Anti-HER4 antibody as well as preparation method and application thereof

Abstract

The present invention provides an anti-HER4 antibody or an antigen binding fragment thereof. The invention further provides bispecific molecules or multispecific molecules comprising the antibody or the antigen-binding fragment thereof, immunoconjugates, as well as nucleic acid molecules encoding the antibody or the antigen-binding fragment thereof, vectors and host cells comprising the nucleic acid molecules. The invention also provides a method for preparing the antibody or the antigen-binding fragment thereof, a pharmaceutical composition containing the antibody or the antigen-binding fragment thereof, and a method and application of using the antibody or the antigen-binding fragment thereof or the pharmaceutical composition thereof in disease treatment. The invention provides a candidate specific antibody drug for treatment and / or prevention of HER4 related diseases or symptoms.

Description

Technical Field

[0001] The present invention relates to an anti-HER4 antibody or an antigen-binding fragment thereof, a polynucleotide encoding the antibody or the antigen-binding fragment thereof, an expression vector and a host cell, a pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof, and a use or method for treating a subject. Background Art

[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 them through dimerization, including RAS-RAF-MEK-ERK, PI3K-AKT and JAK-STAT pathways, and thus participate in the regulation of cell growth, differentiation and survival. HER4 is encoded by the ErbB4 / HER4 gene and is a transmembrane glycoprotein with a molecular weight of approximately 180 kDa. It consists of an extracellular domain (ECD) that binds to a ligand, a transmembrane domain and an intracellular tyrosine kinase domain. HER4 is regulated by ligands including neuregulin-1-4 (NRG-1-4) (JR Toggweiler, Nature 1993, 366: 473-475; KL 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), beta-acetylglobulin (betacellulin, BTC) (DJ Riese et al., Oncogene 1996, 12: 345-353) and epiregulin (T. Komurasaki et al., Oncogene 1997, 15: 2841-2848), and form 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) (Hollmen M et al., Future Oncol 2010, 6: 37-53), many reports show that HER4 expression in tumors is downregulated, or its expression is associated with a good prognosis of the tumor (Srinivasan R et al., Clin Cancer Res 1998, 5: 2877-2883; Witton CJ et al., J Pathol 2003, 200: 290-297).In addition, HER4 is considered as a tumor gene in breast cancer (Wang J et al., Oncotarget 2016, 7: 76693-76703). Therefore, the role of HER4 in the occurrence and progression of tumors needs to be further clarified. At present, HER4 has been shown to play a key role in the development and maintenance of the function of the heart and nervous system (FE Jones 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, it is desirable in the art to develop a neutralizing antibody that can specifically bind to HER4 to more accurately treat HER4-related diseases or conditions. Summary of the invention

[0004] The present invention provides an anti-HER4 antibody and a preparation method thereof and application thereof in HER4-related diseases or symptoms.

[0005] In one aspect, 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 the extracellular domain III of human HER4 and blocks the dimerization of HER4 receptor induced by ligand. In some embodiments, the activity of the antibody or its antigen-binding fragment specifically binding to cells expressing human HER4 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 pathway. In one embodiment, the anti-HER4 antibody or its antigen-binding fragment can inhibit ligand-induced HER4 receptor dimerization and its downstream signal transduction pathway, and the ligand includes NRG-1 to 4, preferably NRG-1. In some embodiments, the inhibitory activity of the antibody or its antigen-binding fragment on NRG-1-induced HER2 / HER4 receptor dimerization is detected by live cell bioluminescence.

[0008] On the other hand, the present invention provides a method for screening and obtaining the anti-HER4 antibody or antigen-binding fragment thereof of the present invention, wherein the method includes but is not limited to hybridoma technology, phage display technology, and single lymphocyte gene cloning technology.

[0009] On the other hand, the present invention provides an isolated nucleic acid molecule (also referred to as a "polynucleotide") encoding an anti-HER4 antibody or an antigen-binding fragment thereof of the present invention, as well as an expression vector comprising the nucleic acid and a host cell comprising the nucleic acid or the expression vector. The present invention also relates to a method for preparing an anti-HER4 antibody or an antigen-binding fragment thereof of the present invention using the host cell, the method comprising culturing the host cell and recovering the antibody or the antigen-binding fragment thereof from the culture medium.

[0010] In another aspect, the present invention provides a bispecific molecule or multispecific molecule, immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus comprising an anti-HER4 antibody or antigen-binding fragment thereof of the present invention.

[0011] On the other hand, the present invention provides a pharmaceutical composition comprising an anti-HER4 antibody or an antigen-binding fragment thereof, a bispecific antibody or a multispecific antibody, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor, or an oncolytic virus of the present invention, and a pharmaceutically acceptable carrier.

[0012] In another aspect, the present invention provides a kit comprising an effective amount of an anti-HER4 antibody or antigen-binding fragment thereof, or a pharmaceutical composition herein, and optionally at least one additional therapeutic agent.

[0013] On the other hand, the present invention provides a use of an anti-HER4 antibody or an antigen-binding fragment thereof of the present invention in preparing a pharmaceutical composition or preparation for treating and / or preventing HER4-related diseases or conditions, or in preparing a diagnostic reagent for diagnosing HER4-related diseases or conditions.

[0014] In another aspect, the present invention provides a method for treating and / or preventing HER4-related diseases or conditions, comprising administering to a subject an effective amount of an anti-HER4 antibody or antigen-binding fragment thereof, a pharmaceutical composition, or a pharmaceutical kit of the present invention.

[0015] On the other hand, the present invention provides a method for inhibiting HER4 receptor and its downstream signal transduction pathway, which comprises using the anti-HER4 antibody or its antigen-binding fragment, pharmaceutical composition or kit of the present invention to inhibit ligand-induced HER4 dimerization in vitro or in vivo, wherein the ligand comprises NRG-1 to 4, preferably NRG-1.

[0016] 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, tissue), comprising: (a) contacting the test sample with the antibody or its antigen-binding fragment of the present invention, and (b) detecting the binding of the antibody or its antigen-binding fragment to HER4 in the test sample.

[0017] The present application is further illustrated by the following drawings and specific embodiments, and other features and advantages disclosed in the present application will be apparent, and these drawings and specific embodiments should not be considered to limit the scope of the present application, and changes that are easily conceived by those skilled in the art will be included in the spirit of the present application and the protection scope of the appended claims. All references cited in this application, including publications, patents and patent applications, are incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 .ELISA was used to detect the binding activity of anti-HER4 antibody Ab4268 with the extracellular domain proteins of human ErbB / HER family protein members (including EGFR, HER3 and HER4) and chimeric HER4 extracellular domain III recombinant protein (HER4_DⅢ).

[0019] Figure 2 Flow cytometry was used to detect the binding activity of antibody Ab4268 variants containing single point mutations and control antibody (parent antibody Ab4268) to HER4-expressing 293T cells.

[0020] Figure 3 Flow cytometry was used to detect the binding activity of antibody Ab4268 variants containing combined mutations and control antibody (parent antibody Ab4268) to HER4-expressing 293T cells.

[0021] Figure 4 Flow cytometry was used to detect the binding activity of variants of antibody Ab4268 (H201-L201 and H202-L201) and control antibody (parent antibody Ab4268) to 293T cells expressing human HER4.

[0022] Figure 5 .The effect of anti-HER4 antibody (Ab4268) on NRG-1-induced HER2 / HER4 receptor dimerization, the control antibody is Pertuzumab. DETAILED DESCRIPTION OF THE INVENTION

[0023] definition

[0024] Unless defined otherwise, 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 the present invention, the following terms are defined below.

[0025] In this article, the terms "HER4", "HER4 receptor" and "HER4 protein" are used interchangeably. The protein is also called ErbB4, c-ErbB-4 or p180erbB4. Unless otherwise specified, the "HER4" expressed in this article refers to human

[0026] Any natural form of HER4 may be an amino acid sequence as shown in SEQ ID NO: 52 and / or a full-length HER4 amino acid sequence as shown in UniProtKB Accession No. Q15303.1, or may be naturally expressed by cells (including cardiomyocytes, breast cells, tumor cells), or expressed by cells transfected with HER4 genes or cDNA. The term includes naturally occurring HER4 allele variants and splice variants, isoforms, homologs, and species homologs. HER4 may be isolated from the human body or may be produced by recombinant or synthetic methods.

[0027] The full-length HER4 protein consists of 1308 amino acids, of which amino acid residues 1-25 are signal peptides, amino acid residues 26-651 are extracellular domains (ECD), amino acid residues 652-675 are transmembrane domains, and amino acid residues 676-7308 are intracellular domains. The extracellular domain consists of 4 subdomains, namely subdomain 1

[0028] (Subdomain 1, abbreviated as D1, approximately amino acid residues 26-210), subdomain 2 (Subdomain 2, abbreviated as D2, approximately amino acid residues 211-332), subdomain 3 (Subdomain 3, abbreviated as D3, approximately amino acid residues 333-501), and subdomain 4 (Subdomain 4, abbreviated as D4, approximately amino acid residues 502-651), among which D1 and D3 are involved in ligand binding, and cysteine-rich D2 and D4 are responsible for receptor dimerization.

[0029] Herein, "cells expressing HER4" may be naturally occurring cells or cell lines (eg, cardiomyocytes, breast cells, tumor cells), or may be recombinantly produced by introducing a nucleic acid encoding HER4 into a host cell.

[0030] In this article, "antigen binding domain" or "antigen binding region" or "epitope binding domain" are used interchangeably, and refer to a specific region on an antibody or antigen binding fragment or derivative thereof, which is directly involved in the specific interaction with the target antigen, such as interacting with the target antigen through binding, steric hindrance, stabilization / destabilization, spatial distribution, etc. to achieve dynamic equilibrium. In the present invention, "antigen binding domain" also refers to a specific region on the antibody or antigen binding fragment or derivative thereof, which interacts with a specific epitope on the HER4 receptor, and achieves dynamic equilibrium between the binding between the two through binding, steric hindrance, stabilization / destabilization, spatial distribution, etc.

[0031] "Antibody" refers to a polypeptide or protein that is generally encoded by an immunoglobulin gene or multiple immunoglobulin genes, or a fragment thereof, and is capable of specifically recognizing and binding an antigen. The recognized immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively, several of which can be further divided into subclasses / isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Typical immunoglobulin (e.g., antibody) structural units include tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminal domain of each chain defines a variable (V) region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The antibody heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH), wherein the heavy chain constant region generally includes three domains, CH1, CH2, and CH3. The light chain consists of a light chain variable region (VL) and a light chain constant domain (CL), wherein the light chain constant domain generally includes one domain, CL. V H and V L The endogenous V L Encoded by gene segments V (variable) and J (joining), and endogenous V H Encoded by V, D (diversity) and J. V L or V HAll include a hypervariable region (Region of Hypervariability) or a complementary 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 a light chain variable region arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the amino terminus to the carboxyl terminus. The term "J region" refers to a subsequence encoding the C-terminal portion of the variable region containing CDR3 and FR4. The V region or J region may be naturally occurring, recombinant or synthetic. In this article, the antibody light chain variable region and / or the antibody heavy chain variable region may sometimes be collectively referred to as an "antibody variable region", and the antibody light chain and / or the antibody heavy chain may be collectively referred to as an "antibody chain". In certain embodiments, the FR of the antibody or its antigen-binding fragment provided herein may be identical to the human germline sequence, or may be naturally or artificially modified.

[0032] The positions of CDRs and FRs can be determined using a variety of definitions known in the art, e.g., Kabat, Chothia, IMGT, and Contact (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. 1991, US 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 1998, 344:877-883; Chothia et al., J Mol Biol 1992, 345:877-883; Chothia et al., J Mol Biol 1992, 346:877-883; Chothia et al., J Mol Biol 1992, 347:877-883; Al-Lazikani et al., J Mol Biol 1997, 348:877-883; Lefranc MP et al., Nucleic Acids Research 1998, 347:877-883; Chothia et al., J Mol Biol 1992, 347:877-883; Chothia et al., J Mol Biol 1992, 347:877-883; Chothia et al 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. The present invention includes any definition method to determine the CDR in the anti-HER4 antibody or antigen-binding fragment thereof of the present application, and Table 1 shows the position number of the antibody CDR amino acid sequence determined using different definition methods. The exact number of amino acid residues covering a specific CDR varies with the sequence of the CDR.When the amino acid sequence of the antibody variable region is known, those skilled in the art can determine the CDR of the antibody by conventional methods including but not limited to the above definition.

[0033] Table 1. CDRs determined by different definition methods 1

[0034]

[0035]

[0036] 1 : The numbering of all CDR definitions in Table 1 is based on the numbering system proposed by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition 1991, US Department of Health and Human Services).

[0037] In addition, Kabat et al. also defined a numbering system for variable region sequences that can be applied to any antibody. A person of ordinary skill in the art can clearly 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 the present application is determined according to the Kabat numbering system.

[0038] Antibodies exist in the form of complete immunoglobulins, or in the form of many fragments produced by digestion with a variety of peptidases. Although various antibody fragments are defined according to the digestion of complete antibodies, it should be understood by the technician that such fragments can be synthesized de novo by chemical cleavage methods or by using recombinant DNA methods. In this article, the "antigen binding fragment" (or simply referred to as "antibody part" or "antibody fragment") of the term antibody refers to an antibody part containing one or more CDRs or any other antibody fragment that can be combined with an antigen (for example, the extracellular domain of HER4 or HER4) but does not have a complete antibody structure. Antigen binding fragments can have the same activity of specific binding to antigens as complete antibodies. In certain embodiments, antigen binding fragments can contain one or more CDRs from a particular human antibody, transferred to the framework region from one or more different human antibodies. Antigen-binding fragments include, but are not limited to: (i) a "Fab" fragment, a monovalent antibody fragment consisting of the VH, VL, CL and CH1 domains; (ii) a "F(ab')2" fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) a "Fv" fragment, consisting of the VL and VH domains of a single antibody arm, and being the smallest antibody fragment containing a complete antigen-binding site; (iv) a "Fd" fragment, consisting of the VH and CH1 domains; (v) a "single-chain Fv antibody (scFv)", "single-chain antibody" or "scFv molecule", refers to an engineered antibody in which the light chain variable region is directly linked to the heavy chain variable region or is linked by a peptide chain (Huston JS et al., Proc Natl Acad Sci USA

[0039] 1988, 85:5879-5883; Bird et al., Science 1988, 242:423-426); in addition, single-chain antibodies also include "linear antibodies" comprising 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; U.S. 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) "diabodies" are bivalent bispecific antibodies 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 the two domains to pair with the complementary domains 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).

[0040] As used herein, the term "Fc region" or "Fc domain" refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region, such as an immunoglobulin heavy chain constant region other than the first constant region (CH1). For IgG, the Fc region may include immunoglobulin domains CH2 and CH3 and a hinge region between CH1 and CH2. The Fc region used herein includes a native sequence Fc region and / or an Fc region variant, and may be a part of the anti-HER4 antibody of the present application. It should be understood in the art that the boundaries of the Fc region may vary, however, the human IgG heavy chain Fc region is generally defined as comprising 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 found in Kabat et al., NIH Publication 1991, 91-3242, National Technical Information Service.

[0041] The term "anti-HER4 antibody" or "antibody that specifically binds to HER4" 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, as long as 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.

[0042] As used herein, the term "specific binding" or "binding specificity" or "specific for" or "binding" refers 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), that is, the binding is selective for the target molecule and can distinguish those undesirable or non-specific interactions. For example, an antibody that specifically binds to a target molecule (which may be an antigen) has a greater affinity, stronger binding activity, is more likely to bind and / or has a longer duration of binding than the antibody binds to other non-target molecules. A variety of immunoassays can be used to select antibodies that specifically bind to a specific protein, such as solid phase ELISA immunoassays, flow cytometry (FACS), cytofluorimetry, or surface plasmon resonance (SPR) technology to determine the binding reaction of the antibody or antigen-binding fragment thereof herein with the target antigen / protein. Typically, under set assay conditions, the specific or selective binding reaction of the antibody or binding agent to the antigen will produce a signal that is at least twice the background, more typically at least 10-100 times the background, and will not essentially bind to other antigens / proteins present in the sample in significant amounts.

[0043] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the antibody population are identical except for natural mutations that may be present in small amounts. Monoclonal antibodies exhibit specificity and affinity for binding to a particular epitope. 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 U.S. Pat. No. 4,816,567), or can be isolated from phage antibody libraries, e.g., by reference to the techniques described in Clackson et al., Nature 1991, 352: 624-628; Marks et al., J Mol Biol 1991, 222: 581-597.

[0044] As used herein, the term "epitope" refers to a protein determinant, a portion of an antigen that can be recognized and specifically bound by an antibody. An epitope is usually composed of surface groups of a molecule such as amino acids or sugar side chains, and usually has specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0045] The term "affinity" or "binding affinity" refers to the intrinsic binding ability of an interaction between a molecule (e.g., a receptor or antigen) and its partner (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 the intrinsic binding affinity used to reflect a one-to-one interaction between members of a binding pair (e.g., a receptor and a ligand or an antigen and an antibody). The affinity of a molecule X for its partner Y can usually be expressed in terms of the equilibrium dissociation constant (K D ) indicates that the equilibrium dissociation constant is the dissociation rate constant (K dis or K off ) and the ratio of the binding rate constant (Ka or Kon). Affinity can be measured by common methods known in the art, including the methods used in the present invention.

[0046] As used herein, the terms "cross-reaction", "cross-reactivity" or "cross-reactivity" are used interchangeably and refer to the ability of an antibody to be specific for one antigen and also to react with a second antigen, a measure of the association between two different antigens. Therefore, an antibody is cross-reactive if it binds to an antigen other than the target antigen. Cross-reactive epitopes typically contain many amino acid sequences that are homologous to the target antigen epitope, or similar structural features, and in some cases may bind to the antibody more closely than the target antigen epitope. If an antibody does not bind to other sequences or structures other than the target antigen epitope and the cross-reactive epitope, the antibody is considered to be "highly specific" for the antigen epitope.

[0047] As used herein, the term "fusion" or "fused" when used for amino acid sequences (e.g., peptides, polypeptides, or proteins) refers to the combination of two or more amino acid sequences into a non-naturally occurring single amino acid sequence by chemical bonding or recombinant means. The fused amino acid sequence can be produced by genetic recombination of two encoding polynucleotide sequences, and can be expressed by introducing a construct containing the recombinant polynucleotide into a host cell.

[0048] As used herein, the term "antibody variant", "antibody variant" or "antibody mutant" refers to an antibody polypeptide sequence containing at least one amino acid mutation in the variable region of a reference antibody. The variant may be substantially homologous or substantially identical to an unmodified antibody. In some embodiments, one, two, three, four, five and / or six CDRs of the anti-HER4 antibody of the invention have amino acid mutations to improve and optimize the performance of the antibody or antigen-binding portion, including but not limited to increasing the degree of humanization, enhancing the specificity for the target protein / antigen (e.g., HER4), binding activity, increasing yield, and / or increasing 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 anti-HER4 antibody of the invention have amino acid mutations to improve and optimize the performance of the antibody or antigen-binding portion, including but not limited to increasing the degree of humanization of the antibody or antigen-binding portion, enhancing the specificity for the target molecule (e.g., HER4), binding activity, increasing yield, and / or increasing 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 made in the CDRs and FRs of the anti-HER4 antibodies of the present application to improve the degree of humanization of the antibody or antigen-binding portion, enhance the specificity for the target molecule (e.g., HER4), binding activity, increase yield, and / or increase 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.

[0049] Among them, amino acid substitutions include conservative amino acid substitutions and non-conservative amino acid substitutions, where conservative amino acid substitutions involve substitutions with another amino acid of the same class (e.g., similar chemical properties or functions), and non-conservative substitutions involve substitutions with amino acids of different classes (dissimilar chemical properties or functions). One of ordinary skill in the art can perform conservative or non-conservative amino acid substitutions based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues involved. For example, (i) non-polar (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); (iv) negatively charged (acidic) amino acids include aspartic acid (Asp, D) and glutamic acid (Glu, E). Generally speaking, conservative amino acid substitutions will not substantially change the functional properties of proteins, while non-conservative amino acid substitutions may cause significant changes in the properties or functions of proteins. Although the sites or regions for introducing amino acid sequence mutations can be predetermined, the potential changes in protein properties or functions brought about by non-conservative substitutions are unpredictable. In some embodiments, the anti-HER4 antibodies of the present invention unexpectedly obtain significant changes in function and performance, such as binding activity, molecular stability, etc., through non-conservative amino acid substitutions.

[0050] In this article, for two or more polypeptide sequences, the terms "identical" or "identity" or "percent identity" or "percent sequence identity" can be used interchangeably, and refer to the percentage rate of the number of amino acid residues in the candidate sequence that are identical to the reference sequence in the total number of amino acid residues in the candidate sequence after introducing the necessary intervals to maximize the number of identical amino acids. The conservative substitution of the amino acid residues may or may not be considered to be the same residue. The sequences can be aligned to determine the percentage sequence identity of the amino acid sequences by tools disclosed in the art, such as BLASTp, ClustalW2 (also see 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. Those skilled in the art can use the default parameters of the tool or appropriately adjust the parameters according to the needs of the comparison, such as by selecting a suitable algorithm to perform sequence alignment.

[0051] The amino acid sequence "reducing or eliminating the risk of deamidation" mentioned herein refers to a sequence in which an amino acid residue that is prone to deamidation is replaced by an amino acid residue that is less prone or not prone to deamidation. 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. Generally, asparagine (Asn) can be converted into aspartic acid or isoaspartic acid, and glutamine (Gln) can be converted into glutamic acid or pyroglutamic acid. The amino acid sequence contains Asn-Gly, Asn-Ser or Asn-Thr sites that are prone to deamidation, and asparagine is more susceptible to deamidation than glutamine. Deamidation of asparagine and / or glutamine can change the structure of the antibody and its stability and / or function (i.e., antibody-antigen binding), and 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 the amino acid residues in the antibody variable region that are susceptible to deamidation (Jasmin F Sydow et al., PLoS ONE 2014, 9(6): e100736), and replacing them with amino acid residues that are less susceptible or not susceptible to deamidation.

[0052] In this article, the term "aspartic acid isomerization" amino acid sequence refers to a sequence containing an aspartic acid residue that is prone to isomerization. Since isomerization can cause changes in antibody conformation, thereby changing the antibody surface charge, resulting in antibody charge heterogeneity. Preferably, the antibody of the present application does not contain an aspartic acid isomerization site. Aspartic acid isomerization is prone to occur on the DG sequence, and it has also been reported that isomerization can also occur on the DH or DS sequence, thereby producing an isoaspartic acid residue. Since this amino acid residue introduces a connecting bond into the polypeptide chain, the stability of the polypeptide chain is reduced (also known as the isoaspartic acid effect), and it may also cause a decrease in antibody-antigen binding affinity.

[0053] In this article, when it comes to protein, the term "isolated" means that the protein is substantially free of other cellular components that are combined with it in the native state, preferably in a homogeneous state, for example, the isolated protein can be removed from a natural or natural environment. The isolated protein can be lyophilized or an aqueous solution. Usually, 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 basically only produces one band in a non-reducing electrophoresis gel. In particular, this means that the protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. For the purpose of the present application, in some embodiments, the recombinant protein expressed in the host cell is then considered to be isolated, and is also applicable to natural or recombinant proteins separated, graded, or partially or substantially purified by any technology 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 antigenic specificities (e.g., an isolated antibody that specifically binds to HER4 is substantially free of antibodies that specifically bind to antigens other than HER4). Moreover, the isolated antibody may be substantially free of other cellular materials and / or chemicals. In some embodiments, the recombinant polynucleotide encoding the present application polypeptide or protein (e.g., anti-HER4 antibody) contained in a vector is considered to be isolated. Other examples of isolated polynucleotides include recombinant polynucleotides contained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.

[0054] Unless the context indicates otherwise, a "derivative" is a polypeptide or fragment thereof having one, or more non-conservative or conservative amino acid substitutions relative to the parent antibody or polypeptide; or a polypeptide or fragment thereof modified by covalent attachment to a second molecule, such as by attachment to a heterologous polypeptide, or by glycosylation, acetylation, phosphorylation, etc. The definition of "derivative" may also include polypeptides with analogs of one or more amino acids (e.g., non-natural amino acids, etc.), as well as other modifications known in the art (natural and non-naturally occurring).

[0055] In this article, the term "expression vector" or "vector" refers to a vehicle into which a polynucleotide or nucleic acid encoding a protein can be operatively inserted and the protein can be expressed. The vector can be used to transform, transduce or transfect host cells so that the genetic material elements it carries can be expressed in the host cells.

[0056] In this article, the term "host cell" refers to a cell into which an exogenous polynucleotide, nucleic acid and / or vector is introduced. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom, without considering the number of generations. Offspring may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. The present invention includes mutant offspring with the same function or biological activity that are screened or selected in the initially transformed cells.

[0057] In this article, the terms "subject", "patient" or "individual" can be used interchangeably, including but not limited to: mammals, such as humans, non-human primates (e.g., monkeys), mice, pigs, dogs, cats, cows, 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, emu or ducks) or fish; and non-mammalian invertebrates. In some embodiments, the subjects and pharmaceutical compositions involved in the uses or methods of the present invention are used to (prophylactically and / or therapeutically) treat non-human animals.

[0058] As used herein, "treating," "treatment," or "to treat" for a disease or symptom refers to alleviating or to alleviate a disease or symptom, reducing the rate of 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.

[0059] As used herein, "HER4-related" disease or symptom refers to any disease or symptom caused, aggravated, or otherwise associated with an increase or decrease in HER4 expression or activity. In certain embodiments, the HER4-related disease is cancer, such as glioma. In certain embodiments, the HER4-related disease is a glial cell proliferative disease.

[0060] The term "therapeutically effective amount", "effective dose" or "effective amount" refers to the amount or concentration of an active ingredient or agent that is effective to prevent or improve symptoms associated with a disease or condition, alleviate or reduce the severity of a disease or condition, and / or delay or stop the progression of a disease, at a desired dose and for a desired period of time. The therapeutically effective amount of a formulation, antibody, or antigen-binding fragment thereof, or composition of the present invention can vary depending on a variety of factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antigen-binding portion to stimulate a desired response in the individual. A therapeutically effective amount can also be considered to be the amount in which the therapeutically beneficial effects of the formulation, antibody, or antigen-binding fragment thereof, or composition significantly outweigh any toxic or deleterious effects caused by it.

[0061] As used herein, the term "pharmaceutically acceptable" or "pharmaceutically acceptable" means that the referenced carriers, vehicles, diluents, excipients and / or salts are generally chemically and / or physically compatible with the other ingredients in the formulation and physiologically compatible with the subject.

[0062] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value, or in one embodiment, a lower limit that is 10% less and an upper limit that is 10% greater, or in another embodiment, a lower limit that is 15% less and an upper limit that is 15% greater, or in another embodiment, a lower limit that is 20% less and an upper limit that is 20% greater.

[0063] The term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0064] As used herein, the terms "comprises" or "includes" or "contains" or "has" or "involving" are used interchangeably, meaning that the elements, integers or steps described are included, but any other elements, integers or steps are not excluded. In this article, when the terms "comprises" or "includes" or "contains" or "has" or "involving" are used, unless otherwise specified, the situation consisting of the elements, integers or steps described is also covered.

[0065] As used herein, the term "optionally" means that the object it modifies is present or absent, for example, "the kit comprises optionally at least one additional therapeutic agent" means that the kit may or may not comprise at least one additional therapeutic agent.

[0066] As used herein, "some embodiments", "one embodiment", "a specific embodiment", or "a specific embodiment" or a combination thereof means that the described particular features, structures, or characteristics associated with the embodiment are included in at least one embodiment of the present application. Therefore, the appearance of the aforementioned terms in various places throughout this specification does not necessarily refer to the same embodiment. In addition, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0067] Unless the context clearly indicates otherwise, singular terms include plural referents and vice versa.

[0068] For the purpose of description and disclosure, all patents, patent applications and other publications are expressly incorporated herein by reference. These publications are provided only because they are disclosed prior to the filing date of the present application. All statements about the dates of these documents or the representation of the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates of these documents or the contents of these documents.

[0069] Various aspects of the application are described in more detail in the following sections.

[0070] 1. Anti-HER4 antibodies and antigen-binding fragments thereof

[0071] On the one hand, the present application provides an isolated anti-HER4 antibody or an antigen-binding fragment thereof, which can specifically recognize and bind to the extracellular domain III of human HER4 and can block the ligand-induced dimerization of the HER4 receptor.

[0072] The anti-HER4 antibody or antigen-binding fragment thereof of the present invention specifically binds to the extracellular domain III of human HER4, and has high binding activity. In some embodiments, the binding activity of the antibody or antigen-binding fragment thereof to 293T cells expressing HER4 is detected by flow cytometry, and the mean fluorescence intensity (MFI) value is high.

[0073] The anti-HER4 antibody or antigen-binding fragment thereof of the present invention has an inhibitory effect on the HER4 receptor and its downstream signal transduction pathway. In some embodiments, the anti-HER4 antibody or antigen-binding fragment thereof can inhibit ligand-induced HER4 receptor dimerization and its downstream signal transduction pathway, and the ligand includes NRG-1 to 4, preferably NRG-1. In some embodiments, the inhibitory activity (IC50 value) of the antibody or antigen-binding fragment thereof on NRG-1-induced HER2 / HER4 receptor dimerization is no more than 10 μg / mL by bioluminescence detection of living cells.

[0074] The anti-HER4 antibody of the present application may also optionally include Fab, Fab', F(ab')2, Fv, scFv, scFv-Fc, single domain antibody (sdAb), or have IgG type. The anti-HER4 antibody of the present application may be a mouse antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a monoclonal antibody, a polyclonal antibody, a monospecific antibody, a bispecific antibody, a multispecific antibody and an antibody fragment, as long as the antibody can specifically recognize and bind to the extracellular domain III of human HER4. In some embodiments, the anti-HER4 antibody is selected from a fully human anti-HER4 antibody or an optimized antibody or antibody fragment thereof.

[0075] On the other hand, the present invention provides an anti-HER4 antibody or antigen-binding fragment thereof as shown in Table 2, including VH and VL. In some embodiments, the anti-HER4 antibody or antigen-binding fragment thereof of the present invention specifically binds to the extracellular domain III of the HER4 receptor, which comprises a VH amino acid sequence as shown in SEQ ID NO: 21, and / or a VL amino acid sequence as shown in SEQ ID NO: 41.

[0076] In some embodiments, the anti-HER4 antibody or antigen-binding fragment thereof of the present invention comprises one or more CDRs in the VH amino acid sequence shown in SEQ ID NO: 21 or the amino acid sequences shown in SEQ ID NOs: 1, 2 and 12 or variants thereof, including optimized antibodies or any other variants described herein.

[0077] In some embodiments, the anti-HER4 antibody or antigen-binding fragment thereof of the present invention comprises one or more CDRs in the VL amino acid sequence shown in SEQ ID NO:41 or the amino acid sequences shown in SEQ ID NOs:13, 19 and 20 or variants thereof, including optimized antibodies or any other variants described herein.

[0078] In some embodiments, the anti-HER4 antibody or antigen-binding fragment thereof of the present invention comprises: one or more CDRs of the VH amino acid sequence shown in SEQ ID NO:21 or the amino acid sequences shown in SEQ ID NOs:1, 2 and 12 or variants thereof, and one or more CDRs in the VL amino acid sequence shown in SEQ ID NO:41 or the amino acid sequences shown in SEQ ID NOs:13, 19 and 20 or variants thereof, said variants including optimized antibodies or any other variants described herein.

[0079] In some specific embodiments, the anti-HER4 antibody or antigen-binding fragment thereof comprises: a HCDR1 amino acid sequence as shown in SEQ ID NO:1, a HCDR2 amino acid sequence as shown in SEQ ID NO:2, and a HCDR3 amino acid sequence as shown in SEQ ID NO:12, and a LCDR1 amino acid sequence as shown in SEQ ID NO:13, a LCDR2 amino acid sequence as shown in SEQ ID NO:19, and a LCDR3 amino acid sequence as shown in SEQ ID NO:20.

[0080] On the other hand, VH and / or VL of the anti-HER4 antibody of the present invention (eg, Ab4268) can be used as starting material for engineering, ie, variants of the parent antibody Ab4268.

[0081] In some embodiments, the anti-HER4 antibody variants of the present invention comprise antibodies or antigen-binding fragments thereof having modified VH and / or VL compared to the VH (SEQ ID NO: 21) and / or VL (SEQ ID NO: 41) of the parent antibody Ab4268. The modifications introduced into the parent antibody include introducing amino acid mutations (including amino acid substitutions, deletions, insertions or any combination thereof) into the heavy chain variable region CDRs, light chain variable region CDRs and / or FRs of the parent antibody, which can improve the degree of humanization, binding activity, and / or reduce or eliminate the risk of aspartic acid isomerization and / or asparagine deacylation of the parent antibody.

[0082] In some specific embodiments, the anti-HER4 antibody variants of the present invention have one or more amino acid mutations relative to the FR region of the parent antibody Ab4268, which can improve the degree of humanization of the parent antibody while retaining or improving the cell binding activity of the parent antibody. For example, one or more amino acid mutations are performed on the FR region (including the heavy chain variable region FR region and / or the light chain variable region FR region) of VH (SEQ ID NO: 21) of Ab4268. Further, amino acid mutations may be introduced into HFR1 and / or HFR2, for example, mutations of the glutamine residue at position 23 of HFR1 include Q23K, mutations of the alanine residue at position 26 include A26G, and mutations of the serine residue at position 27 include S27Y; mutations of the histidine residue at position 1 (corresponding to position 66 of the VH amino acid sequence shown in SEQ ID NO: 21) of HFR3 include H66Q, mutations of the leucine residue at position 4 (corresponding to position 69 of the VH amino acid sequence shown in SEQ ID NO: 21) include L69I, mutations of the valine residue at position 6 (corresponding to position 71 of the VH amino acid sequence shown in SEQ ID NO: 21) include V71A, mutations of the asparagine residue at position 11 (corresponding to position 76 of the VH amino acid sequence shown in SEQ ID NO: 21) include N76S, mutations of the asparagine residue at position 27 (corresponding to position 69 of the VH amino acid sequence shown in SEQ ID NO: 21) include N76S, Mutations of the threonine residue at position 89 of the VH amino acid sequence shown in NO:21 include T89M or T89V; amino acid mutations can also be introduced into LFR2, and the mutations include mutating the valine residue at position 2 of LFR1 to isoleucine (ie, V2I).

[0083] In some specific embodiments, the anti-HER4 antibody variants of the present invention have one or more amino acid mutations relative to the CDR region of the parent antibody Ab4268, which can improve the degree of humanization of the parent antibody while retaining or improving the cell binding activity of the parent antibody. For example, no more than 1, 2, 3, 4 or 5 amino acid residues in the heavy chain variable region CDRs of the parent antibody Ab4286 are mutated, and the mutation can be an amino acid substitution, addition or deletion, preferably an amino acid substitution. Furthermore, amino acid mutations may be introduced into HCDR1 and / or HCDR2, for example, arginine at position 3 (corresponding to position 33 of the VH amino acid sequence shown in SEQ ID NO: 21), valine at position 4 (corresponding to position 34 of the VH amino acid sequence shown in SEQ ID NO: 21) of HCDR1, and / or phenylalanine at position 3 (corresponding to position 52 of the VH amino acid sequence shown in SEQ ID NO: 21), alanine at position 5 (corresponding to position 53 of the VH amino acid sequence shown in SEQ ID NO: 21), and glutamic acid residue at position 16 (corresponding to position 64 of the VH amino acid sequence shown in SEQ ID NO: 21) of HCDR2. The mutation of phenylalanine at position 3 of HCDR1 (corresponding to position 52 of the VH amino acid sequence shown in SEQ ID NO: 21) includes R33W, and the mutation of valine at position 4 (corresponding to position 34 of the VH amino acid sequence shown in SEQ ID NO: 21) includes V34I; the mutation of phenylalanine at position 3 of HCDR2 (corresponding to position 52 of the VH amino acid sequence shown in SEQ ID NO: 21) includes F52Y, the mutation of alanine at position 5 (corresponding to position 53 of the VH amino acid sequence shown in SEQ ID NO: 21) includes A53G, and the mutation of glutamate at position 16 (corresponding to position 64 of the VH amino acid sequence shown in SEQ ID NO: 21) includes E64Q.

[0084] In a specific embodiment, the anti-HER4 antibody variant of the present invention has one or more amino acid mutations relative to the VH amino acid sequence (SEQ ID NO: 21) of the parent antibody Ab4268, including Q23K, A26G, S27Y, R33W, V34I, F52Y, A53G and / or E64Q, and / or has amino acid mutations relative to the VL amino acid sequence (SEQ ID NO: 41) of the parent antibody Ab4268, including V2I, which can significantly improve the degree of humanization of the parent antibody while retaining or improving the cell binding activity of the parent antibody Ab4268.

[0085] In some specific embodiments, the anti-HER4 antibody variants of the present invention have one or more amino acid mutations relative to the CDR region (including heavy chain variable region CDRs and / or light chain variable region CDRs) of the parent antibody Ab4268 to reduce the potential risk of aspartic acid isomerization and / or asparagine deamidation. For example, no more than 1 or 2 amino acid residues in the heavy chain variable region CDRs or light chain variable region CDRs of the parent antibody Ab4268 are mutated, and the mutation can be an amino acid substitution, addition or deletion, preferably an amino acid substitution.

[0086] In a specific embodiment, one or more amino acid mutations are made in the CDR region of the VH (SEQ ID NO: 21) of the parent antibody Ab4268 to perform isomerization modification on the parent antibody. In a preferred embodiment, the anti-HER4 antibody variant of the present invention does not contain an aspartic acid isomerization site, and one or more amino acid mutations can be introduced into the HCDR2 of Ab4268, for example, the aspartic acid at position 6 (corresponding to position 54 of the VH amino acid sequence shown in SEQ ID NO: 21) and the serine at position 7 (corresponding to position 55 of the VH amino acid sequence shown in SEQ ID NO: 21) of HCDR2 can be mutated to reduce or eliminate the aspartic acid isomerization of the amino acid sequence of the parent antibody Ab4268. The mutation of the aspartic acid residue at position 6 of HCDR2 (corresponding to position 54 of the VH amino acid sequence shown in SEQ ID NO: 21) includes D54A, D54F, D54G, D54K, D54S or D54T (preferably D54A, D54F, D54G, D54K or D54S), and the mutation of the serine residue at position 14 (corresponding to position 62 of the VH amino acid sequence shown in SEQ ID NO: 36) includes S55A, S55E, S55N, S55T or S55Y (preferably S55T).

[0087] In a specific embodiment, one or more amino acid mutations are made to the CDR region in the VL (SEQ ID NO: 41) of the parent antibody Ab4268. Further, one or more amino acid mutations can be introduced in LCDR1 so that the variant of the parent antibody comprises an amino acid sequence that reduces or eliminates the risk of deamidation. For example, amino acid mutations can be made to the asparagine at position 10 (corresponding to position 28 of the VL amino acid sequence shown in SEQ ID NO: 41) and the glycine at position 11 (corresponding to position 29 of the VL amino acid sequence shown in SEQ ID NO: 41) of LCDR1. The mutation of the asparagine residue at position 10 of LCDR1 (corresponding to position 28 of the VL amino acid sequence shown in SEQ ID NO:41) includes N28G, N28I, N28L, N28S, N28T or N28V, and the mutation of the glycine residue at position 11 (corresponding to position 29 of the VL amino acid sequence shown in SEQ ID NO:41) includes G29A, G29I, G29N, G29P, G29R, G29T or G29V (preferably, G29G, G29A, G29N or G29R).

[0088] By performing the above-mentioned amino acid mutations on the CDR region and framework region of the parent antibody Ab4268, not only the antibody variant has a higher humanization level and a lower risk of aspartic acid isomerization and / or asparagine deamidation, but also the binding activity of the parent antibody to human HER4 is improved.

[0089] On the other hand, the amino acid sequence numbers of the heavy chain CDR region, light chain CDR region, and heavy chain variable region and light chain variable region of the anti-HER4 antibody (including the parent antibody Ab4268 and its variants) of the present 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 well known in the art, the CDR region can also be defined based on other numbering systems / methods such as Chothia and IMGT, AbM or Contact of the heavy chain / light chain variable region sequence, and the CDR region defined by other numbering systems / methods and the CDR region defined by Kabat used in the present invention are within the scope of protection of the present invention.

[0090] Table 2. CDR regions of anti-HER4 antibodies, as well as amino acid sequence ID numbers of heavy chain variable regions and light chain variable regions

[0091]

[0092]

[0093] In some embodiments, the present invention provides an isolated anti-HER4 monoclonal antibody or an antigen-binding fragment thereof, comprising (1) an amino acid sequence of NYX1 X 2 G(X 1 =R or W; X 2 =V or I; i.e., HCDR1 of SEQ ID NO: 49), preferably, X 1 =R,X 2 =V; (2) the amino acid sequence is IIX 3 PX 4 X 5 X 6 DARYSPSFX 7 G(X 3 =F or Y; X 4 =A or G; X 5 =D,A,F,G,K,S or T;X 6 =S,A,E,N,T or Y;X 7 =E or Q; i.e., HCDR2 of SEQ ID NO: 50), preferably, X 3 =F,X 4 =G,X 5 =A, F, G, K or S, X 6 =S or T, X 7 =Q; (3) the HCDR3 amino acid sequence shown in SEQ ID NO: 12; (4) the amino acid sequence is RSSQSLLHSX 8 X 9 YNYLD(X 8 =N, G, I, L, S, T or V, preferably G or S; X 9 =G, A, I, N, P, R, T or V, preferably G, A, N or R), preferably, X 8 =G or S, X 9 =G, A, N or R; (5) the LCDR2 amino acid sequence as shown in SEQ ID NO:19; and (6) the LCDR3 amino acid sequence as shown in SEQ ID NO:20, 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 said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.

[0094] In some specific embodiments, the above-mentioned anti-HER4 antibody or its antigen-binding fragment comprises: a HCDR1 amino acid sequence as shown in SEQ ID NO: 1, a HCDR2 amino acid sequence as shown in any one of SEQ ID NOs: 2-11, and a HCDR3 amino acid sequence as shown in SEQ ID NO: 12, and a LCDR1 amino acid sequence as shown in any one of SEQ ID NOs: 13-18, a LCDR2 amino acid sequence as shown in SEQ ID NO: 19, and a LCDR3 amino acid sequence as shown in SEQ ID NO: 20.

[0095] In some specific embodiments, the above-mentioned anti-HER4 antibody or its antigen-binding fragment comprises: a HCDR1 amino acid sequence as shown in SEQ ID NO: 1; a HCDR2 amino acid sequence as shown in SEQ ID NO: 2, 3, 4, 10 or 11; and a HCDR3 amino acid sequence as shown in SEQ ID NO: 12; and a LCDR1 amino acid sequence as shown in SEQ ID NO: 13 or 15; a LCDR2 amino acid sequence as shown in SEQ ID NO: 19; and a LCDR3 amino acid sequence as shown in SEQ ID NO: 20.

[0096] In a specific embodiment, the anti-HER4 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3, wherein each CDR comprises:

[0097] (1) the HCDR1 amino acid sequence set forth in SEQ ID NO:1, the HCDR2 amino acid sequence set forth in any one of SEQ ID NOs:2-11, the HCDR3 amino acid sequence set forth in SEQ ID NO:12; and the LCDR1 amino acid sequence set forth in SEQ ID NO:13, the LCDR2 amino acid sequence set forth in SEQ ID NO:19, the LCDR3 amino acid sequence set forth in SEQ ID NO:20, 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 said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively; or

[0098] (2) the HCDR1 amino acid sequence set forth in SEQ ID NO:1, the HCDR2 amino acid sequence set forth in SEQ ID NO:2, the HCDR3 amino acid sequence set forth in SEQ ID NO:12; and the LCDR1 amino acid sequence set forth in any one of SEQ ID NOs:14-18, the LCDR2 amino acid sequence set forth in SEQ ID NO:19, the LCDR3 amino acid sequence set forth in SEQ ID NO:20, 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 said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively; or

[0099] (3) the HCDR1 amino acid sequence as shown in SEQ ID NO:1, the HCDR2 amino acid sequence as shown in SEQ ID NO:11, the HCDR3 amino acid sequence as shown in SEQ ID NO:12; and the LCDR1 amino acid sequence as shown in SEQ ID NO:15, the LCDR2 amino acid sequence as shown in SEQ ID NO:19, the LCDR3 amino acid sequence as shown in SEQ ID NO:20, or 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 to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.

[0100] In one embodiment, the anti-HER4 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3, each CDR comprising: the HCDR1 amino acid sequence as shown in SEQ ID NO:1, the HCDR2 amino acid sequence as shown in SEQ ID NO:2, the HCDR3 amino acid sequence as shown in SEQ ID NO:12, and the LCDR1 amino acid sequence as shown in SEQ ID NO:13, the LCDR2 amino acid sequence as shown in SEQ ID NO:19, the LCDR3 amino acid sequence as shown in SEQ ID NO:20, 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 said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively; or the HCDR1 amino acid sequence as shown in SEQ ID NO:1, the HCDR2 amino acid sequence as shown in SEQ ID NO:11, the HCDR3 amino acid sequence as shown in SEQ ID NO:12 The invention further comprises the following: the HCDR3 amino acid sequence shown in SEQ ID NO:12, the LCDR1 amino acid sequence shown in SEQ ID NO:13 or 15, the LCDR2 amino acid sequence shown in SEQ ID NO:19, the LCDR3 amino acid sequence shown in SEQ ID NO:20, 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 said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.Preferably, the anti-HER4 antibody or antigen-binding fragment thereof 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, the HCDR3 amino acid sequence as shown in SEQ ID NO:12, and the LCDR1 amino acid sequence as shown in SEQ ID NO:13, the LCDR2 amino acid sequence as shown in SEQ ID NO:19, and the LCDR3 amino acid sequence as shown in SEQ ID NO:20, 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; or comprises the HCDR1 amino acid sequence as shown in SEQ ID NO:1, the HCDR2 amino acid sequence as shown in SEQ ID NO:11, the HCDR3 amino acid sequence as shown in SEQ ID NO:12, and the LCDR1 amino acid sequence as shown in SEQ ID NO:15, the LCDR2 amino acid sequence as shown in SEQ ID NO:19, and the LCDR3 amino acid sequence as shown in SEQ ID NO:20. The LCDR2 amino acid sequence shown in NO:19, the LCDR3 amino acid sequence shown in SEQ ID NO:20, 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 embodiments, the anti-HER4 antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain variable region having the HCDR1, HCDR2 and HCDR3 and a light chain variable region having the LCDR1, LCDR2 and LCDR3.

[0102] In some specific embodiments, the anti-HER4 antibody or antigen-binding fragment thereof 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 to the amino acid sequence shown in any one of SEQ ID NOs: 21-40, 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 to the amino acid sequence shown in any one of SEQ ID NOs: 41-48. In some preferred embodiments, the anti-HER4 antibody or antigen-binding fragment thereof of the present invention comprises: a VH having at least 80% identity to the amino acid sequence shown in SEQ ID NOs: 21, 25, 26, 27, 34, 36, 37, 39 or 40, and a VL having at least 80% identity to the amino acid sequence shown in SEQ ID NOs: 41, 42, 44 or 48. In some preferred embodiments, the anti-HER4 antibody or antigen-binding fragment thereof of the present invention comprises: a VH having at least 80% identity with the amino acid sequence shown in SEQ ID NO:39 or 40, and a VL having at least 80% identity with the amino acid sequence shown in SEQ ID NO:48.

[0103] In a specific embodiment, the anti-HER4 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region each comprise 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 VH shown in any one of SEQ ID NOs: 21-40 and the VL shown in SEQ ID NO: 41, respectively; 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 VH shown in SEQ ID NO: 21 and the VL shown in any one of SEQ ID NOs: 42-47, respectively; 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 VH shown in SEQ ID NO: 21 and the VL shown in any one of SEQ ID NOs: 42-47, respectively; The VH shown in NO:39 or 40 and the VL shown in SEQ ID NO:48 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 that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH set forth in SEQ ID NO: 21, 25, 26, 27, 34, 36, 37, 39 or 40 and the VL set forth in SEQ ID NO: 41, 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 VH set forth in SEQ ID NO: 21 and the VL set forth in SEQ ID NO: 42 or 43, 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 VH set forth in SEQ ID NO: 39 or 40 and the VL set forth in SEQ ID NO: NO:48 has 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; More preferably, the heavy chain variable region and the light chain variable region each include 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 VH shown in SEQ ID NO:21 and the VL shown in SEQ ID NO:41, 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 VH shown in SEQ ID NO:39 or 40 and the VL shown in SEQ ID NO:41, respectively. The VL shown in NO:48 has 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.

[0104] In some embodiments, the anti-HER4 antibody or its antigen-binding fragment of the present invention may also include a constant region, the constant region includes a heavy chain constant region and a light chain constant region, the heavy chain constant region includes CH1, hinge and / or CH2-CH3 region. Wherein, the heavy chain constant region includes the natural and mutant protein forms of the Fc region of the human IgG heavy chain constant region, and also includes a hinge region that promotes dimer formation. In certain embodiments, the Fc region includes antibody CH2 and CH3 domains. The fusion protein containing the Fc portion can be purified by protein A or protein G affinity chromatography columns, and the serum half-life can be extended. The preferred Fc region is derived from human IgG, including IgG1, IgG2, IgG3 and IgG4. In this article, the position of specific amino acid residues in the Fc region is determined according to the EU numbering system.

[0105] One function of the Fc region of an antibody is to produce "effector functions" with the immune system when the antibody binds to its target, including the production of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated by the binding of Fc to Fc receptors (FcR) 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 evaluated by various methods, such as Fc receptor binding assays, C1q binding assays, and cell lysis assays.

[0106] The anti-HER4 antibody or antigen-binding fragment thereof of the present invention can specifically bind to the extracellular domain III of human HER4. The anti-HER4 antibody or antigen-binding fragment thereof has a high degree of humanization, a high sequence stability and a high binding activity to human HER4, and can effectively inhibit ligand-induced HER4 dimerization and its downstream signal transduction pathway, especially inhibit NRG-1-induced HER2 / HER4 dimerization and its downstream signal transduction pathway.

[0107] 2. Method for screening and obtaining the anti-HER4 antibody or antigen-binding fragment thereof of the present invention

[0108] The anti-HER4 antibody provided by the present invention can be a mouse antibody, a chimeric antibody, a humanized antibody or a fully human antibody. The method of producing monoclonal antibodies is 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 the present invention to prepare a monoclonal antibody that specifically binds to the extracellular domain of HER4.

[0109] In some embodiments, the present invention separates mononuclear cells (PBMC) from normal human peripheral blood, amplifies antibody variable region genes from PBMC, displays human antibody variable regions on the surface of phages through phage display technology, and constructs a phage display library.

[0110] In some embodiments, a polypeptide of any fragment in the amino acid sequence of the HER4 receptor (preferably the extracellular domain of the HER4 receptor) can be selected as the target antigen for panning, and the interaction between the antibody variable region displayed on the surface of the phage and the target antigen is detected, and the antibody variable region library is screened and amplified by an in vitro selection method, and the in vitro selection is similar to natural selection. In some specific embodiments, the present invention detects and screens the antibody variable region displayed on the surface of the phage that interacts with the target antigen by a variety of methods, including but not limited to flow cytometry (e.g., flow fluorescence-activated cell sorting [fluorescence-activated cell sorting, FACS] detection method), ELISA detection method and cell fluorescence detection method.

[0111] In some embodiments, the present invention constructs a phage display library and performs panning on the target antigen to screen out antibodies that recognize the target antigen from the antibody library. The antibodies of the present invention can be produced using a variety of phage display methods known in the art, for example, U.S. Patent Nos. 5,223,409, 5,622,699 and 6,068,829 disclose methods for preparing phage libraries. Phage display libraries can also be constructed according to the methods described in "Antibody Phage Display: Methods and Protocols (Philippa M. O'Brien, Robert Aitken ed.)". In some embodiments, nucleic acids encoding antibody variable regions can be inserted into phage coat protein genes so that phages display the antibody variable regions on their surfaces, and at the same time, nucleic acids encoding the antibody variable regions are contained inside the phages, thereby realizing the connection between the phenotype and genotype of the antibody variable regions.

[0112] Regarding the screening of antibodies, in phage display, a large library of VH and / or VL regions of antibodies can be expressed on the surface of filamentous phage particles, thereby pairing them to form binding domains. Phage can be screened from the library based on recognition and binding to the target antigen and the binding domains they display.

[0113] In some embodiments, the panning can be achieved by phage infection of host bacteria and propagation and amplification in the host. After the phages secreted by the host bacteria displaying single-chain antibody fragments on their surfaces are collected, multiple rounds of panning can be performed as needed until phages that can selectively or specifically bind to the target antigen are obtained, and 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).

[0114] In a specific embodiment, the present invention uses the HER4 receptor extracellular domain as the target antigen, obtains the human antibody heavy chain variable region family and light chain variable region family gene fragments from PBMC, and prepares the scFv fragment based on this, and connects it to the phage surface structural protein gene III, and uses the co-expression method to make it participate in phage assembly and display it on the phage surface, thereby constructing a phage display library. Using the phage display library, for a specific target antigen (for example, the HER4 receptor extracellular domain), through multiple rounds of adsorption-elution-amplification repetitive processes (panning), phages that can specifically bind to the target antigen can be enriched, and then the corresponding DNA sequence information is obtained through gene sequencing technology, and then the amino acid sequence of the antibody variable region is inferred.

[0115] 3. Polynucleotides, vectors and host cells

[0116] In one aspect, the present invention provides a nucleic acid encoding an anti-HER4 antibody or an antigen-binding fragment thereof. The present application also includes polynucleotide variants encoding the amino acid sequences described herein.

[0117] Those skilled in the art can determine the nucleic acid sequence encoding the antibody of the present application based on the genetic code. Polymerase chain reaction (PCR) can be used to separate and amplify the DNA sequence encoding the anti-HER4 antibody or its antigen-binding fragment of the present application. The oligonucleotide may additionally contain a recognition site for a restriction endonuclease to facilitate the combined insertion of the amplified DNA fragments into the expression vector. PCR technology is described in 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).

[0118] The nucleic acid molecules of the present invention include single-stranded and double-stranded forms of 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 an amount or concentration that can be identified, manipulated and recovered by standard biochemical methods for its component nucleotide sequences (e.g., 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 include open reading frames interrupted by internal non-translated sequences or introns that are not commonly found in eukaryotic genes and / or constructed. The sequence of non-translated DNA can be present 5' or 3' of the open reading frame, wherein the sequence does not interfere with the manipulation or expression of the coding region.

[0119] The anti-HER4 antibody or its antigen-binding fragment of the present invention is prepared by the following steps: using PCR mutagenesis or other techniques known to those skilled in the art to mutagenesis specific site nucleotides in the DNA encoding the anti-HER4 antibody or its antigen-binding fragment to produce DNA encoding variants, and then expressing / amplifying the recombinant DNA in cell culture as outlined herein. In addition, the anti-HER4 antibody or its antigen-binding fragment can also be prepared by in vitro synthesis using established techniques.

[0120] As known to those skilled in the art, due to the degeneracy of the genetic code, the anti-HER4 antibodies or antigen-binding fragments thereof of the present invention are encoded by a very large number of nucleic acids, each of which is within the scope of the present application and can be prepared using standard techniques. Therefore, having identified a specific amino acid sequence, one skilled in the art can prepare many different nucleic acids by simply modifying one or more of the above codons in their respective coding sequences without changing the amino acid sequence of the anti-HER4 antibodies or antigen-binding fragments thereof of the present invention.

[0121] On the other hand, the present invention also provides an expression vector of a nucleic acid encoding the anti-HER4 antibody or antigen-binding fragment thereof described herein.

[0122] The nucleic acid encoding the anti-HER4 antibody or antigen-binding fragment thereof of the present invention can be constructed in a suitable vector to be introduced into a host cell for expression of the target protein. The vector components generally 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 operably connected to the promoter.

[0123] As used herein, the term "operably linked" refers to a functional connection between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, such that the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0124] 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 lambda phage or M13 phage), and animal viruses, etc. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist it in entering the cell, including but not limited to viral particles, liposomes, or protein shells.

[0125] On the other hand, the present invention also provides a host cell comprising a nucleic acid or an expression vector encoding the anti-HER4 antibody or antigen-binding fragment thereof of the present application.

[0126] The cell can be a eukaryotic cell, for example, a mammalian host cell, including but not limited to, SV40 transformed monkey kidney cell CV1 line (COS-7, ATCC, CRL-1651), human embryonic kidney cell line (293 or suspension cultured 293 cell subclone, Graham et al., J Gen Virol 1977, 36:59-74), baby hamster kidney cell (BHK-21, ATCC, CCL-10), Chinese hamster ovary cell (CHO, Urlaub et al., Proc Natl Acad Sci USA 1980, 77:4216-4220), mouse testicular supporting cell (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 liver cells (BRL 3A, ATCC, CRL-1442), human lung cells (W138, ATCC, CCL-75), human hepatoma 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.

[0127] 4. Preparation method of anti-HER4 antibody or antigen-binding fragment thereof of the present invention

[0128] The present invention provides a method for preparing the anti-HER4 antibody or antigen-binding fragment thereof of the present invention using the host cell.

[0129] The method comprises transfecting a nucleic acid or expression vector encoding an anti-HER4 antibody or antigen-binding fragment thereof of the present invention into a host cell, and culturing the host cell in a culture medium for a period of time to express the anti-HER4 antibody or antigen-binding fragment thereof of the present invention. Without limitation, any commercially available culture medium can be used as the culture medium.

[0130] Preferably, the expressed anti-HER4 antibody or antigen-binding fragment thereof can be secreted into the culture medium of the host cell. The antibody is recovered from the culture medium using standard protein purification methods, such as removing impurities by centrifugation or filtration, or purifying the resultant by affinity chromatography; other purification techniques such as anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography can also be used.

[0131] 5. Bispecific / Multispecific Molecules

[0132] The present invention also provides a bispecific molecule, which comprises an anti-HER4 antibody or antigen-binding fragment thereof of the present invention connected to another functional molecule (e.g., another polypeptide or protein such as a Fab' fragment), thereby forming a bispecific molecule or multispecific molecule that binds to multiple binding sites or binding (target) epitopes. For example, the anti-HER4 antibody or antigen-binding fragment thereof of the present invention can be functionally connected to one or more other binding molecules, such as another antibody, antigen-binding fragment, polypeptide or binding mimetic, and the functional connection includes chemical coupling, genetic fusion, non-covalent association or other means).

[0133] In one embodiment, the other functional molecule can be an Fc receptor (FcR), such as human FcγRIII (CD16), FcδRI (CD64) or FcαR (CD89), and thus, the bispecific molecules 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.

[0134] In one embodiment, the other functional molecule is selected from an antibody or an antigen-binding fragment thereof that is specific to a cancer-associated antigen (TAA) or an immune checkpoint protein antigen. The cancer-associated antigen 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 antigen may be selected from PD-1, PD-L1, CTLA-4, CD137, CD47, etc.

[0135] In one embodiment, in addition to the binding specificity and anti-HER4 binding specificity of the above-mentioned functional molecules, the dual-characteristic molecules or multispecific molecules of the present invention may further include a third binding specificity. The third specificity may be an anti-enhancement factor (EF) portion, for example, the molecule may be bound to a surface protein involved in cytotoxic activity, and thereby increase the immune response to the target cell. The anti-enhancement factor portion may be an antibody (including scFv), and the anti-enhancement factor portion may be bound to FcR or a target cell antigen. In addition, the target cell to which the anti-enhancement factor portion is bound may be different from the target cell to which the first and second binding specific molecules are bound, for example, the anti-enhancement factor portion may be bound to a cytotoxic T cell (for example, through CD2, CD3, CD8, CD28, CD4 or ICAM-1) or other immune cells that produce an enhanced immune response to the target cell.

[0136] In this article, the term "target cell" used refers to any undesirable cell in a subject that is targeted by a molecule of the present invention (e.g., an anti-HER4 antibody or its antigen-binding fragment, a bispecific molecule, or a multispecific molecule). In some embodiments, the target cell is a cell that overexpresses HER4, and the cell that overexpresses HER4 includes a tumor cell (e.g., a glioma), or other non-tumor cells.

[0137] The bispecific molecules of the present invention can have many different forms. For example, the bispecific molecule retains the traditional antibody format, but it does not have two binding arms of the same specificity, but two binding arms of different specificities, each of which can be composed of two single-chain antibody fragments (scFv) connected by a peptide chain to construct a bispecific molecule, the so-called Bs(scFv)2 structure. The bispecific molecule can also include two different F(ab) fragments connected 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., supra; Cao and Suresh, Bioconjugate Chemistry, 9(6), 635-644 (1998); and van Spriel et al., Immunology Today, 21(8), 391-397 (2000), and references cited therein.

[0138] 6. Immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or oncolytic viruses

[0139] In one aspect, the present invention provides an immunoconjugate comprising an anti-HER4 antibody or an antigen-binding fragment thereof of the present invention.

[0140] The anti-HER4 antibody or antigen-binding fragment thereof of the present invention can be conjugated with a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include cytotoxins (including paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenotoposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, mitramycin, actinomycin D, 1-dehydroproterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol), alkylating agents (e.g., chloroform, chlorbutiazide, melphalan, carmustine [BSNU] and lomustine [CCNU], cyclophosphamide, butanesulfonimide, dibromonitrobenzene, streptozotocin , mitomycin C and cis-dichlorodiamine platinum (II) [DDP] cisplatin), puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil demethylhydrazine), DNA minor groove binders, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, tubulin binders, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics (e.g., actinomycin, bleomycin, mithramycin and anthramycin [AMC]) and anti-mitotic agents (e.g., vincristine, vinblastine). In ADC, the linker for coupling the antibody and the therapeutic agent can be a cleavable linker, such as a peptide linker, a disulfide bond or a hydrazone linker, or a non-cleavable linker. ADCs may be prepared as described in U.S. Pat. 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; U.S. Pat. Nos. 20060024317, 20060004081, and 20060247295, the disclosures of which are incorporated herein by reference.

[0141] On the other hand, the present invention also provides a chimeric antigen receptor, an engineered T cell receptor, or an oncolytic virus comprising the anti-HER4 antibody or antigen-binding fragment thereof of the present invention.

[0142] 7. Pharmaceutical composition

[0143] The present invention provides a pharmaceutical composition comprising the anti-HER4 antibody or antigen-binding fragment thereof (or immunoconjugate, or bispecific molecule / multispecific molecule, or chimeric antigen receptor, or engineered T cell receptor, or oncolytic virus) of the present invention, and a pharmaceutically acceptable carrier.

[0144] The pharmaceutical composition may include any type of pharmaceutically acceptable carrier. Operable carriers include excipients, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents or combinations thereof. Selection and use of suitable carriers are taught in the following, Gennaro writes, Remington: The Science and Practice of Pharmacy, 20th edition (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0145] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (for example, by injection or infusion). In view of different routes of administration, active ingredient can be coated in material to protect it from acid and other natural conditions that may inactivate it. As used in the present application, the term "parenteral administration" is a non-enteral and local mode of administration, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the antibody of the present application can be administered by non-parenteral route (for example, local, epidermal or mucosal administration route), for example, intranasal, oral, vaginal, rectal, sublingual or topical administration.

[0146] The pharmaceutical compositions can be sterile aqueous solutions or dispersions. They can also be in the form of microemulsions, liposomes, or other ordered structural combinations suitable for high drug concentration.

[0147] 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 mode of administration, and is generally the amount of the composition that can produce a therapeutic effect. Typically, the composition formed with a pharmaceutically acceptable carrier contains 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.

[0148] The dosage regimen is adjusted to provide the best expected response (e.g., a therapeutic response). For example, a single dose may be administered, divided doses may be administered, or the dose may be reduced or increased proportionally depending on the treatment situation. It is particularly advantageous to formulate a composition for parenteral administration in a dosage unit form, which facilitates administration and uniform dosage. As used herein, a dosage unit form refers to a physically discrete unit that is suitable as a unit dose for treating a subject; each unit dose contains a predetermined amount of an active ingredient, and the predetermined amount of the active ingredient is calculated to produce the expected therapeutic effect when administered together with the desired drug carrier. In addition, the anti-HER4 antibody or antigen-binding fragment thereof of the present invention can also be administered as a sustained-release formulation, which can reduce the frequency of administration.

[0149] The dosage of the anti-HER4 antibody or antigen-binding fragment thereof composition can range from about 0.0001 mg / kg to 100 mg / kg body weight, typically 0.001 mg / kg to 50 mg / kg body weight.

[0150] The "therapeutically effective dose" of the anti-HER4 antibody or antigen-binding fragment thereof of the present invention preferably can reduce the severity of disease symptoms, increase the frequency and duration of disease symptom-free progression, or prevent body damage or disability caused by disease distress. For example, relative to untreated subjects, the "therapeutically effective dose" of the subject is preferably able to inhibit 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 therapeutically effective amount of a therapeutic antibody can improve at least one symptom of the subject's condition, and the subject is usually a human or other mammal. The "therapeutically effective dose" can also be determined differently according to various factors, including but not limited to formulation method, administration method, age, body, weight, patient's gender or pathological condition, diet, administration time, administration interval, administration route, excretion rate and reaction sensitivity.

[0151] The pharmaceutical composition can be selected as a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. See, for example, Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0152] The therapeutic drug composition can be delivered by a medical device selected from the group consisting of: (1) needle-free subcutaneous injection devices (e.g., U.S. Pat. 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. Pat. No. 4,487,603); (3) transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion devices (U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Pat. Nos. 4,439,196 and 4,475,196); the disclosures of which are incorporated herein by reference.

[0153] In certain embodiments, the anti-HER4 antibody or antigen-binding fragment thereof of the present invention can be formulated to ensure biodistribution in vivo. For example, in order to ensure that the therapeutic antibody of the present application crosses the blood-brain barrier, it can be formulated into liposomes, which can additionally contain a targeting moiety to enhance selective delivery to specific cells or organs. See, e.g., U.S. Pat. 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.

[0154] 8. Pill Box

[0155] In one aspect, the invention provides a kit comprising an effective amount of a pharmaceutical composition of an anti-HER4 antibody or antigen-binding fragment of the invention, and one or more other therapeutic agents (ie, the kit may or may not contain at least one additional therapeutic agent).

[0156] In some embodiments, the therapeutic agent includes a therapeutic agent targeting reactive glial cell proliferation, including a signaling pathway regulator, a cell cycle inhibitor, and an inflammatory response regulator. In some embodiments, the signaling pathway regulator includes a regulator targeting R1P1K and the downstream VEGFD / VEGFR3 pathway (e.g., Necrostatin [NEC-1] and its analogs), a regulator targeting the PI3K / AKT signaling pathway (e.g., alismatol A24-acetate), a regulator that inhibits transforming growth factor β1 activity and upregulates PPARα expression (e.g., oleoylethanolamine, valproic acid). In some embodiments, the cell cycle inhibitor includes olomocin, salidroside, and carnosine. In some embodiments, the inflammatory response regulator includes Sailuotong capsule, resveratrol, and anti-IL-17A monoclonal antibody (e.g., Sujin monoclonal antibody).

[0157] 9. Therapeutic uses and methods

[0158] On the one hand, the present invention relates to the use of the anti-HER4 antibody or antigen-binding fragment thereof of the present invention in the preparation of a pharmaceutical composition or preparation for treating and / or preventing a HER4-related disease or condition, or in the preparation of a diagnostic agent for diagnosing a HER4-related disease or condition. Alternatively, the present invention relates to a method for treating and / or preventing a HER4-related disease or condition, the method comprising administering to a subject a therapeutically effective amount of the anti-HER4 antibody or antigen-binding fragment thereof, pharmaceutical composition or kit of the present invention.

[0159] Such diseases or conditions include cancer and other HER4-related diseases.

[0160] In some embodiments, the cancer comprises glioma.

[0161] In some embodiments, other HER4-associated diseases include glial proliferative diseases, for example, stroke, Alzheimer's disease, viral encephalitis, traumatic brain injury, multiple sclerosis, epilepsy.

[0162] On the other hand, the present invention provides a method for inhibiting HER4 receptor and its downstream signal transduction pathway, which comprises using the anti-HER4 antibody or its antigen-binding fragment, pharmaceutical composition or kit of the present invention to inhibit ligand-induced HER4 dimerization in vitro or in vivo, wherein the ligand comprises NRG-1 to 4, preferably NRG-1.

[0163] 10. Detection purpose

[0164] 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 methods for screening patients with HER4-related diseases who are responsive to treatment with the anti-HER4 antibodies or antigen-binding fragments thereof of the present invention.

[0165] In some embodiments, the anti-HER4 antibody or antigen-binding fragment thereof can be used to diagnose a disease or condition in which HER4 is abnormally expressed (e.g., overexpressed, underexpressed, or lacking in expression) to facilitate determination of a treatment regimen. For example, the antibody can be conjugated with a detectable marker or reporter molecule and the labeled antibody can be contacted with a sample obtained from a patient to diagnose and determine the HER4 expression level. The detectable marker or reporter molecule can be a radioactive isotope, such as 3 H. 14 C. 32 P. 33 P. 35 S. 123 I. 125 I. 131 I. 111 In or 188 Rh; a fluorescent material, for example, umbelliferone, luciferin, rhodamine, fluorescein isothiocyanate, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; a chemiluminescent material, for example, luminol; a bioluminescent material, for example, luciferase, luciferin or aequorin; or an enzyme, for example, alkaline phosphatase, β-galactosidase, acetylcholinesterase, horseradish peroxidase or luciferase.

[0166] In some embodiments, the method for detecting or measuring the content or expression level of HER4 in a sample includes: (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 in the sample that is bound to the antibody or its antigen-binding fragment (quantitative or qualitative). Wherein, the control sample includes a positive control and a negative control, the positive control can be a known sample capable of evaluating a disease or condition, and the negative control can be a sample of a healthy subject. Compared to the control, the expression level of HER4 in the sample is higher and has a statistical difference, indicating the presence of a disease or condition related to HER4. Specific exemplary assays that can be used to detect or measure the expression level of HER4 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immune PET (e.g., 89Zr, 64Cu, etc.) and fluorescence activated cell sorting (FACS).

[0167] In some embodiments, the present invention provides a method for detecting, diagnosing or monitoring a HER4-related disease or condition in a subject, the method comprising: (1) contacting a test sample obtained from the subject with an anti-HER4 antibody or an antigen-binding fragment thereof of the present invention, and detecting the content or expression level of HER4 in the test sample (quantitatively or qualitatively); (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 equivalent to that of the normal cells), wherein if the test sample expresses a higher level of HER4 than the control sample, it can be judged that the subject suffers from a HER4-related disease or condition, or that the subject has a condition of a HER4-related disease or condition.

[0168] 11. Technical Effects

[0169] The anti-HER4 antibody or antigen-binding fragment thereof of the present invention can specifically bind to the extracellular domain III of human HER4, and the binding activity is relatively high. The anti-HER4 antibody or antigen-binding fragment thereof provided by the present invention has an inhibitory effect on ligand-induced HER4 dimerization, indicating that the anti-HER4 antibody or antigen-binding fragment thereof of the present invention can block the signal transduction pathway mediated by ligand-induced HER4, and the antibody or antigen-binding fragment thereof can reduce the risk of cardiac toxicity caused by existing cancer therapeutics (e.g., Pertuzumab) when used as a cancer therapeutic.

[0170] In summary, the anti-HER4 antibodies or antigen-binding fragments thereof provided by the present invention can be used as candidates for drugs for treating and / or preventing HER4-related diseases or conditions, and are also suitable for development into bispecific molecules or multispecific molecules.

[0171] The application is further illustrated by the following examples, which should not be construed as further limiting. The contents of all drawings and all references, patents and patent applications cited in the application in full are expressly incorporated herein by reference. Unless otherwise indicated, the materials, reagents and devices involved in the following examples are commercially available. Example

[0172] Example 1 Construction of anti-human HER4 phage immune library and screening of antibodies

[0173] 1.1 Construction of phage immune library

[0174] Total RNA was extracted from human peripheral blood mononuclear cells using the TriZOL method and reverse transcription kit (PrimeScript TMII 1st Strand cDNA Synthesis Kit, Takara), and the extracted RNA was used as a template for reverse transcription to obtain cDNA. Subsequently, cDNA was used as a template, and specific primers were used to amplify the heavy chain variable region family and light chain variable region family gene fragments of the full set of human antibodies by PCR method, and then the light and heavy chain variable region gene fragments were spliced ​​and amplified by overlapping PCR (SOE-PCR) to obtain single-chain antibody variable region gene fragments (scFv). The scFv sequence was connected to the phagemid display vector by enzyme digestion, and then the connection product was electroporated into TG1 competent cells. After the bacterial liquid was infected with helper phage M13KO7, a fully human phage library was obtained.

[0175] 1.2 Preliminary screening and sequence analysis of antibodies

[0176] The obtained scFv library was added to an immunotube coated with a recombinant protein of the human HER4 extracellular domain (amino acid sequence at positions 26-651 as shown in SEQ ID NO:52), incubated at 4°C overnight, blocked, washed and eluted, neutralized with 1M Tris (pH8.0), and infected with TG1 Escherichia coli cells in the logarithmic growth phase. After culture, the bacterial cells were collected and infected with M13KO7 helper phage, and 3-5 rounds of "adsorption-elution-amplification" were repeated to enrich the anti-human HER4 phage display antibody library, pick a single colony for expansion and culture, and perform bacterial liquid PCR identification and monoclonal phage-ELISA identification, followed by gene sequencing to obtain a scFv that can specifically bind to the antigen, the amino acid sequence of VH is shown in SEQ ID NO:21, and the amino acid sequence of VL is shown in SEQ ID NO:41.

[0177] 1.3 Methods for preparing anti-HER4 antibodies

[0178] The VH and VL of the scFv obtained in the above steps were cloned into the human IgG1 heavy chain constant region (shown in SEQ ID NO:53) and the human κ light chain constant region (shown in SEQ ID NO:54), respectively, that is, the nucleotide sequence encoding VH and the human IgG1 heavy chain constant region and the nucleotide sequence encoding VL and the human κ light chain constant region were constructed into a heavy chain expression vector and a light chain expression vector, respectively, and the heavy chain expression vector and the light chain expression vector were mixed at a ratio of 1:1, transiently transfected into Expi-CHOS cells in the logarithmic growth phase, and cultured at 32°C for 10 days. The cell supernatant was collected, the cells were centrifuged and precipitated, filtered, and the antibody was purified by Protein A affinity chromatography, and the antibody was named Ab4268.

[0179] Example 2 Antigen Binding Epitope Analysis of Antibody Ab4268

[0180] The binding epitope of antibody Ab4268 on HER4 was detected by ELISA. The specific method is as follows: After antibody Ab4268 was diluted to 1 μg / mL with PBS, it was added to a 96-well ELISA plate at 100 μL / well and coated overnight at 4°C. After washing the plate with PBS, 200 μL of blocking solution (PBST containing 1% BSA [PBS+0.1% Tween-20]) was added to each well and incubated at room temperature for 1 hour. After washing the plate 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) diluted 1:200 in blocking solution was added to each well and incubated at room temperature for 2 hours. The plate was washed with PBST, and then 100 μL of anti-6×His-HRP antibody (Proteintech) diluted 1:10,000 in blocking solution was added to each well and incubated at room temperature for 1 hour. After washing with PBST, 100 μL of TMB colorimetric solution (Thermo) was added to each well, and incubated at room temperature in the dark for 3-10 minutes, and then 50 μL of stop solution (2M HCl) was added to each well, and then the OD450 value was read using a multifunctional microplate reader (Varioskan, Thermo). The raw data was analyzed using GraphPad Prism 9 software. The test results are shown in Figure 2. Figure 1 As shown, the recognition epitope of Ab4268 is located in the HER4 extracellular domain III.

[0181] Example 3 Optimization of antibody Ab4268

[0182] 3.1 Single-point mutation optimization of Ab4268

[0183] Using the online software abYsis (http: / / abysis.org / abysis / index.html) and MOE software, the amino acid residues with low humanization degree, potential stability risk sites, and amino acid residues with low binding activity in the light and heavy chain variable region sequences of antibody Ab4268 were analyzed, and a total of 42 single-point mutations were designed to increase the degree of humanization and / or reduce the stability risk of the antibody molecule. Based on antibody Ab4268, 42 designed single-point mutations were introduced respectively, and then the antibody light and heavy chain expression plasmid combinations were transiently transfected into ExpiCHO-S cells to express each single-point mutant antibody. The expression level of each single-point mutant antibody and the parent antibody Ab4268 in the culture supernatant after 6 days of transient culture was detected using a molecular interaction instrument (ForteBio, model Oke). Specifically, the culture supernatant was harvested after transient expression in ExpiCHO-S cells and added to a 96-well black board at 200 μL / well. The sample to be tested and the Protein A probe were placed in the ForteBio instrument, and the binding rate of each antibody molecule in the culture supernatant and the Protein A probe was detected by the preset quantitative program of the machine. After each cycle, the probe was immersed in 10mM glycine buffer (pH 1.5) to regenerate the probe and start the next cycle detection. After the detection is completed, the analysis software is used to calculate according to the standard curve to obtain the expression level of each mutant.

[0184] The binding activity of the above mutants to 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) and added to a 96-well U-shaped plate at 50 μL / well. After each mutant to be tested was diluted to 20 μg / mL with FACS buffer, 50 μL per well was added to the above well plate, mixed and placed on ice for 1 hour. Centrifuge to remove the supernatant and wash 3 times with FACS buffer. The secondary antibody AF488-labeled goat anti-human IgG (H+L) (Jackson Immunoresearch) was diluted in FACS buffer at a volume ratio of 1:1000, added to the above well plate at 100 μL per well, mixed and placed on ice for 40 minutes. Centrifuge to remove the supernatant and wash 3 times with FACS buffer. The cells were resuspended in 100 μL / well FACS buffer and detected using a flow cytometer (NovoCyte 3005, Agilent).

[0185] The mutation sites and mutant amino acids, transient expression levels, and FACS assay results of cell binding activity of each mutant are shown in Tables 1 and Figure 2As shown. Compared with the parent antibody Ab4268, the mutations that improve the degree of humanization of the parent antibody, retain or improve the cell binding activity of the parent antibody, and / or significantly reduce the risk of aspartate isomerization and / or asparagine deamidation of the antibody molecule include 17 single point mutations in the antibody heavy chain (H101, H102, H103, H104, H107, H109, ​​H110, H111, H112, H113, H118, H120, H122, H123, H126, H127 and H128), and 6 single point mutations in the antibody light chain (L101, L102, L105, L108, L110 and L112).

[0186] Table 1. Single point mutation, transient expression level and binding activity of Ab4268 to 293T-HER4 cells

[0187]

[0188]

[0189] H is heavy chain; L is light chain; MFI is mean fluorescence intensity

[0190] 3.2 Combination mutation optimization of Ab4268 antibody

[0191] Seven single point mutations in the heavy chain of the above-mentioned antibody (i.e., H104, H107, H109, ​​H122, H123, H126 and H127) and two single point mutations in the light chain of the antibody (i.e., L101 and L105) were selected and introduced into the variable region sequence of antibody Ab4268 in combination to construct antibody molecules containing different combination mutations. Antibody samples were prepared according to the method described in Item 3.1 above, and the expression levels of each combination mutant and the control parent antibody Ab4268 were detected by ForteBio, and the single-point binding activity of each combination mutant to 293T-HER4 cells was detected by flow cytometry.

[0192] The mutation sites, transient expression levels, and binding activities of each combined mutant molecule to 293T-HER4 cells are shown in Tables 2 and Figure 3 The expression levels of each combination mutant were significantly higher than that of the parent antibody Ab4268; except for L201, the cell binding activity of the other combination mutants was comparable to that of the parent antibody.

[0193] Table 2. Combination mutations, transient expression levels and binding activity of Ab4268 to 293T-HER4 cells

[0194]

[0195] MFI: Mean fluorescence intensity

[0196] Example 4 Binding activity of Ab4268 variants to 293T-HER4 cells

[0197] According to the method described in Item 3.1 above, the binding activity of Ab4268 variants H201-L201 and H202-L201 to 293T-HER4 cells was detected by flow cytometry.

[0198] Test results such as Figure 4 As shown in Table 3, it was shown that the cell binding activity of antibodies H201-L201 and H202-L201 was comparable to that of the parent antibody Ab4268.

[0199] Table 3. Binding activity of Ab4268 variants to 293T-HER4 cells

[0200] Antibody ID Cell binding activity EC50 (μg / mL) H201-L201 0.02684 H202-L201 0.03157 Ab4268 0.02834

[0201] Example 5 Effect of Ab4268 on NRG-1-induced HER2 / HER4 receptor dimerization

[0202] Promega's NanoBiT structural complementation reporter system was used to further verify whether Ab4268 would affect NRG-1-induced HER2 / HER4 receptor dimerization. The NanoBiT system consists of a LgBiT subunit and a SmBiT subunit, which can be fused with the test protein respectively; if the two test proteins interact to form a dimer, LgBiT will complement the SmBiT structure to form a functional luciferase, which will react with the substrate to produce a bright luminescent signal. In this example, the nucleotide sequences encoding the extracellular domain and transmembrane domain of human HER2 and HER4 were first inserted into the NanoBiT system pBiT1.3-C and pBiT2.3-C vectors by molecular cloning technology, and then the two plasmids were co-transfected into U2OS cells; the cells were collected 24 hours after transfection, plated in a 96-well white-walled plate, and cultured overnight at 37°C. Antibody Ab4268 and control antibody Pertuzumab were diluted in series, and then mixed with NRG-1 at a volume ratio of 1:1 for later use (final concentration of NRG-1 was 1 nM). Nano-Glo Live Cell Reagent (Promega) was added to a 96-well white-walled plate, and the chemiluminescence value at this time was read as the background; then the prepared samples were added to each well, and the chemiluminescence value was read after incubation at 37°C for 6 minutes. The results are as follows Figure 5As shown in Table 4, both antibody Ab4268 and control antibody Pertuzumab can inhibit NRG-1-induced HER2 / HER4 receptor dimerization, but the inhibitory activity of Ab4268 is significantly lower than that of control antibody Pertuzumab. This result shows that antibody Ab4268 can affect the normal biological function of HER4 receptor by inhibiting NRG-1-induced HER4 receptor dimerization, for example, inhibiting the downstream signal transduction pathway of HER4 receptor.

[0203] Table 4. Inhibitory activity of Ab4268 on NRG-1-induced HER4 receptor dimerization

[0204] Ab4268 Pertuzumab IC50(μg / mL) 8.639 0.3341

[0205] Although the present invention has been described by one or more embodiments, it should be understood that the present invention is not limited to these embodiments, and the present invention description is intended to cover all substitutions, modifications and changes that fall within the spirit and broad scope of the appended claims. All references cited in the present invention are incorporated into the present invention by reference as a whole.

Claims

1. An isolated anti-HER4 antibody or antigen-binding fragment thereof, characterized in that: The anti-HER4 antibody or antigen-binding fragment thereof comprises at least one of the following characteristics: (1) the anti-HER4 antibody or antigen-binding fragment thereof specifically binds to the extracellular domain III of human HER4; and (2) The anti-HER4 antibody or its antigen-binding fragment can inhibit ligand-induced HER4 receptor dimerization and its downstream signal transduction pathway.

2. The anti-HER4 antibody or antigen-binding fragment thereof according to claim 1, comprising the following heavy chain variable region HCDR1, HCDR2 and HCDR3 and light chain variable region LCDR1, LCDR2 and LCDR3: (1) HCDR1 having an amino acid sequence of NYX1X2G (X1=R or W; X2=V or I; i.e., as shown in SEQ ID NO:49), preferably, X1=R, X2=V; (2) HCDR2 having an amino acid sequence of IIX3PX4X5X6DARYSPSFX7G (X3=F or Y; X4=A or G; X5=D, A, F, G, K, S or T; X6=S, A, E, N, T or Y; X7=E or Q; i.e., as shown in SEQ ID NO:50), preferably, X3=F, X4=G, X5=A, F, G, K or S, X6=S or T, X7=Q; (3) NO:12; (4) a LCDR1 having an amino acid sequence of RSSQSLLHSX8X9YNYLD (X8 = N, G, I, L, S, T or V, preferably G or S; X9 = G, A, I, N, P, R, T or V, preferably G, A, N or R), preferably, X8 = G or S, X9 = G, A, N or R; (5) a LCDR2 amino acid sequence as shown in SEQ ID NO:19; and (6) a LCDR3 amino acid sequence as shown in SEQ ID NO:20, 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 said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.

3. An anti-HER4 antibody or antigen-binding fragment thereof as described in claim 1 or 2, comprising: (1) a HCDR1 amino acid sequence as shown in SEQ ID NO: 1; (2) a HCDR2 amino acid sequence as shown in any one of SEQ ID NOs: 2-11; and (3) a HCDR3 amino acid sequence as shown in SEQ ID NO: 12; and (4) a LCDR1 amino acid sequence as shown in any one of SEQ ID NOs: 13-18; (5) a LCDR2 amino acid sequence as shown in SEQ ID NO: 19; and (6) a LCDR3 amino acid sequence as shown in SEQ ID NO:

20.

4. The anti-HER4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising the following heavy chain variable region HCDR1, HCDR2 and HCDR3 and light chain variable region LCDR1, LCDR2 and LCDR3, wherein each CDR comprises: (1) the HCDR1 amino acid sequence set forth in SEQ ID NO:1, the HCDR2 amino acid sequence set forth in any one of SEQ ID NOs:2-11, the HCDR3 amino acid sequence set forth in SEQ ID NO:12; and the LCDR1 amino acid sequence set forth in SEQ ID NO:13, the LCDR2 amino acid sequence set forth in SEQ ID NO:19, the LCDR3 amino acid sequence set forth in SEQ ID NO:20, 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 said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively; or (2) the HCDR1 amino acid sequence set forth in SEQ ID NO:1, the HCDR2 amino acid sequence set forth in SEQ ID NO:2, the HCDR3 amino acid sequence set forth in SEQ ID NO:12; and the LCDR1 amino acid sequence set forth in SEQ ID NO:14-18, the LCDR2 amino acid sequence set forth in SEQ ID NO:19, the LCDR3 amino acid sequence set forth in SEQ ID NO:20, or 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 to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively; or (3) the HCDR1 amino acid sequence shown in SEQ ID NO:1, the HCDR2 amino acid sequence shown in SEQ ID NO:11, the HCDR3 amino acid sequence shown in SEQ ID NO:12; and the LCDR1 amino acid sequence shown in SEQ ID NO:15, the LCDR2 amino acid sequence shown in SEQ ID NO:19, the LCDR3 amino acid sequence shown in SEQ ID NO:20, or 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 to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.

5. The anti-HER4 antibody or antigen-binding fragment thereof of claims 1-4, comprising: a HCDR1 amino acid sequence as set forth in SEQ ID NO: 1, a HCDR2 amino acid sequence as set forth in SEQ ID NO: 2, a HCDR3 amino acid sequence as set forth in SEQ ID NO: 12; and a LCDR1 amino acid sequence as set forth in SEQ ID NO: 13, a LCDR2 amino acid sequence as set forth in SEQ ID NO: 19, a LCDR3 amino acid sequence as set forth in SEQ ID NO: 20, 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 said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively; or the HCDR1 amino acid sequence shown in SEQ ID NO:1, the HCDR2 amino acid sequence shown in SEQ ID NO:11, the HCDR3 amino acid sequence shown in SEQ ID NO:12, and the LCDR1 amino acid sequence shown in SEQ ID NO:13 or 15, the LCDR2 amino acid sequence shown in SEQ ID NO:19, the LCDR3 amino acid sequence shown in SEQ ID NO:20, or 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 to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.

6. An anti-HER4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising a heavy chain variable region VH having said HCDR1, HCDR2 and HCDR3, and a light chain variable region VL having said LCDR1, LCDR2 and LCDR3, wherein the antibody or antigen-binding fragment thereof comprises: a VH having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NOs: 21 to 40, and a VL having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NOs: 41 to 48.

7. The anti-HER4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising the following 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 the VH and VL comprises: (1) 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 VH set forth in any one of SEQ ID NOs: 21-40 and the VL set forth in SEQ ID NO: 41, respectively; or (2) 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 VH set forth in SEQ ID NO:21 and the VL set forth in any one of SEQ ID NOs:42-47, respectively; or (3) 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 VH set forth in SEQ ID NO:39 or 40 and the VL set forth in any one of SEQ ID NO:48, respectively; Preferably, 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 VH shown in SEQ ID NO: 21, 25, 26, 27, 34, 36, 37, 39 or 40 and the VL shown in SEQ ID NO: 41, respectively; 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 VH shown in SEQ ID NO: 21 and the VL shown in SEQ ID NO: 42 or 43, respectively.

8. A nucleic acid encoding the anti-HER4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. An expression vector capable of expressing the nucleic acid according to claim 8. 10 . A host cell comprising the nucleic acid of claim 8 or the expression vector of claim 9 .

11. A method for preparing the anti-HER4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 using the host cell according to claim 10, comprising: (i) expressing the anti-HER4 antibody or antigen-binding fragment thereof in the host cell, and (ii) isolating the anti-HER4 antibody or antigen-binding fragment thereof from the host cell or cell culture.

12. A bispecific molecule or a multispecific molecule, comprising an anti-HER4 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7, and having binding specificity to an Fc receptor, or a cancer-related antigen or an immune checkpoint protein antigen, wherein the cancer-related antigen is selected from HER2, HER3, VEGF, VEGFR, c-Met, TFR (transferrin receptor), IGF-1R (human insulin-like growth factor I receptor); the immune checkpoint protein antigen is selected from PD-1, PD-L1, CTLA-4, CD137, CD47.

13. The bispecific antibody or multispecific antibody of claim 12, further comprising a third binding specificity, which may be an anti-enhancement factor (EF) portion.

14. An immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus, characterized in that: It comprises the anti-HER4 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-7, or the bispecific antibody or multispecific antibody of claim 12 or 13, or the immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus of claim 14, and a pharmaceutically acceptable carrier.

16. A drug kit comprising the pharmaceutical composition of claim 15, and one or more other therapeutic agents selected from the group consisting of signaling pathway regulators, cell cycle inhibitors, and inflammatory response regulators.

17. Use of an anti-HER4 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7 for preparing a pharmaceutical composition or formulation for treating or preventing a HER4-related disease or condition.

18. A method for treating or preventing a HER4-related disease or condition, the method comprising administering to a subject the anti-HER4 antibody or antigen-binding fragment of any one of claims 1 to 7, or the pharmaceutical composition of claim 15, or the drug kit of claim 16.

19. The use according to claim 17 and the method according to claim 18, wherein The diseases or conditions include cancer and other HER4-related diseases, the cancer includes glioma, and the other HER4-related diseases include glial proliferative diseases, for example, stroke, Alzheimer's disease, viral encephalitis, traumatic brain injury, multiple sclerosis, epilepsy; the subject may be a human, a non-human primate or other mammal (such as a dog), etc.

20. The method of claim 18 or 19, wherein: The method can inhibit ligand-induced HER4 dimerization in the subject, wherein the ligand includes NRG-1 to 4, preferably NRG-1.

21. A method for detecting and / or measuring HER4 or HER4-expressing cells in a sample, and a method for screening patients with HER4-related diseases who are responsive to treatment with an anti-HER4 antibody or antigen-binding fragment thereof as described in any one of claims 1-7, comprising incubating the anti-HER4 antibody or antigen-binding fragment thereof as described in any one of claims 1-7 with the sample or a biological sample isolated from the patient, and detecting whether the antibody or antigen-binding fragment thereof binds to the sample or biological sample.

Images