hand, the present invention provides a kit containing an effective amount of the pharmaceutical composition of the present invention’s anti-HER4 antibody or antigen-binding fragment, together with one or more other therapeutic agents (i.e., the kit may contain or not contain at least one other therapeutic agent).

[0167] In some embodiments, the therapeutic agents include those targeting reactive glial cell proliferation, including signaling pathway modulators, cell cycle inhibitors, and inflammatory response modulators. In some specific embodiments, the signaling pathway modulators include modulators targeting R1P1K and downstream VEGFD / VEGFR3 pathways (e.g., tumor suppressor [Necrostatin-1, NEC-1] and its analogues), modulators targeting the PI3K / AKT signaling pathway (e.g., alismacosolvan A24-acetate), modulators inhibiting transforming growth factor β1 activity, and modulators upregulating PPARα expression (e.g., oleoylethanolamine, valproic acid). In some specific embodiments, the cell cycle inhibitors include olomosin, rhodioloside, and carnosine. In some specific embodiments, the inflammatory response modulators include serotropin capsules, resveratrol, and anti-IL-17A monoclonal antibodies (e.g., secukinumab).

[0168] 9. Therapeutic uses and methods

[0169] On one hand, this invention relates to the use of the anti-HER4 antibody or its antigen-binding fragment of the present invention in the preparation of pharmaceutical compositions or formulations for treating and / or preventing HER4-related diseases or conditions, and in the preparation of diagnostic reagents for diagnosing HER4-related diseases or conditions. Alternatively, this invention relates to a method for treating and / or preventing HER4-related diseases or conditions, the method comprising administering to a subject a therapeutically effective amount of the anti-HER4 antibody or its antigen-binding fragment of the present invention, a pharmaceutical composition, or a kit.

[0170] The diseases or conditions mentioned include cancer and other HER4-related diseases.

[0171] In some implementations, the cancer includes glioma.

[0172] In some implementations, other HER4-related diseases include glial proliferative disorders such as stroke, Alzheimer's disease, viral encephalitis, traumatic brain injury, multiple sclerosis, and epilepsy.

[0173] On the other hand, the present invention provides a method for inhibiting the HER4 receptor and its downstream signal transduction pathways, comprising using the anti-HER4 antibody of the present invention or its antigen-binding fragment, pharmaceutical composition or kit to inhibit ligand-induced HER4 dimerization in vitro or in vivo, wherein the ligands include NRG-1 to 4, preferably NRG-1.

[0174] 10. Testing applications

[0175] The present invention also relates to the use of anti-HER4 antibodies or antigen-binding fragments thereof for detecting and / or measuring HER4 or HER4-expressing cells in samples (e.g., biological samples, such as serum, tissues), and to methods for screening patients with HER4-related conditions who respond to treatment with the anti-HER4 antibodies or antigen-binding fragments thereof of the present invention.

[0176] In some embodiments, the anti-HER4 antibody or its antigen-binding fragment can be used to diagnose diseases or conditions of abnormal HER4 expression (e.g., overexpression, underexpression, or lack of expression) to facilitate the determination of treatment regimens. For example, the antibody can be conjugated to a detectable marker or reporter molecule, and the labeled antibody can be contacted with a sample obtained from a patient to diagnose HER4 expression levels. The detectable marker or reporter molecule can be a radioactive isotope, for example... 3 H, 14 C 32 P, 33 P, 35 S, 123 I, 125 I, 131 I, 111 In or 188 Rh; fluorescent materials, such as umbelliferone, luciferin, rhodamine, luciferin isothiocyanate, dichlorotriazineamine luciferin, dansyl chloride, or phycoerythrin; chemiluminescent materials, such as luminol; bioluminescent materials, such as luciferase, luciferin, or jellyfish luminescent protein; or enzymes, such as alkaline phosphatase, β-galactosidase, acetylcholinesterase, horseradish peroxidase, or luciferase.

[0177] In some implementations, methods for detecting or measuring the content or expression level of HER4 in a sample include: (a) contacting the sample or control sample with the anti-HER4 antibody or its antigen-binding fragment, and (b) detecting the content or expression level of HER4 bound to the antibody or its antigen-binding fragment in the sample (quantitatively or qualitatively). The control sample includes a positive control and a negative control; the positive control may be a sample known to assess a disease or condition, and the negative control may be a sample from a healthy subject. A statistically significant increase in HER4 expression level in a sample compared to a control indicates the presence of a HER4-related disease or condition. Specific exemplary assays for detecting or measuring HER4 expression levels in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoPET (e.g., 89Zr, 64Cu, etc.), and fluorescence activated cell sorting (FACS).

[0178] In some embodiments, the present invention provides a method for detecting, diagnosing, or monitoring HER4-related diseases or conditions in a subject, the method comprising: (1) contacting a test sample obtained from the subject with an anti-HER4 antibody or its antigen-binding fragment of the present invention to detect the content or expression level of HER4 in the test sample (quantitative or qualitative); (2) comparing the content or expression level of HER4 in the test sample with the content or expression level of HER4 in a control sample (e.g., normal cells derived from the same tissue as the test sample, or cells with a HER4 expression level comparable to that of the normal cells), wherein a higher level of HER4 expression in the test sample compared to the control sample can be used to determine that the subject has a HER4-related disease or condition, or that the subject has a HER4-related disease or condition.

[0179] 11. Technical effect

[0180] The anti-HER4 antibody or its antigen-binding fragment of the present invention can specifically bind to the extracellular domain III of human HER4 with high binding activity and does not cross-react with other human ErbB / HER receptor tyrosine kinases (e.g., EGFR, HER2, HER3). The anti-HER4 antibody or its antigen-binding fragment provided by the present invention inhibits ligand-induced HER4 dimerization, indicating that the anti-HER4 antibody or its antigen-binding fragment of the present invention can block the ligand-induced HER4-mediated signal transduction pathway.

[0181] In summary, the anti-HER4 antibody or its antigen-binding fragment provided by this invention can serve as a candidate for drugs to treat and / or prevent HER4-related diseases or conditions, and is also suitable for development into a bispecific or multispecific molecule.

[0182] The present application is further illustrated by the following examples, which should not be construed as further limiting. All figures and references, patents and patent applications cited throughout this application are expressly incorporated herein by reference. Unless otherwise stated, the materials, reagents and apparatus involved in the following examples are commercially available. Example

[0183] Example 1: Construction of anti-human HER4 phage immune library and screening of antibodies

[0184] 1.1 Construction of phage immune libraries

[0185] Total RNA was extracted from human peripheral blood mononuclear cells using the TriZOL method and then reverse transcribed using a PrimeScript reverse transcription kit. TMThe II 1st Strand cDNA Synthesis Kit (Takara) was used to obtain cDNA through reverse transcription using extracted RNA as a template. Subsequently, using the cDNA as a template, specific primers were used to amplify the complete set of human antibody heavy chain variable region and light chain variable region gene fragments separately via PCR. Then, overlap PCR (SOE-PCR) was used to splice and amplify the light and heavy chain variable region gene fragments separately to obtain single-chain antibody variable region gene fragments (scFv). The scFv sequence was ligated into a phage display vector via enzyme digestion. The ligation product was then electroporated into TG1 competent cells. After infection with helper phage M13KO7, a fully human phage library was obtained.

[0186] 1.2 Preliminary screening and sequence analysis of antibodies

[0187] The obtained scFv library was added to an immunotube coated with recombinant human HER4 extracellular domain recombinant protein (amino acid sequence 26-651 as shown in SEQ ID NO:82). The tube was incubated overnight at 4°C. After blocking, washing, and elution, the protein was neutralized with 1M Tris (pH 8.0) and used to infect TG1 Escherichia coli cells in logarithmic growth phase. After culturing, bacterial cells were collected and infected with M13KO7 helper phage. The "adsorption-elution-amplification" process was repeated 3-5 times to enrich the anti-human HER4 phage display antibody library. Single colonies were picked and expanded, and identified by bacterial PCR and single-clone phage-ELISA. Gene sequencing was then performed to obtain scFv that specifically binds to the antigen. The amino acid sequence of VH is shown in SEQ ID NO:38, and the amino acid sequence of VL is shown in SEQ ID NO:63.

[0188] 1.3 Methods for preparing anti-HER4 antibodies

[0189] The VH and VL of the scFv obtained in the above steps were cloned into the human IgG1 heavy chain constant region (SEQ ID NO: 87) and the human κ light chain constant region (SEQ ID NO: 88), respectively. Specifically, nucleotide sequences encoding VH and the human IgG1 heavy chain constant region, and nucleotide sequences encoding VL and the human κ light chain constant region, were used to construct heavy chain expression vectors and light chain expression vectors, respectively. The heavy chain and light chain expression vectors were mixed at a 1:1 ratio and transiently transfected into Expi-CHOS cells in logarithmic growth phase, and cultured at 32°C for 10 days. The cell supernatant was collected, the cells were centrifuged, filtered, and the antibody was purified by Protein A affinity chromatography. This antibody was named Ab4261.

[0190] Example 2: Antigen-binding epitope analysis of antibody Ab4261

[0191] The binding epitope of antibody Ab4261 on HER4 was detected by ELISA. The specific method is as follows: Antibody Ab4261 was diluted to 1 μg / mL with PBS, and 100 μL was added to each well of a 96-well ELISA plate. The plate was incubated overnight at 4°C. After washing with PBS, 200 μL of blocking buffer (PBST [PBS + 0.1% Tween-20] containing 1% BSA) was added to each well, and the plate was incubated at room temperature for 1 hour. After washing with PBST, 100 μL of human EGFR extracellular domain protein, HER3 extracellular domain protein, HER4 extracellular domain protein, or chimeric HER4 extracellular domain III protein (i.e., a chimeric recombinant protein constructed by replacing EGFR extracellular domain III with HER4 extracellular domain III) was added to each well, and the plate was incubated at room temperature for 2 hours. Wash the plate with PBST, then add 100 μL of anti-6×His-HRP antibody (Proteintech) diluted 1:10,000 in blocking buffer to each well and incubate at room temperature for 1 hour. After washing with PBST, add 100 μL of TMB chromogenic solution (Thermo) to each well and incubate at room temperature in the dark for 3-10 minutes. Then add 50 μL of stop solution (2M HCl) to each well and read the OD450 value using a multi-functional microplate reader (Varioskan, Thermo). Analyze the raw data using GraphPad Prism 9 software. The detection results are as follows: Figure 1 As shown, the recognition epitope of Ab4261 is located in the extracellular domain III of HER4.

[0192] Example 3: Optimization of antibody Ab4261

[0193] 3.1 Single-point mutation optimization of Ab4261

[0194] Using the online software abYsis (http: / / abysis.org / abysis / index.html) and MOE software, we analyzed the amino acid residues with low humanization levels, potential stability risk sites, and amino acid residues with low binding activity in the light and heavy chain variable regions of antibody Ab4261. A total of 154 single-point mutations were designed to improve humanization, enhance affinity, and / or reduce the stability risk of the antibody molecule. Based on antibody Ab4261, the 154 designed single-point mutations were introduced, and the light and heavy chain expression plasmids were transiently transfected into ExpiCHO-S cells to express each single-point mutated antibody. The expression levels of each single-point mutated antibody and the parent antibody Ab4261 in the culture supernatant after 6 days of transient culture were detected using a ForteBio molecular interaction analyzer (model Oke). Specifically, after transient transfection of ExpiCHO-S cells, the culture supernatant was harvested and added at 200 μL / well to a 96-well black plate. The samples to be tested and the Protein A probe were placed in a ForteBio analyzer, and the binding rate of each antibody molecule in the culture supernatant to the Protein A probe was detected using the machine's preset quantitative program. After each cycle, the probe was immersed in 10 mM glycine buffer (pH 1.5) to regenerate the probe, and the next cycle of detection began. After the detection was completed, the expression level of each mutant was calculated using analysis software based on a standard curve.

[0195] The binding activity of the above mutants with HER4-expressing 293T cells (293T-HER4) was detected by flow cytometry. The specific method is as follows: 293T-HER4 cells were collected and resuspended in FACS buffer (PBS containing 1% BSA) at 50 μL / well in a 96-well U-shaped plate. Each mutant to be tested was diluted to 20 μg / mL with FACS buffer and added to each well at 50 μL. After mixing, the plate was incubated on ice for 1 hour. The supernatant was discarded by centrifugation, and the plate was washed three times with FACS buffer. AF488-labeled goat anti-human IgG (H+L) (Jackson Immunoresearch) was diluted 1:1000 in FACS buffer and added to each well at 100 μL. After mixing, the plate was incubated on ice for 40 minutes. The supernatant was discarded by centrifugation, and the plate was washed three times with FACS buffer. Cells were resuspended in 100 μL / well FACS buffer and analyzed using a flow cytometer (NovoCyte 3005, Agilent).

[0196] The mutation sites and mutated amino acids, transient expression levels, and FACS-detected cell binding activity of each mutant are shown in Table 1. Compared with the maternal antibody Ab4261, the mutants showed improved humanization, enhanced binding activity with HER4-expressing cells, and / or significantly reduced the risk of aspartic acid isomerization and / or asparagine deamidation in the antibody molecule. These mutations included 22 single-point mutations in the antibody heavy chain (G101, G102, G103, G112, G114, G115, G116, G119, G122, G122, G126 ... G125, G126, G127, G128, G132, G135, G136, G139, G142, G143, G145, G147 and G161), and 13 single-point mutations in the antibody light chain (K101, K103, K104, K105, K118, K119, K120, K128, K132, K141, K149, K150 and K161).

[0197] Table 1. Single point mutation, transient expression level, and binding activity of Ab4261 with 293T-HER4 cells.

[0198]

[0199]

[0200]

[0201]

[0202] H represents the heavy chain; L represents the light chain; MFI represents the average fluorescence intensity.

[0203] 3.2 Optimization of Combination Mutations for Ab4261 Antibody

[0204] Six single-point mutations (G101, G102, G103, G135, G143, and G147) from the antibody heavy chain and seven single-point mutations (K101, K103, K104, K105, K120, K128, and K149) from the antibody light chain were selected and introduced into the variable region sequence of antibody Ab4261 to construct antibody molecules containing different combinations of mutations. Antibody samples were prepared according to the method described in section 3.1 above. The expression levels of each mutant and the control parent antibody Ab4261 were detected using ForteBio, and the binding activity of each mutant with 293T-HER4 cells was detected using flow cytometry.

[0205] The mutation sites, transient expression levels, and binding activity with 293T-HER4 cells contained in each combined mutant are shown in Table 2 and 2. Figure 2As shown, the expression levels of most combined mutants were comparable to those of the maternal antibody Ab4261, and the cell-binding activities of each combined mutant were superior to those of the maternal antibody. Among them, the cell-binding activities of the combined mutants G201-K203 and G202-K201 were significantly superior to those of the maternal antibody and other mutants.

[0206] Table 2. Results of Ab4261 combinatorial mutations, transient expression levels, and binding activity with 293T-HER4 cells.

[0207]

[0208] MFI: Mean fluorescence value

[0209] Example 4: Antigen binding specificity and species cross-binding activity of the optimized molecule Ab4261

[0210] 4.1 Optimized molecular binding activity of Ab4261 to ErbB / HER family protein members

[0211] The binding activity of the optimized Ab4261 molecule G202-K201 to members of the human ErbB / HER family of proteins was detected using an ELISA method. The specific steps were as follows: The HER4 recombinant protein and other members of the human ErbB / HER family (EGFR, HER2, and HER3) were diluted with PBS to 1 μg / mL, and then 100 μL / well was added to a 96-well ELISA plate and incubated overnight at 4°C. After washing with PBS, 200 μL of blocking buffer (PBST [PBS + 0.1% Tween-20] containing 1% BSA) was added to each well, and the plate was incubated at room temperature for 1 hour. After washing with PBST, antibody samples serially diluted 4-fold from 5 μg / mL in the blocking buffer were added at 100 μL / well, and the plate was incubated at room temperature for 2 hours. Wash the plate with PBST, then add 100 μL of anti-human IgG(H+L)-HRP antibody (Jackson Immuno) diluted 1:5,000 in blocking buffer to each well and incubate at room temperature for 1 hour. After washing with PBST, add 100 μL of TMB chromogenic solution (Thermo) to each well and incubate at room temperature in the dark for 3-10 minutes. Then add 50 μL of stop solution (2M HCl) to each well and read the OD450 value using a multi-functional microplate reader (Varioskan, Thermo). Analyze the raw data using GraphPad Prism 9 software. The results are as follows: Figure 3 As shown, antibody G202-K201 binds only to HER4 (EC50 value is approximately 0.01827 μg / mL) and does not bind to other ErbB / HER family protein members.

[0212] 4.2 Optimized molecular binding activity of Ab4261 with HER4 from different species

[0213] Following the above method, the binding activity of the optimized Ab4261 molecule G202-K201 with HER4 from different species was detected. The antigens included recombinant HER4 proteins derived from humans, mice, rats, and dogs. The results are as follows: Figure 4 As shown, the binding activity of antibody G202-K201 with mouse and rat HER4 is comparable to that with human HER4, while its binding activity with canine HER4 is slightly lower than that with human HER4.

[0214] Example 5: Optimized molecular binding activity of Ab4261 to 293T-HER4 cells

[0215] Following the method described in section 3.1 above, the binding activity of the optimized Ab4261 molecules G201-K203 and G202-K201 to 293T-HER4 cells was detected by flow cytometry.

[0216] Test results as follows Figure 5 As shown in Table 3, the cell-binding activities of antibodies G201-K203 and G202-K201 were significantly higher than those of the parent antibody Ab4261, and the cell-binding activity of G202-K201 was slightly better than that of G201-K203.

[0217] Table 3. Binding activity of the optimized Ab4261 molecule with 293T-HER4 cells.

[0218] Antibody number Cell binding activity EC50 (μg / mL) G201-K203 0.02967 G202-K201 0.02398 Ab4261 1.726

[0219] Example 6: Effect of the optimized antibody Ab4261 on NRG-1-induced HER2 / HER4 receptor dimerization

[0220] The Promega NanoBiT structural complementarity reporter molecule system was used to further verify whether the Ab4261 optimized molecule G202-K201 would affect NRG-1-induced HER2 / HER4 receptor dimerization. The NanoBiT system consists of an LgBiT subunit and an SmBiT subunit, which can fuse with the target protein. If the two target proteins interact to form a dimer, LgBiT will structurally complement SmBiT to form a functional luciferase, thereby reacting with the substrate to produce a bright luminescent signal. In this embodiment, the nucleotide sequences encoding the extramembrane domains and transmembrane domains of human HER2 and HER4 were first inserted into the NanoBiT system vectors pBiT1.3-C and pBiT2.3-C using molecular cloning technology. These two plasmids were then co-transfected into U2OS cells. Cells were collected 24 hours after transfection and seeded in 96-well white-walled plates, incubated overnight at 37°C. The parent antibody Ab4261, its optimized antibody G202-K201, and the control antibody Pertuzumab were serially diluted and then mixed with NRG-1 at a 1:1 volume ratio (the final concentration of NRG-1 was 1 nM). Nano-Glo Live CellReagent (Promega) was added to each well of a 96-well white-walled plate, and the chemiluminescence value at this point was recorded as the background. The prepared sample was then added to each well, incubated at 37°C for 6 minutes, and the chemiluminescence value was recorded again. The results are as follows: Figure 6 As shown in Table 4, both antibody G202-K201 and the control antibody Pertuzumab significantly inhibited NRG-1-induced HER2 / HER4 receptor dimerization, while the inhibitory activity of the parent antibody Ab4261 was significantly weaker than that of G202-K201 and the control antibody Pertuzumab. This result indicates that antibody G202-K201 can affect the normal biological function of the HER4 receptor by inhibiting NRG-1-induced HER4 receptor dimerization, for example, by inhibiting downstream signaling pathways of the HER4 receptor.

[0221] Table 4. Inhibitory activity of optimized Ab4261 molecules against NRG-1-induced HER4 receptor dimerization

[0222] G202-K201 Ab4261 Pertuzumab IC50 (μg / mL) 0.3217 14.74 0.3341

[0223] Although the invention has been described through one or more embodiments, it should be understood that the invention is not limited to these embodiments, and the specification is intended to cover all alternatives, modifications, and variations falling within the spirit and broad scope of the appended claims. All references cited in this invention are incorporated herein by reference in their entirety.

Claims

1. An isolated anti-HER4 antibody or its antigen-binding fragment, characterized in that, The anti-HER4 antibody or its antigen-binding fragment contains at least one of the following characteristics: (1) The anti-HER4 antibody or its antigen-binding fragment specifically binds to human HER4 extracellular domain III; and (2) The anti-HER4 antibody or its antigen-binding fragment can inhibit ligand-induced HER4 receptor dimerization and its downstream signal transduction pathway; The anti-HER4 antibody or its antigen-binding fragment comprises the HCDR1 amino acid sequence shown in SEQ ID NO: 1, the HCDR2 amino acid sequence shown in SEQ ID NO: 22, the HCDR3 amino acid sequence shown in SEQ ID NO: 23, the LCDR1 amino acid sequence shown in SEQ ID NO: 28, the LCDR2 amino acid sequence shown in SEQ ID NO: 34, and the LCDR3 amino acid sequence shown in SEQ ID NO:

36.

2. The anti-HER4 antibody or its antigen-binding fragment as claimed in claim 1, comprising a heavy chain variable region VH having the HCDR1, HCDR2 and HCDR3 and a light chain variable region VL having the LCDR1, LCDR2 and LCDR3, wherein each of VH and VL comprises an amino acid sequence having at least 85% identity with the VH shown in SEQ ID NO: 62 and the VL shown in SEQ ID NO: 77, respectively.

3. A nucleic acid encoding an anti-HER4 antibody or an antigen-binding fragment thereof as described in claim 1 or 2.

4. An expression vector capable of expressing the nucleic acid of claim 3.

5. A host cell comprising the nucleic acid of claim 3 or the expression vector of claim 4.

6. A method for preparing the anti-HER4 antibody or its antigen-binding fragment of claim 1 or 2 using the host cell of claim 5, comprising: (i) expressing the anti-HER4 antibody or its antigen-binding fragment in the host cells, and (ii) isolating the anti-HER4 antibody or its antigen-binding fragment from the host cells or cell cultures.

7. A pharmaceutical composition comprising the antiHER4 antibody of claim 1 or the antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

8. Use of the anti-HER4 antibody or its antigen-binding fragment as described in claim 1 or 2 in the preparation of a kit for detecting the content or expression level of HER4 in a test sample.

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