CN119823275A - Anti-HER 4 antibody and preparation method and application thereof - Google Patents

Abstract

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

Description

Anti-HER 4 antibody and preparation method and application thereof

Technical Field

The present invention relates to anti-HER 4 antibodies or antigen-binding fragments thereof, polynucleotides encoding the antibodies or antigen-binding fragments thereof, expression vectors and host cells, pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof, and uses or methods thereof for treating a subject.

Background

The receptor tyrosine kinase ErbB/HER family includes ErbB1 (also known as EGFR or HER 1), erbB2 (HER 2, c-Neu), erbB3 (HER 3) and ErbB4 (HER 4). ErbB/HER family receptors activate their mediated downstream signaling pathways through dimerization, including RAS-RAF-MEK-ERK, PI3K-AKT and JAK-STAT pathways, and are thus involved 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 about 180kDa and consists of an extracellular domain (Extracellular Domain, ECD), a transmembrane domain (Transmembrane Domain) and an intracellular tyrosine kinase domain (Intracellular Tyrosine Kinase Domain) that bind to the ligand. HER4 is composed of ligands including neuregulin 1-4 (neuregulin-1-4, NRG-1-4) (J.R. Toggweiler, nature 1993,366:473-475; K.L. Carraway et al, nature 1997,387:512-516; D.Zhang et al, proc NATL ACADSCI USA,1997,94:9562-9567; D.Harari et al, oncogene 1999, 18:2681-2689), heparin binding epidermal growth factor (HB-EGF) (K.Elenius et al, EMBO J1997, 16:1268-1278), beta-acetylglobulin (betacellulin, BTC) (D.J. Riese et al, oncogene 1996, 12:345-353) and epidermal regulatory protein (epiregulin) (T.Komuraasaki et al, oncogene 1997, 15:2841-2848) activate and form homodimers or heterodimers with other ErbB/HER family member proteins. Although HER4 is expressed in a variety of tumors (e.g., blastoma, breast, lung, melanoma, pancreatic, gastric, colorectal, ovarian, and bladder cancers) (Hollm e n M et al, future Oncol 2010, 6:37-53), several reports have shown that HER4 is down-regulated in tumors, or that its expression is associated with a good prognosis in tumors (SRINIVASAN R et al, CLIN CANCER RES 1998,5:2877-2883; witton CJ et al, J Pathol 2003, 200:290-297). Furthermore, HER4 is considered a tumor suppressor gene in breast cancer (Wang J et al Oncotarget 2016, 7:76693-76703). Thus, the role of HER4 in the development and progression of tumors remains to be further elucidated. At present, HER4 has been shown to play a key role in the development and maintenance of function of the heart and nervous system (F.E.Jones et al, J Cell Biol 1999,147:77-87; H.Tidcombe et al, proc NATL ACAD SCI USA 2003,100:8281-8286; T.Juntila et al, trends Cardiovasc Med 2000,10:304-310; A.Buonanno et al, curr Opin Neurobiol 2001, 11:287-296).

Accordingly, it is desirable in the art to develop a neutralizing antibody capable of specifically binding HER4 to more accurately treat HER 4-related diseases or conditions.

Disclosure of Invention

The invention provides an anti-HER 4 antibody, a preparation method thereof and application thereof in diseases or symptoms related to HER 4.

In one aspect, the invention provides an antibody or antigen binding fragment capable of specifically binding HER 4.

In some embodiments, the anti-HER 4 antibody or antigen-binding fragment thereof specifically binds to human HER4 extracellular domain III and blocks ligand-induced activation of HER4 receptor. In some embodiments, the activity of the antibody or antigen binding fragment thereof to specifically bind to a cell expressing human HER4 is detected by flow cytometry.

In some embodiments, the anti-HER 4 antibody or antigen-binding fragment thereof has an inhibitory effect on HER4 receptor and downstream signal transduction pathways thereof. In one embodiment, the anti-HER 4 antibody or antigen binding fragment thereof is capable of inhibiting ligand-induced HER4 receptor dimerization and downstream signaling pathways thereof, said ligand comprising NRG-1-4, preferably NRG-1. In some embodiments, the antibody or antigen binding fragment thereof has an NRG-1 induced HER2/HER4 receptor dimerization inhibition activity (IC 50 value) of no more than 1 μg/mL, preferably no more than 0.5 μg/mL, as measured by living cell bioluminescence.

In some embodiments, the anti-HER 4 antibody or antigen binding fragment thereof is not cross-reactive with other members of the human ErbB/HER family, such as EGFR, HER2, and/or HER 3.

In some embodiments, the HER4 antibody or antigen binding fragment thereof is capable of cross-reacting with HER4 of other species (e.g., mouse, rat, canine).

In another aspect, the invention provides methods of screening for an anti-HER 4 antibody or antigen-binding fragment thereof of the invention, including, but not limited to, hybridoma technology, phage display technology, single lymphocyte gene cloning technology.

In another aspect, the invention provides an isolated nucleic acid molecule (also referred to as a "polynucleotide") encoding an anti-HER 4 antibody or antigen-binding fragment thereof of the invention, as well as expression vectors comprising said nucleic acid and host cells comprising the nucleic acid or expression vector. The invention also relates to a method of producing an anti-HER 4 antibody or antigen-binding fragment thereof of the invention using the host cell, the method comprising culturing the host cell and recovering the antibody or antigen-binding fragment thereof from the culture medium.

In another aspect, the invention provides a bispecific or multispecific molecule, immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus comprising an anti-HER 4 antibody or antigen binding fragment thereof of the invention.

In another aspect, the invention provides a pharmaceutical composition comprising an anti-HER 4 antibody or antigen-binding fragment thereof, a bispecific or multispecific antibody, an immunoconjugate, a chimeric antigen receptor, an engineered T cell receptor, or an oncolytic virus of the invention, and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a kit comprising an effective amount of an anti-HER 4 antibody of the invention, or an antigen-binding fragment thereof, or a pharmaceutical composition herein, and optionally at least one additional therapeutic agent.

In a further aspect, the invention provides the use of an anti-HER 4 antibody or antigen binding fragment thereof of the invention in the manufacture of a pharmaceutical composition or formulation for the treatment and/or prophylaxis of a HER4 related disease or disorder, in the manufacture of a diagnostic reagent for the diagnosis of a HER4 related disease or disorder.

In another aspect, the invention provides a method of treating and/or preventing a HER 4-related disease or disorder, the method comprising administering to a subject an effective amount of an anti-HER 4 antibody or antigen-binding fragment thereof, a pharmaceutical composition, or a kit of parts of the invention.

In another aspect, the invention provides a method of inhibiting HER4 receptor and its downstream signal transduction pathway comprising inhibiting ligand-induced dimerization of HER4 in vitro or in vivo using an anti-HER 4 antibody or antigen binding fragment, pharmaceutical composition or kit of the invention, said ligand comprising NRG-1-4, preferably NRG-1.

In another aspect, the invention provides a method of detecting the level of HER4 in a test sample (e.g., a biological sample, e.g., serum, tissue) comprising (a) contacting the test sample with an antibody or antigen-binding fragment thereof of the invention, and (b) detecting binding of the antibody or antigen-binding fragment thereof to HER4 in the test sample.

Other features and advantages of the present disclosure will be apparent from the following drawings and detailed description, which should not be taken as limiting the scope of the application, and modifications, which will occur to those skilled in the art, are intended to be included within the spirit of the application and the scope of the appended claims. All references, including publications, patents, and patent applications cited in this disclosure are incorporated by reference in their entirety.

Drawings

Figure 1. Detection of the binding activity of anti-HER 4 antibody Ab4261 to human ErbB/HER family protein members (including EGFR, HER3 and HER 4) ectodomain proteins and chimeric HER4 ectodomain III recombinant proteins (her4_diii) using ELISA.

Figure 2. Binding activity of antibody Ab4261 optimized molecules containing the combinatorial mutations and control antibodies (maternal antibody Ab 4261) to HER4 expressing 293T cells was detected using flow cytometry.

FIG. 3 detection of the binding activity of the optimized molecule (G202-K201) of antibody Ab4261 to human ErbB/HER family protein members (including EGFR, HER2, HER3 and HER 4) by ELISA.

FIG. 4 detection of the binding activity of the optimized molecule (G202-K201) of antibody Ab4261 to HER4 of different species (including human, mouse, rat and canine) by ELISA.

Fig. 5 the binding activity of the optimized molecules of antibody Ab4261 (G201-K203 and G202-K201) and the control antibody (maternal antibody Ab 4261) to HER4 expressing 293T cells was detected using flow cytometry.

FIG. 6 effect of optimized molecule (G202-K201) of antibody Ab4261 on NRG-1 induced dimerization of HER2/HER4 receptor, control antibodies include maternal antibodies Ab4261 and Pertuzumab.

Detailed Description

Definition of the definition

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.

The terms "HER4", "HER4 receptor" and "HER4 protein" are used interchangeably herein, and the proteins are also referred to as ErbB4, c-ErbB-4 or p180ErbB4. Unless otherwise indicated that it is from a non-human species, "HER4" as expressed herein refers to a human

Any native form of HER4, which may be the amino acid sequence shown as SEQ ID No. 86 and/or the full length HER4 amino acid sequence shown as UniProtKB accession No. Q15303.1, may be expressed naturally by cells (including cardiomyocytes, breast cells, tumor cells) or by cells transfected with the HER4 gene or cDNA. The term includes naturally occurring HER4 allelic 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.

Full length HER4 protein consists of 1308 amino acids, wherein amino acid residues 1-25 are signal peptides, amino acid residues 26-651 are extracellular domains (extracellular domain, 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, subdomain 1

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

Herein, a "HER 4 expressing cell" may be a naturally occurring cell or cell line (e.g., cardiomyocyte, breast cell, tumor cell) or may be recombinantly produced by introducing a nucleic acid encoding HER4 into a host cell.

"Antigen binding domain" or "antigen binding region" or "epitope binding domain" are used interchangeably herein to refer to a specific region on an antibody or antigen binding fragment or derivative thereof that is directly involved in specific interactions with a target antigen, e.g., by binding, steric hindrance, stabilization/destabilization, spatial distribution, etc., to interact with the target antigen until dynamic equilibrium is reached. The term "antigen binding domain" as used herein also refers to a specific region on the antibody or antigen binding fragment or derivative thereof that interacts with a specific epitope on the HER4 receptor to achieve dynamic equilibrium of binding between the two by binding, steric hindrance, stabilization/destabilization, spatial distribution, and the like.

An "antibody" refers to a polypeptide or protein encoded by substantially an immunoglobulin gene or genes, or fragments thereof, that is capable of specifically recognizing and binding an antigen. Putative immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. The heavy chains are classified as gamma, mu, alpha, delta or epsilon, which in turn define immunoglobulin classes (class) IgG, igM, igA, igD and IgE, respectively, several of which may be further classified as subclasses (subclasses)/isotypes (isotypes), e.g., igG1, igG2, igG3, igG4, igA1, igA2. typical immunoglobulin (e.g., antibody) building blocks include tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" (about 50-70 kD) chain. 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 typically comprises 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 typically comprises one domain, CL. Pairing of V _H and V _L together forms a single antigen binding site. Endogenous V _L is encoded by gene segments V (variable) and J (joined), and endogenous V _H is encoded by V, D (diversity) and J. v _L or V _H each include a hypervariable Region (Region of Hypervariability) or complementarity determining regions (Complementarity Determining Region, CDRs) and Framework Regions (FR). The term "variable region" or "V region" is used interchangeably and refers to a heavy chain variable region or a light chain variable region arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from amino-terminus to carboxy-terminus. The term "J region" refers to a subsequence encoding the C-terminal portion of the variable region comprising CDR3 and FR 4. The V or J region may be naturally occurring, recombinant or synthetic. The antibody light chain variable region and/or antibody heavy chain variable region may sometimes be collectively referred to herein as an "antibody variable region," and the antibody light chain and/or antibody heavy chain may be collectively referred to herein as an "antibody chain. In certain embodiments, the FR of an antibody or antigen binding fragment thereof provided herein may be identical to a human germline sequence, or may be modified naturally or artificially.

The positions of the CDRs and FRs can be determined using a variety of definition methods well known in the art, for example, kabat, chothia, IMGT and contacts (see, e.g., kabat et al, sequences of Proteins of Immunological Interest,1991 fifth edition, U.S. device of HEALTH AND Human Services, NIH publication No. 91-3242; johnson et al, nucleic Acids Res2001,29:205-206;Chothia&Lesk,J Mol Biol 1987,196:901-917; chothia et al, nature 1989,342:877-883; chothia et al, J Mol Biol 1992,227:799-817; al-Lazikani et al, J Mol Biol 1997,273:927-748; lefranc MP et al, nucleic ACIDS RESEARCH 1999,27:209-212; macllum RM et al, J Mol Biol 1996, 262:732-745). The definition of antigen binding sites is also described in 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, 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, edit Protein Structure Prediction, oxford University Press, oxford 1996,141-172. The present application encompasses any of the definition methods for determining the CDRs in an anti-HER 4 antibody or antigen-binding fragment thereof of the application and table 1 shows the position numbers of the antibody CDR amino acid sequences determined using the different definition methods. The exact number of amino acid residues covering a particular CDR varies with the sequence of the CDR. Where the amino acid sequence of the antibody variable region is specified, one skilled in the art can determine the CDRs of the antibody by conventional methods including, but not limited to, the definition.

TABLE 1 CDR's determined by different definition methods ¹

¹ All CDR definitions in Table 1 are numbered according to the numbering system proposed by Kabat et al (Kabat et al Sequences of Proteins of Immunological Interest, fifth edition 1991, U.S. section of HEALTH AND Human Services).

Furthermore, kabat et al define a numbering system for variable region sequences that is applicable to any antibody. One of ordinary skill in the art can explicitly apply this "Kabat numbering" system to variable region sequences of any antibody without relying on any experimental data other than the antibody sequence itself to determine the variable region sequence. Unless otherwise indicated, the numbering of specific amino acid residue positions in the variable region of the antigen binding domain of an anti-HER 4 antibody of the application is determined according to the Kabat numbering system.

Antibodies exist as intact immunoglobulins or as a number of fragments produced by digestion with various peptidases. Although various antibody fragments are defined in terms of digestion of intact antibodies, the skilled artisan will appreciate that such fragments may be synthesized de novo by chemical cleavage methods or by using recombinant DNA methods. Herein, the term "antigen binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment") refers to an antibody fragment consisting of an antibody portion containing one or more CDRs or any other antibody fragment capable of binding an antigen (e.g., HER4 or extracellular domain of HER 4) but not having an intact antibody structure. Antigen binding fragments may have the same activity as whole antibodies to specifically bind antigen. In certain embodiments, an antigen binding fragment may contain one or more CDRs from a particular human antibody that are grafted to framework regions 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 disulfide bonds in the hinge region, (iii) an "Fv" fragment, consisting of the VL and VH domains of an antibody single arm, which is the smallest antibody fragment containing the complete antigen binding site, (iv) an "Fd" fragment, consisting of the VH and CH1 domains, (v) "single chain Fv antibody (scFv)", "single chain antibody" or "scFv molecule", referring to an engineered antibody consisting of the light chain variable region and the heavy chain variable region linked directly or via a single peptide chain (Huston JS et al, proc NATL ACAD SCI USA)

1988,85:5879-5883; Bird et al, science 1988, 242:423-426), in addition, single chain antibodies include "linear antibodies" comprising a pair of Fv segments in tandem (VH-CH 1-VH-CH 1) that together with a complementary light chain polypeptide form a pair of antigen binding regions (Zapata et al, protein Eng 1995,8:1057-1062; U.S. Pat. No. 5,641,870); (vi) a "dAb" fragment (Ward et al, nature 1989,341:544-546; PCT publication No. WO 90/05144A 1) comprising a single variable domain, e.g., a VH domain. Single domain antibodies (sdabs) are independent immunoglobulin domains, (vii) "diabodies" are bivalent bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but the peptide linker used is too short to pair the two domains on the same chain, thus forcing the two domains to pair with the complementary domains of the other chain, forming two antigen binding sites (Holliger P et al, proc NATL ACAD SCI USA 1993,90:6444-6448; poljak et al, structures 1994,2:1121-1123; patent EP404097; PCT publication No. WO 93/11161).

As used herein, the term "Fc region" or "Fc domain" refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region, such as an immunoglobulin heavy chain constant region other than the first constant region (CH 1). For IgG, the Fc region may comprise immunoglobulin domains CH2 and CH3 and a hinge region between CH1 and CH 2. The Fc region as used herein includes native sequence Fc regions and/or Fc region variants, and may be part of an anti-HER 4 antibody of the application. It is understood in the art that the boundaries of the Fc region may vary, however, a 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 seen in Kabat et al, NIH Publication1991,91-3242,National Technical Information Service.

The term "anti-HER 4 antibody" or "antibody that specifically binds HER 4" refers to any form of antibody or fragment thereof that specifically binds HER4, and encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and biofunctional antibody fragments, so long as the fragment specifically binds HER 4. The anti-HER 4 antibodies of the invention are preferably antibodies or antibody fragments that specifically bind to the extracellular domain of HER 4.

In this context, the term "specific binding" or "binding specificity" or "pair of" means that the binding reaction 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 distinguishes between those undesired or non-specific interactions. For example, an antibody that specifically binds to a target molecule (which may be an antigen) is one that has greater affinity, greater binding activity, easier binding, and/or longer binding duration when bound to the target molecule than when bound to other non-target molecules. A variety of immunoassay methods can be used to select antibodies that specifically bind to a particular protein, such as solid phase ELISA immunoassays, flow cytometry fluorescence sorting (FACS), cytofluorimetry, or surface plasmon resonance (Surface Plasmon Resonance, abbreviated SPR) techniques to determine the binding of an antibody or antigen binding fragment thereof herein to a target antigen/protein. Typically, the specific or selective binding reaction of the antibody or binding agent with the antigen will produce a signal at least twice background, more typically at least 10-100 times background, under the set assay conditions and will not substantially bind in significant amounts to other antigens/proteins present in the sample.

Herein, the term "monoclonal antibody" refers to an antibody obtained from a population of antibodies that are substantially homogeneous, i.e., the individual antibodies that make up the population are identical except for natural mutations that may be present in minor amounts. Monoclonal antibodies exhibit specificity and affinity for binding to a particular epitope. Monoclonal antibodies can be prepared by the hybridoma method described first by Kohler et al, nature 1975,256:495, can also be prepared by recombinant DNA methods (see U.S. Pat. No. 4,816,567), and can also be isolated from phage antibody libraries, for example, by the techniques described in Clackson et al, nature 1991,352:624-628; marks et al, J Mol Biol 1991, 222:581-597.

The term "epitope" as used herein refers to the protein determinant (protein determinant), an antigen moiety capable of being recognized by an antibody and specifically bound. Epitopes are generally composed of surface groups of molecules such as amino acids or sugar side chains, and generally have specific three-dimensional structural features, as well as specific charge characteristics.

The term "affinity" or "binding affinity" refers to the inherent binding capacity of an interaction between a molecule (e.g., a receptor or antigen) and its counterpart (e.g., a ligand or antibody), i.e., the strength of the sum of all non-covalent interactions. As used herein, unless otherwise stated, "binding affinity" is an intrinsic binding affinity that is used to reflect one-to-one interactions 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 counterpart Y can be generally expressed by the equilibrium dissociation constant (K _D), which is the ratio of the dissociation rate constant (K _dis or K _off) to the association rate constant (Ka or Kon). Affinity can be measured by common methods known in the art, including the methods used in the present invention.

The terms "cross-reactive", "cross-reactive" or "cross-reactive" are used interchangeably herein to 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. Thus, antibodies are cross-reactive if they bind to antigens other than the target antigen. Cross-reactive epitopes typically contain a number of amino acid sequences that are homologous to the target epitope, or similar structural features, and in some cases may bind antibodies more closely than the target epitope. An antibody is considered "highly specific" for a target epitope if it does not bind to sequences or structures other than the epitope.

As used herein, the term "fused" or "fused" when used with respect to an amino acid sequence (e.g., a peptide, polypeptide, or protein) refers to the combination of two or more amino acid sequences into a single amino acid sequence that does not occur in nature by chemical bonding or recombinant means. The fusion amino acid sequence may be produced by gene recombination of two encoding polynucleotide sequences and may be expressed by a method of introducing a construct containing the recombinant polynucleotide into a host cell.

Herein, the term "antibody variant" or "antibody variant" refers to an antibody polypeptide sequence that contains at least one amino acid mutation in a reference antibody variable region. The variant may be substantially homologous or substantially identical to the unmodified antibody. In some embodiments, one, two, three, four, five, and/or six CDRs of an inventive anti-HER 4 antibody 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, increasing the specificity for a target protein/antigen (e.g., HER 4), 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 an anti-HER 4 antibody of the application 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, increasing the specificity for a target molecule (e.g., HER 4), 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-HER 4 antibodies of the application to increase the degree of humanization of the antibody or antigen-binding portion, to increase specificity for a target molecule (e.g., HER 4), to bind activity, to increase yield, and/or to increase stability (e.g., to reduce or eliminate the risk of aspartic acid isomerization and/or asparagine deacylation). In some embodiments, the amino acid mutation comprises an amino acid substitution, deletion, insertion, or any combination thereof.

Where amino acid substitutions include conservative amino acid substitutions that involve substitution with another amino acid in the same class (e.g., chemically or functionally similar), and non-conservative amino acid substitutions that involve substitution with a different class of amino acid (chemically or functionally dissimilar). One of ordinary skill in the art may make conservative or non-conservative amino acid substitutions based on the similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, (I) nonpolar (hydrophobic) amino acids include alanine (Ala, A), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), proline (Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W) and methionine (Met, M), (ii) polar neutral amino acids include glycine (Gly, G), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N) and glutamine (Gln, Q), (iii) positively charged (basic) amino acids include arginine (Arg, R), lysine (Lys, K) and histidine (His, H), and (iv) negatively charged (acidic) amino acids include aspartic acid (Asp, D) and glutamic acid (Glu, E). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein, and non-conservative amino acid substitutions may result in a substantial change in the properties or function of the protein. Although the site or region at which the mutation in the amino acid sequence is introduced may be predetermined, the potential change in protein properties or function due to non-conservative substitution is not anticipated. In some embodiments, the anti-HER 4 antibodies of the invention unexpectedly achieve significant changes in function and performance, such as binding activity, molecular stability, etc., through non-conservative amino acid substitutions.

In this context, the terms "identical" or "identity" or "percent sequence identity" are used interchangeably with respect to two or more polypeptide sequences, and refer to the percentage of the number of amino acid residues in a candidate sequence that are identical to the reference sequence over the total number of amino acid residues in the candidate sequence when amino acid sequence alignments are performed and when the necessary spacing is introduced to maximize the number of identical amino acids. Conservative substitutions of the amino acid residues may or may not be considered the same residue. The sequences may be aligned to determine percent sequence identity of amino acid sequences by means disclosed in the art, for example BLASTp, clustalW2 (see also HIGGINS DG et al, methods enzymes 1996,266:383-402; larkin MA et al, bioinformation 2007, 23:2947-2948) and ALIGN or Megalign (DNASTAR) software. The person skilled in the art can use the default parameters of the tool or adjust the parameters appropriately according to the alignment requirements, for example by choosing an appropriate algorithm for sequence alignment.

Reference herein to an "amino acid sequence that reduces or eliminates the risk of deamidation" refers to a sequence comprising amino acid residues that are prone to deamidation replaced by amino acid residues that are less prone or not prone to deamidation. Deamidation is a chemical reaction in which the amide function in the asparagine or glutamine side chain is removed or converted to another function. Typically, asparagine (Asn) can be converted to aspartic acid or isoaspartic acid, and glutamine (Gln) can be converted to glutamic acid or pyroglutamic acid. Deamidation is easily performed by inclusion of Asn-Gly, asn-Ser or Asn-Thr sites in the amino acid sequence, and asparagine is more easily deamidated than glutamine. Deamidation of asparagine and/or glutamine can alter the structure of an antibody and its stability and/or function (i.e., antibody-antigen binding), and thus, there is a need 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 variable region of the antibody that are susceptible to deamidation (Jasmin F Sydow et al, PLoS ONE 2014,9 (6): e 100736), and substituting amino acid residues that are less susceptible or less susceptible to deamidation.

As used herein, the term "aspartate isomerized" amino acid sequence refers to a sequence comprising aspartate residues which are susceptible to isomerization. Isomerization causes conformational changes in the antibody, thereby altering the surface charge of the antibody, resulting in the development of antibody charge heterogeneity. Preferably, the antibodies of the application do not comprise an aspartic acid isomerisation site. Aspartic acid isomerisation is readily seen in D-G sequences, and it has been reported that isomerisation may also occur in D-H or D-S sequences, resulting in iso-aspartic acid residues, which may lead to a reduced stability of the polypeptide chain due to the introduction of a linkage to the polypeptide chain (also known as iso-aspartic acid effect), and possibly also to a reduced antibody-antigen binding affinity.

As used herein, the term "isolated" when referring to a protein means that the protein is substantially free of other cellular components to which it is associated in its native state, preferably in a homogenous state, e.g., the isolated protein may be removed from its natural or natural environment. The isolated protein may be lyophilized or in aqueous solution. Typically, purity and uniformity can be determined using analytical chemistry techniques (e.g., polyacrylamide gel electrophoresis or high performance liquid chromatography). The protein is substantially purified in the isolated preparation. The term "purified" means that the protein produces substantially only one band in the 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 purposes of the present application, recombinant proteins expressed in host cells are considered isolated in some embodiments, as well as natural or recombinant proteins that are separated, fractionated (fractionate), or partially or substantially purified by any technique well known to those of skill in the art. In some embodiments, an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds HER4 is substantially free of antibodies that specifically bind antigens other than HER 4). Furthermore, the isolated antibodies may be substantially free of other cellular material and/or chemicals. In some embodiments, recombinant polynucleotides encoding a polypeptide or protein of the application (e.g., an anti-HER 4 antibody) contained in a vector are considered isolated. Other examples of isolated polynucleotides include recombinant polynucleotides contained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.

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 that is modified by covalent attachment of a second molecule, e.g., by attachment of a heterologous polypeptide, or by glycosylation, acetylation, phosphorylation, or the like. The definition of "derivative" may also include polypeptides of analogs of one or more amino acids (e.g., unnatural amino acids, etc.), as well as other modifications (natural and non-naturally occurring) known in the art.

As used herein, the term "expression vector" or "vector" refers to a vehicle into which a polynucleotide or nucleic acid encoding a protein can be operably inserted and expressed. Vectors may be used to transform, transduce or transfect host cells such that elements of genetic material carried thereby are expressed within the host cells.

As used herein, the term "host cell" refers to a cell that is an exogenous polynucleotide, nucleic acid, and/or vector. Host cells include "transformants" and "transformed cells" which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may comprise the mutation. Mutant progeny having the same function or biological activity, selected or selected in the initially transformed cells, are encompassed by the present invention.

The terms "subject," "patient," or "individual" are used interchangeably herein and include, but are 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, emus, or ducks) or fish, and non-mammalian invertebrates. In some embodiments, the subject and pharmaceutical compositions to which the use or method of the invention relates are for (prophylactically and/or therapeutically) treating a non-human animal.

In this context, "treating" or "treatment" of a disease or condition refers to alleviating (alleviating or to alleviate) a disease or condition, reducing the rate at which a disease or condition develops or the risk of developing a disease or condition, or delaying the development of a condition associated with a disease or condition, reducing or terminating a condition associated with a disease or condition, producing a complete or partial reversal of a disease or condition, curing a disease or condition, or a combination thereof.

As used herein, a "HER 4-related" disease or condition refers to any disease or condition caused by, exacerbated by, or otherwise associated with an increase or decrease in HER4 expression or activity. In certain embodiments, the HER 4-related disease is cancer, such as glioma. In certain embodiments, the HER 4-related disease is a gliosis disease.

The term "therapeutically effective amount", "effective dose" or "effective amount" refers to the amount or concentration of an active ingredient or agent effective to achieve prevention or amelioration of symptoms associated with a disease or disorder, alleviation or relief of the severity of a disease or disorder, and/or delay or stop of progression of a disease, at a desired dosage and for a desired period of time. The therapeutically effective amount of the formulations, antibodies or antigen-binding fragments thereof, or compositions of the invention may 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 elicit a desired response in the individual. A therapeutically effective amount may also be considered to be a therapeutically beneficial effect of a formulation, antibody or antigen-binding fragment thereof, or composition significantly exceeding any toxic or detrimental effect that it causes.

As used herein, the term "pharmaceutically acceptable" or "pharmaceutically acceptable" refers to carriers, vehicles, diluents, excipients and/or salts as indicated, which are generally compatible chemically and/or physically with the other ingredients in the formulation, and physiologically compatible with the subject.

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

The term "and/or" is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.

As used herein, the terms "comprises" or "comprising" or "includes" or "having" or "including" are used interchangeably to mean including the recited elements, integers or steps, but not excluding any other elements, integers or steps. In this document, when the terms "comprises" or "comprising" or "includes" or "having" or "involving" are used, unless otherwise indicated, the terms "comprising" or "including" or "having" or "including" are also intended to cover the circumstance that the recited elements, integers or steps are included.

As used herein, the term "optional" means the presence or absence of a subject whose modification, e.g., "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.

Reference herein to "some embodiments," "one embodiment," "one particular embodiment," or "a particular embodiment," or a combination thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the application. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

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

All patents, patent applications, and other publications are expressly incorporated herein by reference for the purpose of description and disclosure. These publications are provided solely for their disclosure prior to the filing date of the present application. All statements as to the date or representation as to 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 or contents of these documents.

Various aspects of the application will be described in more detail in the following sections.

1. Anti-HER 4 antibodies and antigen binding fragments thereof

In one aspect, the application provides an isolated anti-HER 4 antibody or antigen-binding fragment thereof that is capable of specifically recognizing and binding to extracellular domain III of human HER4 and is capable of blocking ligand-induced dimerization of HER4 receptors.

The anti-HER 4 antibody or antigen binding fragment thereof specifically binds to the extracellular domain of human HER4 and has high binding activity. In some embodiments, the binding activity of the antibody or antigen binding fragment thereof to HER4 expressing 293T cells is detected using flow cytometry with a high Mean Fluorescence Intensity (MFI) value.

The anti-HER 4 antibodies or antigen binding fragments thereof of the invention have an inhibitory effect on HER4 receptor and its downstream signaling pathways. In some embodiments, the anti-HER 4 antibody or antigen binding fragment thereof is capable of inhibiting ligand-induced HER4 receptor dimerization and downstream signaling pathways thereof, the ligand comprising NRG-1-4, preferably NRG-1. In some embodiments, the antibody or antigen binding fragment thereof has an NRG-1 induced HER2/HER4 receptor dimerization inhibition activity (IC 50 value) of no more than 1 μg/mL, preferably no more than 0.5 μg/mL, as measured by living cell bioluminescence.

The anti-HER 4 antibodies or antigen binding fragments thereof of the invention are not cross-reactive with other members of the human ErbB/HER family (including EGFR, HER2 and HER 3). The extent of binding of the anti-HER 4 antibody or antigen binding fragment thereof to a non-target protein (e.g., EGFR, HER2, or HER 3) is more than about 10% less than the binding of the antibody or antigen binding fragment thereof to HER4, as determined by methods known in the art, such as ELISA, fluorescence Activated Cell Sorting (FACS) analysis, or Radioimmunoassay (RIA).

The anti-HER 4 antibodies or antigen binding fragments thereof of the invention have cross-reactivity with HER4 of other species (e.g., mouse, rat, canine) which is comparable to the binding activity of human HER 4.

The anti-HER 4 antibodies of the application may also optionally comprise Fab, fab ', F (ab') 2, fv, scFv, scFv-Fc, single domain antibodies (sdabs), or of IgG type. The anti-HER 4 antibodies of the application may be murine, chimeric, humanized, fully human, monoclonal, polyclonal, monospecific, bispecific, multispecific, and antibody fragments so long as the antibodies specifically recognize and bind to the human HER4 extracellular domain and have no cross-reactivity with other members of the ErbB/HER family. In some embodiments, the anti-HER 4 antibody is selected from a fully human anti-HER 4 antibody or an optimized antibody or antibody fragment thereof.

In another aspect, the invention provides an anti-HER 4 antibody or antigen-binding fragment thereof shown in table 2 comprising VH and VL. In some embodiments, an anti-HER 4 antibody or antigen-binding fragment thereof of the invention specifically binds to HER4 receptor extracellular domain III, which comprises the VH amino acid sequence shown in SEQ ID No. 36, and/or the VL amino acid sequence shown in SEQ ID No. 59.

In some embodiments, an anti-HER 4 antibody or antigen binding fragment thereof of the invention comprises one or more CDRs in the VH amino acid sequence set forth in SEQ ID NO:36 or the amino acid sequences set forth in SEQ ID NOs:1, 2 and 21, or variants thereof, including optimized antibodies or any other variants described herein.

In some embodiments, an anti-HER 4 antibody or antigen binding fragment thereof of the invention comprises one or more CDRs in the VL amino acid sequence set forth in SEQ ID NO:59 or the amino acid sequences set forth in SEQ ID NOs:23, 30 and 34, or variants thereof, including optimized antibodies or any other variants described herein.

In some embodiments, an anti-HER 4 antibody or antigen binding fragment thereof of the invention comprises one or more CDRs of the VH amino acid sequence set forth in SEQ ID NO:36 or amino acid sequences set forth in SEQ ID NOs:1, 2 and 21, or variants thereof, and one or more CDRs of the VL amino acid sequence set forth in SEQ ID NO:59 or amino acid sequences set forth in SEQ ID NOs:23, 30 and 34, or variants thereof, including optimized antibodies or any other variants described herein.

In some embodiments, the anti-HER 4 antibody or antigen binding fragment thereof comprises an HCDR1 amino acid sequence as shown in SEQ ID NO. 1, an HCDR2 amino acid sequence as shown in SEQ ID NO. 2 and an HCDR3 amino acid sequence as shown in SEQ ID NO. 21, and an LCDR1 amino acid sequence as shown in SEQ ID NO. 23, an LCDR2 amino acid sequence as shown in SEQ ID NO. 30 and an LCDR3 amino acid sequence as shown in SEQ ID NO. 34.

Alternatively, VH and/or VL of an anti-HER 4 antibody of the invention (e.g., ab 4261) can be used as starting materials for engineering to make an optimized antibody more suitable for the human body as described herein, i.e., the parent antibody Ab 4261.

In some embodiments, the anti-HER 4 optimized antibodies of the invention comprise antibodies or antigen-binding fragments thereof having a modified VH and/or VL as compared to the VH (SEQ ID NO: 36) and/or VL (SEQ ID NO: 59) of parent antibody Ab 4261. Modifications introduced in the parent antibody include the introduction of amino acid mutations (including amino acid substitutions, deletions, insertions, or any combination thereof) in the heavy chain variable region CDRs, light chain variable region CDRs, and/or FRs of the parent antibody, which may increase 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.

In some embodiments, the anti-HER 4 optimized antibodies of the invention have one or more amino acid mutations relative to the FR region of parent antibody Ab4261 to increase the degree of humanization of the parent antibody. For example, the FR region (including the heavy chain variable region FR region and/or the light chain variable region FR region) of the VH (SEQ ID NO: 36) of Ab4261 may be subjected to one or more amino acid mutations, and further, amino acid mutations may be introduced in HFR1, HFR3 and/or HFR4, for example, a threonine residue at position 7 of HFR1 may be mutated, a glycine at position 23 of HFR3 (corresponding to position 85 of the VH amino acid sequence shown in SEQ ID NO: 36) and/or a methionine residue at position 6 of HFR4 (corresponding to position 108 of the VH amino acid sequence shown in SEQ ID NO: 36) may be mutated, and amino acid mutations may also be introduced in LFR2 and/or LFR3, for example, a methionine at position 14 of LFR2 (corresponding to position 48 of the VL amino acid sequence shown in SEQ ID NO: 59) and/or a serine at position 15 (corresponding to position 49 of the VL amino acid sequence shown in SEQ ID NO: 59) and/or a valine at position 29 of LFR2 (corresponding to position 85 of the amino acid sequence shown in SEQ ID NO: 59).

In a specific embodiment, the anti-HER 4 optimized antibodies of the invention have one or more amino acid mutations in the HFR region, including T7S, G E and/or M108L, relative to the VH amino acid sequence of parent antibody Ab4261 (SEQ ID NO: 36), and/or one or more amino acid mutations in the LFR region, including M48I, S49Y and/or I85V, relative to the VL amino acid sequence of parent antibody Ab4261 (SEQ ID NO: 59), capable of significantly increasing the degree of humanization of parent antibody Ab4261 while retaining cell-binding activity with parent antibody Ab 4261.

In some embodiments, the anti-HER 4 optimized antibodies of the application have one or more amino acid mutations relative to the CDR regions of parent antibody Ab4261 (including the heavy chain variable region CDRs and/or the light chain variable region CDRs) to increase the binding activity of the parent antibody to cells expressing HER 4. For example, the CDRs of the VH (SEQ ID NO: 36) and/or VL (SEQ ID NO: 59) of Ab4261 are subjected to one or more amino acid mutations. Further, one or more amino acid mutations may be introduced into HCDR2 and/or HCDR3, for example, amino acid mutations may be made in alanine at position 1 of HCDR2 (corresponding to position 50 of the VH amino acid sequence shown in SEQ ID NO: 36), serine at position 8 (corresponding to position 56 of the VH amino acid sequence shown in SEQ ID NO: 36), and/or glycine at position 2 of HCDR3 (corresponding to position 96 of the VH amino acid sequence shown in SEQ ID NO: 36), glycine at position 3 (corresponding to position 97 of the VH amino acid sequence shown in SEQ ID NO: 36). Mutations of alanine residues at position 1 of HCDR2 (corresponding to position 50 of the VH amino acid sequence shown in SEQ ID NO: 36) include A50E, A50E, A50E, A50E, A50E, A50E, A50E, A50W or A50Y, mutations of serine residues at position 8 (corresponding to position 56 of the VH amino acid sequence shown in SEQ ID NO: 36) include S56E, A56E, A56V or S56Y (preferably S56E, A56R or S56Y); mutations of glycine residues at position 2 (corresponding to position 96 of the VH amino acid sequence shown in SEQ ID NO: 36) of HCDR3 include G96E, A96E, A96E, A96E, A96E, A96E, A96E, A96E, A96E, A96E, A96E, A96W or G96Y (preferably, G96Y), and mutations of glycine residues at position 3 (corresponding to position 97 of the VH amino acid sequence shown in SEQ ID NO: 36) include G97E, A97E, A97E, A97E, A97E, A97E, A97E, A97E, A97E, A97E, A97E, A97E, A97E, A97W or G97Y.

One or more amino acid mutations may also be introduced into LCDR1, LCDR2 and/or LCDR3, for example, histidine at position 8 of LCDR1 (corresponding to position 27d of the VL amino acid sequence shown in SEQ ID NO: 59), tyrosine at position 12 (corresponding to position 30 of the VL amino acid sequence shown in SEQ ID NO: 59), histidine at position 13 (corresponding to position 31 of the VL amino acid sequence shown in SEQ ID NO: 59), aspartic acid at position 16 (corresponding to position 34 of the VL amino acid sequence shown in SEQ ID NO: 59), leucine at position 1 of LCDR2 (corresponding to position 50 of the VL amino acid sequence shown in SEQ ID NO: 59), Asparagine at position 4 (corresponding to position 53 of the VL amino acid sequence shown in SEQ ID NO: 59), methionine at position 1 of LCDR3 (corresponding to position 89 of the VL amino acid sequence shown in SEQ ID NO: 59), alanine at position 3 (corresponding to position 91 of the VL amino acid sequence shown in SEQ ID NO: 59), glutamine at position 5 (corresponding to position 93 of the VL amino acid sequence shown in SEQ ID NO: 59) were subjected to amino acid mutations. The mutation of histidine at position 8 (corresponding to position 27D of the VL amino acid sequence shown in SEQ ID NO: 59) of LCDR1 comprises H27Y, the mutation of tyrosine at position 12 (corresponding to position 30 of the VL amino acid sequence shown in SEQ ID NO: 59) comprises Y30D, Y30G, Y30K, Y N or Y30R, the mutation of histidine at position 13 (corresponding to position 31 of the VL amino acid sequence shown in SEQ ID NO: 59) comprises H31D, H31K, H N, H31 5631 6257 31T or H31Y (preferably H31K or H31T), the mutation of aspartic acid at position 16 (corresponding to position 34 of the VL amino acid sequence shown in SEQ ID NO: 59) comprises D34A, the mutation of leucine residue at position 1 of LCDR2 (corresponding to position 50 of the VL amino acid sequence shown in SEQ ID NO: 59) comprises L50A, L50E, L50K, L50Q, L50R, L50S, L50V, L W or L50Y (preferably L50A, L50W or L50Y), the mutation of asparagine residue at position 4 (corresponding to position 53 of the VL amino acid sequence shown in SEQ ID NO: 59) comprises N53D, N E, The mutation of methionine residue at position 1 of LCDR3 (corresponding to position 89 of the VL amino acid sequence shown in SEQ ID NO: 59) comprises M89Q, M A, M H, M L or M89S (preferably M89L), the mutation of alanine residue at position 3 (corresponding to position 91 of the VL amino acid sequence shown in SEQ ID NO: 59) comprises A91D, A91F, A91G, A91H, A91L, A R, A91S, A91T, A91W or A91Y, and the mutation of glutamine residue at position 5 (corresponding to position 93 of the VL amino acid sequence shown in SEQ ID NO: 59) comprises Q93D, Q93G, Q93H, Q93I, Q93R, Q S or Q93T.

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

In a specific embodiment, the CDR regions in the VH (SEQ ID NO: 36) and/or VL (SEQ ID NO: 59) of Ab4261 are subjected to one or more amino acid mutations, further one or more amino acid mutations may be introduced at HCDR2 and/or LCDR1 such that the mutant of the parent antibody comprises an amino acid sequence that reduces or eliminates the risk of deamidation. For example, amino acid mutations may be made at the asparagine at position 3 (corresponding to position 52 of the VH amino acid sequence shown in SEQ ID NO: 36), at position 4 (corresponding to position 52a of the VH amino acid sequence shown in SEQ ID NO: 36), at position 5 (corresponding to position 53 of the VH amino acid sequence shown in SEQ ID NO: 36), and at position 6 (corresponding to position 54 of the VH amino acid sequence shown in SEQ ID NO: 36) of the glycine residue. HCDR 2aT position 3 (corresponding to SEQ ID NO:36 comprises a mutation aT position 52 of the VH amino acid sequence shown in SEQ ID NO: 36) comprising a N52Y, N S, N52W, N S, N G or a N52H (preferably, N52S), a mutation aT position 4 (corresponding to position 52a of the VH amino acid sequence shown in SEQ ID NO: 36) comprising a N53 Aa, N52aF, N52aH, N52aP, N52aQ, N52aR, N52aT, N52aW, N52aY or N52aG (preferably, N52aF, N52aH, N52aP, N52aS, N52aY, N52aW or N52 aG), a mutation aT position 5 (corresponding to position 53 of the VH amino acid sequence shown in SEQ ID NO: 36) comprising a mutation aT position 53E, N4253 5353Y, N G or a N53S (preferably, N53I, N3453Y or N53S) comprising a mutation aT position 54G (corresponding to position 54A VH amino acid sequence shown in SEQ ID NO: 36).

Amino acid mutations may also be made in the asparagine at position 10 of LCDR1 (corresponding to position 28 of the VL amino acid sequence shown in SEQ ID NO: 59) and in the glycine at position 11 (corresponding to position 29 of the VL amino acid sequence shown in SEQ ID NO: 59). Mutations of asparagine residues at position 10 of LCDR1 (corresponding to position 28 of the VL amino acid sequence shown in SEQ ID NO: 59) include N28G, N28H, N28I, N28Y, N28L, N28S, N T and N28V, and mutations of glycine residues at position 11 (corresponding to position 29 of the VL amino acid sequence shown in SEQ ID NO: 59) include G29A, G29I, G29N, G29P, G3529R, G S, G T or G29V (preferably G29R, G S or G29T).

In a specific embodiment, the CDR regions in the VH (SEQ ID NO: 36) of parent antibody Ab4261 are subjected to one or more amino acid mutations to isomerise the parent antibody. In a preferred embodiment, the anti-HER 4 optimized antibodies of the invention do not comprise an aspartic acid isomerisation site, and one or more amino acid mutations may be introduced in HCDR2 of Ab4261, e.g. mutations may be made in aspartic acid at position 13 (corresponding to position 61 of the VH amino acid sequence shown in SEQ ID NO: 36), serine at position 14 (corresponding to position 62 of the VH amino acid sequence shown in SEQ ID NO: 36) to reduce or eliminate aspartic acid isomerisation of the amino acid sequence of maternal antibody Ab 4261. The mutation of aspartic acid residue at position 13 (corresponding to position 61 of the VH amino acid sequence shown in SEQ ID NO: 36) of HCDR2 comprises D61Q or D61P (preferably, D61P), and the mutation of serine residue at position 14 (corresponding to position 62 of the VH amino acid sequence shown in SEQ ID NO: 36) comprises S62K.

By performing the above amino acid mutations in the CDR and framework regions of the parent antibody Ab4261, not only is the optimized antibody provided with a higher level of humanization, lower risk of aspartic acid isomerization and/or asparagine deamidation, but also the binding activity of the parent antibody to human HER4 is improved.

In another aspect, the amino acid sequence numbers of the heavy chain CDR regions, light chain CDR regions, and heavy and light chain variable regions of an anti-HER 4 antibody of the application (including parent antibody Ab4261 and its optimized antibody) are summarized in table 2, wherein the heavy and light chain variable region CDRs are defined by the Kabat numbering system. However, as is well known in the art, CDR regions may be defined based on other heavy/light chain variable region sequences such as Chothia and IMGT, abM or Contact numbering systems/methods, and CDR regions defined by other numbering systems/methods are within the scope of the present application as well as those defined by Kabat employed in the present application.

TABLE 2 amino acid sequence ID numbers of CDR regions, heavy chain variable region and light chain variable region of anti-HER 4 antibodies

In some embodiments, the invention provides an isolated anti-HER 4 monoclonal antibody, or antigen-binding fragment thereof, comprising (1) an HCDR1 amino acid sequence as shown in SEQ ID No. 1; (2) the amino acid sequence is X₁IX₂X₃X₄X₅GX₆TYYAX₇X₈VKG(X₁＝A,E,G,I,R,S,V,W or Y; X ₂ =n, Y, S, W, S, G or H; X ₃ = N, a, F, H, P, Q, R, S, T, W or G, X ₄ = N, E, H, I, R, Y, G or S, X ₅ = G, a, F or K, X ₆ = S, D, N, R, V or Y, X ₇ = D, Q or P, X ₈ = S or K, i.e. as shown in SEQ ID NO: 81), HCDR2, preferably X ₁＝A,X₂ = N or S, X ₃ = a, F, H, P, S, Y, W or G, X ₄ = I, R, Y or S, X ₅ = G or a, X ₆ = S, N, R or Y, X ₇＝P,X₈ = S, the amino acid sequence GX ₉X₁₀AFDI(X₉ = G, a, D, E, F, H, I, K, Q, R, S, V, W or Y, X ₁₀ = G, a, D, E, K, H, N, V, W or Y, I, X43952 = G, a, X43943 = S, X, Y, X4634 = G or G, X, V, X ₇＝P,X₈ = 3, X, V, X, V, X, N, X, V, X, V-V, d, K, N, Q, S, T or Y; X ₁₆ = D, a, G, H, N, Q, S or Y; i.e. as shown in SEQ ID NO: 83), preferably X ₁₁＝H,X₁₂＝N,X₁₃ = G, R, S or T, X ₁₄＝Y,X₁₅ = H, K or T, X ₁₆ = D; 5) amino acid sequence X ₁₇GSX₁₈RAS(X₁₇ = L, a, E, G, K, Q, R, S, V, W or Y; X ₁₈ = N, D, E, K, Q, R, S or T; i.e. as shown in SEQ ID NO: 84), LCDR2, preferably X ₁₇ = L, a, W or Y, X ₁₈ = N; 6) amino acid sequence X ₁₉QX₂₀LX₂₁TTRT(X₁₉ = M, Q, a, H, L or S; X ₂₀ =a, D, F, G, H, L, R, S, T, W or Y, X ₂₁ =q, D, G, H, I, R, S, T, i.e. as shown in SEQ ID NO: 85), preferably X ₁₉＝L,X₂₀＝A,X₂₁ =q, or respectively with said HCDR1, HCDR, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 have 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.

In some embodiments, the anti-HER 4 antibody or antigen binding fragment thereof comprises an HCDR1 amino acid sequence as shown in SEQ ID NO. 1, an HCDR2 amino acid sequence as shown in any of SEQ ID NO. 2-22, and an HCDR3 amino acid sequence as shown in either SEQ ID NO. 23 or 24, and an LCDR1 amino acid sequence as shown in any of SEQ ID NO. 25-31, an LCDR2 amino acid sequence as shown in any of SEQ ID NO. 32-35, and an LCDR3 amino acid sequence as shown in either SEQ ID NO. 36 or 37.

In some specific embodiments, the anti-HER 4 antibody or antigen binding fragment thereof comprises an HCDR1 amino acid sequence as shown in SEQ ID NO. 1, an HCDR2 amino acid sequence as shown in SEQ ID NO. 2, 14, 18, 20, 21 or 22, and an HCDR3 amino acid sequence as shown in SEQ ID NO. 23, and an LCDR1 amino acid sequence as shown in SEQ ID NO. 25, 28, 29 or 31, an LCDR2 amino acid sequence as shown in SEQ ID NO. 32 or 34, and an LCDR3 amino acid sequence as shown in SEQ ID NO. 36.

In a specific embodiment, the anti-HER 4 antibody or antigen-binding fragment thereof of the invention comprises HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3, each CDR comprising:

(1) An HCDR1 amino acid sequence as set forth in SEQ ID NO. 1, an HCDR2 amino acid sequence as set forth in any one of SEQ ID NO. 2-22, an HCDR3 amino acid sequence as set forth in SEQ ID NO. 23, and an LCDR1 amino acid sequence as set forth in SEQ ID NO. 25, an LCDR2 amino acid sequence as set forth in SEQ ID NO. 32, an LCDR3 amino acid sequence as set forth in SEQ ID NO. 36, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, or

(2) An HCDR1 amino acid sequence as set forth in SEQ ID NO. 1, an HCDR2 amino acid sequence as set forth in SEQ ID NO. 2, an HCDR3 amino acid sequence as set forth in SEQ ID NO. 24, and an LCDR1 amino acid sequence as set forth in SEQ ID NO. 25, an LCDR2 amino acid sequence as set forth in SEQ ID NO. 32, an LCDR3 amino acid sequence as set forth in SEQ ID NO. 36, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, or

(3) An HCDR1 amino acid sequence as set forth in SEQ ID NO. 1, an HCDR2 amino acid sequence as set forth in SEQ ID NO. 2, an HCDR3 amino acid sequence as set forth in SEQ ID NO. 23, and an LCDR1 amino acid sequence as set forth in any one of SEQ ID NO. 26-30, an LCDR2 amino acid sequence as set forth in SEQ ID NO. 32, an LCDR3 amino acid sequence as set forth in SEQ ID NO. 36, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, or

(4) An HCDR1 amino acid sequence as set forth in SEQ ID NO. 1, an HCDR2 amino acid sequence as set forth in SEQ ID NO. 2, an HCDR3 amino acid sequence as set forth in SEQ ID NO. 23, and an LCDR1 amino acid sequence as set forth in SEQ ID NO. 25, an LCDR2 amino acid sequence as set forth in SEQ ID NO. 33 or 35, an LCDR3 amino acid sequence as set forth in SEQ ID NO. 36, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, or

(5) An HCDR1 amino acid sequence as set forth in SEQ ID NO. 1, an HCDR2 amino acid sequence as set forth in SEQ ID NO. 2, an HCDR3 amino acid sequence as set forth in SEQ ID NO. 23, and an LCDR1 amino acid sequence as set forth in SEQ ID NO. 25, 28 or 31, an LCDR2 amino acid sequence as set forth in SEQ ID NO. 34, an LCDR3 amino acid sequence as set forth in SEQ ID NO. 36, or amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, or

(6) An HCDR1 amino acid sequence as set forth in SEQ ID NO. 1, an HCDR2 amino acid sequence as set forth in SEQ ID NO. 2, an HCDR3 amino acid sequence as set forth in SEQ ID NO. 23, and an LCDR1 amino acid sequence as set forth in SEQ ID NO. 25, an LCDR2 amino acid sequence as set forth in SEQ ID NO. 32, an LCDR3 amino acid sequence as set forth in SEQ ID NO. 37, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, or

(7) An HCDR1 amino acid sequence as set forth in SEQ ID NO. 1, an HCDR2 amino acid sequence as set forth in SEQ ID NO. 21 or 22, an HCDR3 amino acid sequence as set forth in SEQ ID NO. 23, and an LCDR1 amino acid sequence as set forth in SEQ ID NO. 28 or 31, an LCDR2 amino acid sequence as set forth in SEQ ID NO. 34, an LCDR3 amino acid sequence as set forth in SEQ ID NO. 36, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.

In one embodiment, an anti-HER 4 antibody or antigen binding fragment thereof of the invention comprises HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3, each CDR comprising an HCDR1 amino acid sequence as shown in SEQ ID NO.1, an HCDR2 amino acid sequence as shown in SEQ ID NO. 21 or 22, an HCDR3 amino acid sequence as shown in SEQ ID NO. 23, and an LCDR1 amino acid sequence as shown in SEQ ID NO. 28 or 31, an LCDR2 amino acid sequence as shown in SEQ ID NO. 34, an LCDR3 amino acid sequence as shown in SEQ ID NO. 36, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively. Preferably, the anti-HER 4 antibody or antigen binding fragment thereof comprises an HCDR1 amino acid sequence as shown in SEQ ID No.1, an HCDR2 amino acid sequence as shown in SEQ ID No. 22, an HCDR3 amino acid sequence as shown in SEQ ID No. 23, and an LCDR1 amino acid sequence as shown in SEQ ID No. 28, an LCDR2 amino acid sequence as shown in SEQ ID No. 34, an LCDR3 amino acid sequence as shown in SEQ ID No. 34, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

In some embodiments, the anti-HER 4 antibodies or antigen-binding fragments thereof of the invention further comprise a heavy chain variable region having the HCDR1, HCDR2, and HCDR3 and a light chain variable region having the LCDR1, LCDR2, and LCDR 3.

In some embodiments, an anti-HER 4 antibody or antigen-binding fragment thereof of the 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 set forth in any one of SEQ ID NOS.38-62, and a light chain variable region VL having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity to the amino acid sequence set forth in any one of SEQ ID NOS.63-80. In some preferred embodiments, an anti-HER 4 antibody or antigen binding fragment thereof of the invention comprises a VH having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:38, 39, 40, 41, 53, 57 or 59, and a VL having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:63, 64, 65, 66, 67, 70, 71 or 74. In some preferred embodiments, an anti-HER 4 antibody or antigen-binding fragment thereof of the invention comprises a VH having at least 80% identity to the amino acid sequence shown in SEQ ID NO. 61 or 62, and a VL having at least 80% identity to the amino acid sequence shown in SEQ ID NO. 77, 78, 79 or 80.

In a specific embodiment, an anti-HER 4 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, each comprising 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 a VH shown in any of SEQ ID NOS: 38-62 and a VL shown in SEQ ID NO:63, respectively, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% or 100% identical to a VH shown in SEQ ID NO:38 and a VL shown in any of SEQ ID NOS: 64-80, respectively, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98% or 100% identical to a VL shown in any of SEQ ID NOS: 61 or 62, respectively. Preferably, the heavy and light chain variable regions each comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a VH shown in SEQ ID No. 61 and a VL shown in SEQ ID No. 79, respectively, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a VH shown in SEQ ID No. 62 and a VL shown in SEQ ID No. 77, respectively, more preferably the heavy and light chain variable regions each comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a VH shown in SEQ ID No. 62 and a VL shown in SEQ ID No. 77, respectively.

In some embodiments, an anti-HER 4 antibody or antigen-binding fragment thereof of the invention may further comprise constant regions comprising a heavy chain constant region comprising a CH1, hinge, and/or CH2-CH3 region and a light chain constant region. Wherein the heavy chain constant region comprises the natural and mutant forms of the Fc region of a human IgG heavy chain constant region, and further comprises a hinge region that promotes dimer formation. In certain embodiments, the Fc region comprises the CH2 and CH3 domains of the antibody. Fusion proteins comprising an Fc moiety may be purified by protein a or protein G affinity chromatography columns and may extend serum half-life. Preferred Fc regions are derived from human IgG, including IgG1, igG2, igG3, and IgG4. Herein, the position of a particular amino acid residue of the Fc region is determined according to the EU numbering system.

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 antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated by Fc binding to Fc receptors (fcrs) on the surface of immune cells, and CDC is mediated by Fc binding to proteins of the complement system, e.g., C1 q. Effector function may be assessed by various methods, such as Fc receptor binding assays, C1q binding assays, and cell lysis assays.

The anti-HER 4 antibodies or antigen binding fragments thereof of the invention are capable of specifically binding to human HER4 extracellular domain III, have no cross-reactivity with other members of the human HER4 family (including EGFR, HER2 and HER 3), and have cross-reactivity with other species (e.g., mouse, rat, canine) HER4, have a higher degree of humanization, higher sequence stability, and higher binding activity with human HER4, and are effective in inhibiting ligand-induced dimerization of HER4 and its downstream signaling pathways.

2. Method for screening for obtaining an anti-HER 4 antibody or antigen-binding fragment thereof of the invention

The anti-HER 4 antibody provided by the invention can be a murine antibody, or can be a chimeric antibody, a humanized antibody or a fully human antibody. Methods of producing monoclonal antibodies are known in the art, and any of the known methods (e.g., hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.) may be used in the present invention to produce monoclonal antibodies that specifically bind to the extracellular domain of HER 4.

In some embodiments, the invention isolates mononuclear cells (PBMC) from normal human peripheral blood, and amplifies antibody variable region genes from the PBMC, displays human antibody variable regions on phage surfaces by phage display techniques, and constructs phage display libraries.

In some embodiments, a polypeptide of any fragment of the HER4 receptor amino acid sequence, preferably the HER4 receptor extracellular domain, may be selected for panning (screening) as the antigen of interest, the interaction of the antibody variable region displayed on the phage surface with said antigen of interest is detected, and the library of antibody variable regions is screened and amplified by an in vitro selection method, which is similar to the natural selection. In some embodiments, the invention detects, screens for, and uses in a variety of methods for, antibody variable regions displayed on the surface of phage that interact with the antigen of interest, including, but not limited to, flow cytometry (e.g., flow-fluorescence-activated cell sorting (FACS) assay), ELISA assay, and cytofluorescence assay.

In some embodiments, the invention screens antibody libraries for antibodies that recognize an antigen of interest by constructing a phage display library and panning on the antigen of interest. Various phage display methods known in the art can be used to generate antibodies of the invention, for example, U.S. Pat. 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 method described in "anti-body PHAGE DISPLAY: methods and Protocols (Philippa M.O' Brien, robert Aitken edit)". In some embodiments, nucleic acid encoding the antibody variable region may be inserted into a phage coat protein gene such that the phage displays the antibody variable region on its surface, while nucleic acid encoding the antibody variable region is contained within the phage, thereby effecting a link between the antibody variable region phenotype and genotype.

With respect to screening of antibodies, in phage display, a large library of VH and/or VL regions of the antibody can be expressed on the surface of a filamentous phage particle, thereby pairing them to form a binding domain. Phage may be screened from the library based on recognition and binding to the antigen of interest and the binding domain displayed thereby.

In some embodiments, the panning may be achieved by phage infection of host bacteria and propagation amplification in the host. After collection of phage secreted by the host bacteria and having single chain antibody fragments displayed on their surface, multiple rounds of panning may be performed as needed until phage are obtained that are capable of selectively or specifically binding to the target antigen, and finally the amino acid sequence of the antibody variable region is obtained by sequencing the antibody genes in the phage genome (Arap et al, science 1998,279:377-380; smith et al, science 1985,228: 1315-1317).

In a specific embodiment, the invention utilizes the extracellular domain of HER4 receptor as a target antigen, human antibody heavy chain variable region gene fragment and light chain variable region gene fragment are obtained from PBMC, scFv fragments are prepared based on the human antibody heavy chain variable region gene fragment and the light chain variable region gene fragment and are connected to phage surface structural protein gene III, and the human antibody heavy chain variable region gene fragment and the scFv fragments are involved in phage assembly and displayed on the surface of phage by utilizing a co-expression mode, so that a phage display library is constructed and obtained. Phage display libraries are used to enrich for phage that specifically bind to a target antigen (e.g., HER4 receptor extracellular domain) by repeated rounds of adsorption-elution-amplification (panning), and corresponding DNA sequence information is obtained by gene sequencing techniques to infer the amino acid sequence of the antibody variable region.

3. Polynucleotides, vectors and host cells

In one aspect, the application provides a nucleic acid encoding an anti-HER 4 antibody or antigen-binding fragment thereof. The application also includes polynucleotide variants encoding the amino acid sequences described herein.

The person skilled in the art can determine the nucleic acid sequence encoding the antibody of the application based on the genetic code. The Polymerase Chain Reaction (PCR) can be used to isolate and amplify DNA sequences encoding the anti-HER 4 antibodies or antigen binding fragments thereof of the application. The oligonucleotides may additionally contain recognition sites for restriction endonucleases to facilitate insertion of amplified DNA fragment combinations into expression vectors. PCR techniques are described In Saiki et al, science 1988,239:487-491;Recombinant DNA Methodology,Wu et al, ACADEMIC PRESS, in c., san Diego (1989), pages 189-196, PCR Protocols: A Guide to Methods and Applications, innis et al, ACADEMIC PRESS, inc. (1990).

The nucleic acid molecules of the invention include single and double stranded forms of DNA and RNA, and the corresponding complementary sequences. The nucleic acid molecules of the invention also include isolated nucleic acid molecules, preferably derived from purification, and the amount or concentration of DNA or RNA of their constituent nucleotide sequences can be identified, manipulated and recovered by standard biochemical methods (e.g., as described in Sambrook et al, molecular Cloning: ALaboratory Manual, 2 nd edition, cold Spring Harbor Laboratory, cold Spring Harbor, N.Y. (1989)). Preferably, such sequences include those provided and/or constructed in open reading frames with internal untranslated sequences or intron breaks that are not commonly found in eukaryotic genes. The sequence of the non-translated DNA may be present 5 'or 3' to the open reading frame, wherein the sequence does not interfere with manipulation or expression of the coding region.

The anti-HER 4 antibodies or antigen-binding fragments thereof of the invention are prepared by mutagenesis of specific site nucleotides in DNA encoding the anti-HER 4 antibody or antigen-binding fragment thereof using PCR mutagenesis or other techniques known to those of ordinary skill in the art to produce DNA encoding the variant, after which the recombinant DNA is expressed/amplified in cell culture as outlined herein. In addition, anti-HER 4 antibodies or antigen-binding fragments thereof may also be prepared by in vitro synthesis using established techniques.

As known to those skilled in the art, due to the degeneracy of the genetic code, an anti-HER 4 antibody or antigen binding fragment thereof of the application is encoded by an extremely large number of nucleic acids, each of which is within the scope of the application and can be made using standard techniques. Thus, identifying a particular amino acid sequence, one skilled in the art can prepare many different nucleic acids by simply modifying one or more of the above codons for their respective coding sequences in a manner that does not alter the amino acid sequence of the anti-HER 4 antibodies or antigen binding fragments thereof of the application.

In another aspect, the invention also provides an expression vector encoding a nucleic acid of an anti-HER 4 antibody or antigen-binding fragment thereof herein.

The nucleic acid encoding the anti-HER 4 antibodies or antigen-binding fragments thereof of the invention may be constructed in a suitable vector for introduction into a host cell for expression of the protein of interest. The vector components typically include, but are not limited to, one or more of 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 protein of interest in the vector is operably linked to a promoter.

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

Suitable vectors include plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages (e.g., lambda or M13 phages), animal viruses, and the like. Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-virus-vacuolated viruses (e.g., SV 40). The vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. The vector may also include components that assist in its entry into the cell, including but not limited to viral particles, liposomes, or protein shells.

In another aspect, the application also provides a host cell comprising a nucleic acid or expression vector encoding an anti-HER 4 antibody or antigen-binding fragment thereof of the application.

The cell may be a eukaryotic cell, for example, a mammalian host cell, including but not limited to, SV40 transformed monkey kidney 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 mouse 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 testis support cell (TM 4, mather, biol red 1980, 23:243-251), monkey kidney cell (CV 1, ATCC, CCL-70), african green monkey kidney cell (VERO-76, ATCC, CRL-1587), human cervical cancer cell (HELA, ATCC, CCL-2), canine kidney cell (MDCK, ATCC, CCL-34), butrorat liver cell (BRL 3A, ATCC, L-1442), human hamster ovary cell (ATCC, urlaub et al, proc NATL ACAD SCI USA 1980, 77:4216-4220), mouse testis support cell (TM 4, mather, biol red 1980, 23:243-251), monkey kidney cell (CV 1, ATCC, CCL-70), african green monkey kidney cell (VERO-76, ATCC, CRL-1587), human cervical cancer cell (HELA, ATCC, CCL-2), dog kidney cell (MDCK, ATCC, CCL, butro-34), butro rat liver cell (BRL-4, ATCC, SCL-138, fleet-4, mouse tumor cell (ATCC, fleet-4, mouse tumor cell, or the like).

4. Method for preparing anti-HER 4 antibody or antigen binding fragment thereof

The invention provides methods of using the host cells to make the anti-HER 4 antibodies or antigen-binding fragments thereof of the invention.

The method comprises transfecting a nucleic acid or expression vector encoding an anti-HER 4 antibody or antigen-binding fragment thereof of the invention into a host cell, and culturing the host cell in a culture medium for a period of time to express the anti-HER 4 antibody or antigen-binding fragment thereof of the invention. Without limitation, commercially available media may be used as the medium.

Preferably, the expressed anti-HER 4 antibody or antigen-binding fragment thereof is secreted into the medium in which the host cells are cultured. Antibodies are recovered from the culture medium using standard protein purification methods, such as removal of impurities by centrifugation or filtration, or purification of the resultant by affinity chromatography, other purification techniques such as anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography may also be used.

5. Bispecific molecule-multispecific molecules

The invention also provides a bispecific molecule comprising an anti-HER 4 antibody or antigen-binding fragment thereof of the invention linked to another functional molecule (e.g. another polypeptide or protein such as a Fab' fragment) to form a bispecific or multispecific molecule that binds to multiple binding sites or binding (target) epitopes. For example, an anti-HER 4 antibody or antigen-binding fragment thereof of the invention may be functionally linked to one or more other binding molecules, such as another antibody, antigen-binding fragment, polypeptide, or binding mimetic, including chemical coupling, gene fusion, non-covalent association, or other means).

In one embodiment, the other functional molecule may be an Fc receptor (FcR), such as human fcγriii (CD 16), fcδri (CD 64) or fcαr (CD 89), and thus the bispecific or multispecific molecule of the present invention may be targeted to both fcγ R, fc αr or fcεr expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells [ PMN ]) and HER4 expressing tumor cells.

In one embodiment, the further functional molecule is selected from an antibody or antigen binding fragment thereof specific for a cancer-associated antigen (TAA) which may be selected from HER2, HER3, VEGF, VEGFR, c-Met, TFR (transferrin receptor), IGF-1R (human insulin-like growth factor I receptor), etc., or an immune checkpoint protein antigen which may be selected from PD-1, PD-L1, CTLA-4, CD137, CD47, etc.

In one embodiment, the bispecific or multispecific molecules of the present invention may further comprise a third binding specificity in addition to the binding specificity and anti-HER 4 binding specificity of the functional molecules described above. The third specificity may be an anti-Enhancement Factor (EF) moiety, for example, the molecule may bind to a surface protein involved in cytotoxic activity and thereby increase the immune response to the target cell. The anti-enhancer moiety may be an antibody (including scFv), and the anti-enhancer moiety may bind to FcR or target cell antigen. In addition, the target cells to which the anti-enhancer moiety binds may be different from the target cells to which the first and second binding specificity molecules bind, e.g., the anti-enhancer moiety may bind to cytotoxic T cells (e.g., via CD2, CD3, CD8, CD28, CD4, or ICAM-1) or other immune cells that produce an enhanced immune response to the target cells.

The term "target cell" as used herein refers to a cell targeted by a molecule of the invention (e.g., an anti-HER 4 antibody or antigen-binding fragment thereof, a bispecific molecule, or a multispecific molecule) to any disorder in a subject. In some embodiments, the target cell is a cell that overexpresses HER4, the cell that overexpresses HER4 comprises a tumor cell (e.g., glioma), or other non-tumor cell.

Bispecific molecules of the present invention can take many different forms. For example, a bispecific molecule retains the traditional antibody format, except that it does not have two binding arms of the same specificity, but two binding arms of different specificity, each of which may consist of two single chain antibody fragments (scFv) linked by a peptide chain, to construct a bispecific molecule, the so-called Bs (scFv) 2 structure. Bispecific molecules may also include two different F (ab) fragments linked by a peptide-based linker. These and other forms of bispecific molecules can be prepared by genetic engineering, somatic hybridization, or chemical synthesis methods. See, e.g., 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.

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

In one aspect, the invention provides an immunoconjugate comprising the anti-HER 4 antibody or antigen-binding fragment thereof of the invention.

The anti-HER 4 antibodies or antigen-binding fragments thereof of the invention may be conjugated to a therapeutic agent to form an immunoconjugate, e.g., an antibody-drug conjugate (ADC). Suitable therapeutic agents include cytotoxins (including paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, ipecine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, milteframycin, actinomycin D, 1-dehydroproteinsterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol), alkylating agents (e.g., chloroform, chloro Ding Liuniao, melphalan, carmustine [ BSNU ] and lomustine [ CCNU ], cyclophosphamide, butylsulfonated imide, dibromonitrobenzene, streptozotocin, mitomycin C and cisplatin (II) [ DDP ] cisplatin), puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil norhydrazine), DNA minor groove binders, DNA intercalators, DNA cross-linkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, tubulin binders, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics (such as actinomycin, bleomycin, milteframycin, and anthracycline [ AMC ]), and antimitotics (such as vincristine, vinblastine). In ADC, the linker to which the antibody and therapeutic agent are conjugated may be selected from cleavable linkers (Cleavable linker), such as peptidyl linkers, disulfide bonds, or hydrazone linkers, and Non-cleavable linkers (Non-cleavable linker). ADCs may be prepared as described in U.S. patent nos. 7,087,600, 6,989,452, and 7,129,261, pct publications WO 02/096910, WO 07/038,658, WO 07/051,081, WO07/059,404, WO 08/083,312, and WO 08/103,693, and U.S. patent publications 20060024317, 20060004081, and 20060247295, the disclosures of which are incorporated herein by reference.

In another aspect, the invention also provides a chimeric antigen receptor, an engineered T cell receptor, or an oncolytic virus comprising an anti-HER 4 antibody or antigen-binding fragment thereof of the invention.

7. Pharmaceutical composition

The present invention provides pharmaceutical compositions comprising an anti-HER 4 antibody or antigen-binding fragment thereof (or an immunoconjugate, or a bispecific/multispecific molecule, or a chimeric antigen receptor, or an engineered T cell receptor, or an oncolytic virus) of the invention, and a pharmaceutically acceptable carrier.

The pharmaceutical composition may comprise any kind of pharmaceutically acceptable carrier. Carriers that may be used include excipients, surfactants, thickeners or emulsifiers, solid binders, dispersing or suspending aids, solubilizers, colorants, flavorants, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonicity agents or combinations thereof. The selection and use of suitable vectors is taught below, gennaro et al, remington: THE SCIENCE AND PRACTICE of Pharmacy, 20 th edition (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). In view of the different routes of administration, the active ingredient may be encapsulated in a material to protect it from acids and other natural conditions that may inactivate it. As used herein, the term "parenteral administration" is a non-enteral and topical mode of administration and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the antibodies of the application may be administered by a non-parenteral route (e.g., topical, epidermal, or mucosal route of administration), e.g., intranasal, oral, vaginal, rectal, sublingual, or topical.

The pharmaceutical composition may be a sterile aqueous solution or dispersion. They are also capable of forming microemulsions, liposomes, or other ordered structural combinations suitable for high drug concentrations.

The amount of active ingredient that can be combined with the carrier material to form a single dosage form is determined according to the subject and the particular mode of administration and is also generally the amount of the composition that is capable of producing a therapeutic effect. Generally, the compositions formed with pharmaceutically acceptable carriers comprise from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of the active ingredient.

The dosage regimen is adjusted to provide the best expected response (e.g., therapeutic response). For example, the dosage may be administered in a single dose, may be administered in several doses, or may be proportionally reduced or increased depending on the treatment situation. It is particularly advantageous to formulate compositions for parenteral administration in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the treatment of subjects, each unit dosage containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect upon administration of the active ingredient in association with the required pharmaceutical carrier. In addition, the anti-HER 4 antibodies or antigen-binding fragments thereof of the application may also be administered as a sustained release formulation, which may reduce the frequency of administration.

The amount of the composition of the anti-HER 4 antibody or antigen-binding fragment thereof administered may range from about 0.0001mg/kg to 100mg/kg body weight, typically from 0.001mg/kg to 50mg/kg body weight.

The "therapeutically effective dose" of the anti-HER 4 antibodies or antigen-binding fragments thereof of the invention preferably results in a decrease in severity of disease symptoms, an increase in the frequency and duration of disease symptoms in the absence of progression, or prevention of damage or disability to the body caused by the affliction of the disease. For example, a "therapeutically effective dose" of a subject is preferably capable of inhibiting progression of a condition by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80%, relative to an untreated subject. A therapeutically effective amount of the therapeutic antibody may ameliorate at least one symptom of a disorder in a subject, typically a human or other mammal. The "therapeutically effective dose" may also be determined differently depending on various factors including, but not limited to, the method of formulation, the method of administration, the age, body, weight, sex or pathological condition of the patient, diet, time of administration, interval of administration, route of administration, rate of excretion, and sensitivity of reaction.

The pharmaceutical compositions may be selected from controlled release formulations including implants, transdermal patches and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used. See, for example, sustained and Controlled Release Drug DELIVERY SYSTEMS, J.R.ROBINSON, editions, MARCEL DEKKER, inc., new york, 1978.

The therapeutic pharmaceutical composition may be delivered by a medical device selected from (1) needleless 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) microinjection pumps (U.S. Pat. No. 4,487,603), (3) transdermal devices (U.S. Pat. No. 4,486,194), (4) infusion apparatuses (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.

In certain embodiments, the anti-HER 4 antibodies or antigen-binding fragments thereof of the application may be formulated to ensure in vivo biodistribution. For example, to ensure that therapeutic antibodies of the application cross the blood brain barrier, they may be formulated into liposomes that may additionally contain targeting moieties to enhance selective transport to specific cells or organs. See, for example, 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 Physiol1995,268: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.

8. Medicine box

In one aspect, the invention provides a kit comprising an effective amount of a pharmaceutical composition of an anti-HER 4 antibody or antigen-binding fragment of the invention, and one or more additional therapeutic agents (i.e., the kit may or may not comprise at least one additional therapeutic agent).

In some embodiments, the therapeutic agent comprises a therapeutic agent that targets glioblastoma, including signal pathway modulators, cell cycle inhibitors, and inflammatory response modulators. In some embodiments, the signaling pathway modulator comprises a modulator that targets R1P1K and downstream VEGFD/VEGFR3 pathway (e.g., oncostatin-1, nec-1, and analogs thereof), a modulator that targets PI3K/AKT signaling pathway (e.g., alisol a 24-acetate), a modulator that inhibits transforming growth factor β1 activity, and upregulates pparα expression (e.g., oleoylethanolamide, valproic acid). In some embodiments, the cell cycle inhibitor comprises olomoucine, salidroside, carnosine. In some embodiments, the inflammatory response modifier comprises a siroton capsule, resveratrol, anti-IL-17A mab (e.g., secukinumab).

9. Therapeutic uses and methods

In one aspect, the invention relates to the use of an anti-HER 4 antibody or antigen binding fragment thereof of the invention for the preparation of a pharmaceutical composition or formulation for the treatment and/or prevention of a HER4 related disease or disorder, for the preparation of a diagnostic reagent for the diagnosis of a HER4 related disease or disorder. Or the invention relates to a method of treating and/or preventing a HER 4-related disease or disorder, comprising administering to a subject a therapeutically effective amount of an anti-HER 4 antibody or antigen-binding fragment thereof, a pharmaceutical composition or a kit of parts of the invention.

Such diseases or conditions include cancer and other HER4 related diseases.

In some embodiments, the cancer comprises glioma.

In some embodiments, other HER 4-related diseases include gliosis diseases, e.g., stroke, alzheimer's disease, viral encephalitis, traumatic brain injury, multiple sclerosis, epilepsy.

In another aspect, the invention provides a method of inhibiting HER4 receptor and its downstream signal transduction pathway comprising inhibiting ligand-induced dimerization of HER4 in vitro or in vivo using an anti-HER 4 antibody or antigen binding fragment, pharmaceutical composition or kit of the invention, said ligand comprising NRG-1-4, preferably NRG-1.

10. Detection use

The invention also relates to the use of an anti-HER 4 antibody or antigen-binding fragment thereof for detecting and/or measuring HER4 or HER4 expressing cells in a sample (e.g., a biological sample, e.g., serum, tissue), and methods for screening patients for a HER 4-related disorder responsive to treatment with an anti-HER 4 antibody or antigen-binding fragment thereof of the invention.

In some embodiments, the anti-HER 4 antibodies or antigen-binding fragments thereof may be used to diagnose diseases or conditions of aberrant expression (e.g., over-expression, under-expression, or under-expression) of HER4 to facilitate determination of a treatment regimen. For example, an antibody may be conjugated to a detectable label or reporter molecule and the labeled antibody contacted with a sample obtained from the patient to diagnostically determine HER4 expression levels. The detectable label or reporter may be a radioisotope, such as ³H、¹⁴C、³²P、³³P、³⁵S、¹²³I、¹²⁵I、¹³¹I、¹¹¹ In or ¹⁸⁸ Rh, a fluorescent material, such as umbelliferone, luciferin, rhodamine, fluorescein isothiocyanate, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin, a chemiluminescent material, such as luminol, a bioluminescent material, such as luciferase, luciferin or aequorin, or an enzyme, such as alkaline phosphatase, beta-galactosidase, acetylcholinesterase, horseradish peroxidase or luciferase.

In some embodiments, a method that can be used to detect or measure the level of HER4 in a sample comprises (a) contacting a sample or control sample with the anti-HER 4 antibody or antigen-binding fragment thereof, and (b) detecting the level of HER4 bound to the antibody or antigen-binding fragment thereof in the sample (quantitative or qualitative). Wherein the control sample comprises a positive control, which may be a sample known to be capable of assessing a disease or condition, and a negative control, which may be a sample of a healthy subject. Higher levels of HER4 expression in the sample, and statistical differences, compared to the control, indicate the presence of HER 4-related disease or disorder. Specific exemplary assays that can be used to detect or measure the level of HER4 expression in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunopet (e.g., 89Zr, 64Cu, etc.), and Fluorescence Activated Cell Sorting (FACS).

In some embodiments, the invention provides a method of detecting, diagnosing, or monitoring a HER 4-associated disease or disorder in a subject, the method comprising (1) contacting a test sample obtained from the subject with an anti-HER 4 antibody or antigen-binding fragment thereof of the invention, detecting the level of HER4 in the test sample (either quantitatively or qualitatively), and (2) comparing the level of HER4 in the test sample to the level of HER4 in a control sample (e.g., a normal cell derived from the same tissue as the test sample, or a cell comparable to the level of HER4 expression of the normal cell), wherein a test sample expressing a higher level of HER4 than the control sample can then be determined as the subject having a disease or disorder associated with HER4, or the subject having a condition associated with HER 4.

11. Technical effects

The anti-HER 4 antibodies or antigen binding fragments thereof of the invention can specifically bind to human HER4 extracellular domain III, have high binding activity, and do not cross react with other human ErbB/HER receptor tyrosine kinases (e.g., EGFR, HER2, HER 3). The anti-HER 4 antibody or the antigen binding fragment thereof provided by the invention has an inhibition effect on ligand-induced HER4 dimerization, which indicates that the anti-HER 4 antibody or the antigen binding fragment thereof can block a ligand-induced HER4 mediated signal transduction pathway.

In summary, the anti-HER 4 antibodies or antigen binding fragments thereof provided herein may be used as candidates for drugs for the treatment and/or prevention of HER 4-related diseases or disorders, and are also suitable for development as bispecific or multispecific molecules.

The application is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, patents and patent applications cited throughout this application are expressly incorporated herein by reference. Unless otherwise indicated, the materials, reagents and devices referred to in the examples below are commercially available.

Examples

Example 1 construction of anti-human HER4 phage immune library and screening of antibodies

1.1 Construction of phage immune library

Total RNA was extracted from human peripheral blood mononuclear cells by TriZOL method, and a reverse transcription reaction was performed using the extracted RNA as a template using a reverse transcription kit (PRIMESCRIPT ^TM II 1st Strand cDNA Synthesis Kit,Takara) to obtain cDNA. And then, respectively amplifying by using cDNA as a template and utilizing a specific primer through a PCR method to obtain a complete set of human antibody heavy chain variable region gene fragment and a complete set of human antibody light chain variable region gene fragment, respectively splicing and amplifying the light chain variable region gene fragment and the heavy chain variable region gene fragment through overlap PCR (SOE-PCR) to obtain single-chain antibody variable region gene fragments (scFv), connecting the scFv sequences into a phagemid display vector through enzyme digestion, then electrically transferring the connection products into TG1 competent cells, and infecting bacterial liquid through helper phage M13KO7 to obtain a complete humanized phage library.

1.2 Preliminary screening and sequence analysis of antibodies

Adding the obtained scFv library into an immune tube coated by a recombinant protein of an extracellular domain of human HER4 (26-651 amino acid sequence shown as SEQ ID NO: 82), standing at 4 ℃ for incubation overnight, blocking, cleaning, eluting, neutralizing with 1M Tris (pH8.0), infecting TG1 E.coli cells in logarithmic growth phase, culturing, collecting bacterial cells, adding M13KO7 helper phage for infection, repeating 3-5 rounds of 'adsorption-elution-amplification' panning, enriching to obtain an anti-human HER4 phage display antibody library, picking single colony for amplification culture, carrying out bacterial liquid PCR identification and monoclonal phage-ELISA identification, and carrying out gene sequencing to obtain scFv capable of specifically binding with the antigen, wherein the amino acid sequence of VH is shown as SEQ ID NO:38, and the amino acid sequence of VL is shown as SEQ ID NO: 63.

1.3 Methods of making anti-HER 4 antibodies

VH and VL of scFv obtained in the above steps were cloned into a human IgG1 heavy chain constant region (shown in SEQ ID NO: 87) and a human kappa light chain constant region (shown in SEQ ID NO: 88), respectively, i.e., a nucleotide sequence containing a sequence encoding VH and human IgG1 heavy chain constant region and a nucleotide sequence containing a sequence encoding VL and human kappa light chain constant region were constructed as 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 in a ratio of 1:1, transiently transfected into Expi-CHOS cells in a logarithmic growth phase, and cultured at 32 ℃ for 10 days. Cell supernatants were collected, cells were pelleted by centrifugation, filtered, and antibodies were purified by Protein A affinity chromatography and designated Ab4261.

Example 2 antigen binding epitope analysis of antibody Ab4261

The binding epitope of antibody Ab4261 on HER4 was detected by ELISA. The specific method is that antibody Ab4261 is diluted to 1. Mu.g/mL with PBS, added to a 96-well ELISA plate at 100. Mu.L/well, and coated overnight at 4 ℃. After washing the plate with PBS, 200. Mu.L of blocking solution (PBST [ PBS+0.1% Tween-20] containing 1% BSA) was added to each well, and the plate was left to stand at room temperature for incubation for 1 hour. After PBST plate wash, 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., chimeric recombinant protein constructed by HER4 extracellular domain III in place of EGFR extracellular domain III) diluted 1:200 in blocking solution was added per well and incubated at room temperature for 2 hours. PBST plates were washed and 100. Mu.L of anti-6 XHis-HRP antibody (Proteintech) diluted 1:10,000 in blocking solution was added to each well and incubated at room temperature for 1 hour. After PBST wash plates 100. Mu.L TMB chromogenic solution (Thermo) was added to each well, after incubation for 3-10 min at RT and light-proof 50. Mu.L stop solution (2M HCl) was added to each well, and then OD450 values were read using a multifunctional microplate reader (Varioskan, thermo). Raw data was analyzed using GRAPHPAD PRISM software. The detection results are shown in fig. 1, where the recognition epitope of Ab4261 is located in HER4 extracellular domain III.

Example 3 optimization of antibody Ab4261

3.1Ab4261 Single Point mutation optimization

Using on-line software abYsis (http:// analysis. Org/abysis/index. Html) and MOE software, a total of 154 single point mutations were designed to increase the degree of humanization, improve affinity, and/or reduce the risk of stability of the antibody molecule after analysis of the light and heavy chain variable region sequences of antibody Ab4261 for amino acid residues with a lower degree of humanization, potential stability risk sites, and amino acid residues with lower binding activity. Based on antibody Ab4261, 154 single point mutations are introduced, and then the expression plasmid combination of light and heavy chain of the antibody is transiently transferred ExpiCHO-S cells to express the single point mutation antibodies. The expression levels of each single-point mutant antibody and parent antibody Ab4261 in the culture supernatant after 6 days of transient culture were examined by a molecular interaction instrument (ForteBio, model Oke). Specifically, culture supernatants were harvested after ExpiCHO-S cells were transiently expressed, and 200. Mu.L/well of the culture supernatants were added to a 96-well blackboard, samples to be tested and Protein A probes were placed in a ForteBio-action apparatus, and the binding rates of each antibody molecule in the culture supernatants and the Protein A probes were detected by a quantitative program preset by the machine. After each cycle, the probe was regenerated by immersing it in 10mM glycine buffer (pH 1.5) and the next cycle was started. And after the detection is finished, calculating according to a standard curve by using analysis software to obtain the expression quantity of each mutant.

The binding activity of each of the above mutants to HER4 expressing 293T cells (293T-HER 4) was examined by flow cytometry by collecting 293T-HER4 cells and adding them to a 96-well U-plate at 50 μl/well after resuspension with FACS buffer (PBS containing 1% BSA). After diluting each mutant to be tested to 20. Mu.g/mL with FACS buffer, 50. Mu.L of each well was added to the above-mentioned well plate, and the mixture was left to stand on ice for incubation for 1 hour. The supernatant was centrifuged off and washed 3 times with FACS buffer. Secondary anti-AF 488-labeled goat anti-human IgG (H+L) (Jackson Immunoresearch) was diluted 1:1000 by volume in FACS buffer, added to the above well plate at 100. Mu.L per well, mixed well and placed on ice for 40 min of incubation. The supernatant was removed by centrifugation and washed 3 times with FACS buffer. Cells were resuspended in 100 μl/well FACS buffer and examined using a flow cytometer (NovoCyte 3005, agilent).

The results of cell binding activity of the mutant sites and mutant amino acids, transient expression levels, and FACS detection of each mutant are shown in Table 1. Mutations that increase the degree of humanization of the parent antibody, increase the binding activity of the parent antibody to HER 4-expressing cells, and/or significantly reduce the risk of aspartic acid isomerization and/or asparagine deamidation of the antibody molecule, as compared to parent antibody Ab4261, including 22 single point mutations (G101、G102、G103、G112、G114、G115、G116、G119、G122、G125、G126、G127、G128、G132、G135、G136、G139、G142、G143、G145、G147 and G161 in the antibody heavy chain), and 13 single point mutations (K101, K103, K104, K105, K118, K119, K120, K128, K132, K141, K149, K150, and K161) in the antibody light chain.

TABLE 1 Single point mutation, transient expression level of Ab4261 and binding Activity results with 293T-HER4 cells

H is a heavy chain, L is a light chain, and MFI is the average fluorescence intensity

3.2 Combinatorial mutation optimization of Ab4261 antibodies

The 6 single point mutations in the heavy chain of the antibody (i.e., G101, G102, G103, G135, G143, and G147) and the 7 single point mutations in the light chain of the antibody (i.e., K101, K103, K104, K105, K120, K128, and K149) were selected for combination and introduced into the variable region sequence of antibody Ab4261 to construct antibody molecules containing different combinations of mutations. Antibody samples were prepared according to the method described in item 3.1 above, using ForteBio to detect the expression levels of each mutant and control parent antibody Ab4261, and using flow cytometry to detect the binding activity of each mutant to 293T-HER4 cells.

The mutation sites, transient expression levels, and binding activities to 293T-HER4 cells contained in each combination mutant are shown in Table 2 and FIG. 2. The expression level of most of the combined mutants was comparable to that of the parent antibody Ab4261, and the cell binding activity of each combined mutant was superior to that of the parent antibody, with the combined mutants G201-K203 and G202-K201 being significantly superior to that of the parent antibody and other mutants.

TABLE 2 combined mutation, transient expression levels of Ab4261 and binding Activity results with 293T-HER4 cells

MFI mean fluorescence value

Example 4 antigen binding specificity and Cross-species binding Activity of optimized molecules of Ab4261

4.1 Binding Activity of optimized molecules of Ab4261 and ErbB/HER family protein Member

The binding activity of the optimized molecule G202-K201 of Ab4261 to a member of the human ErbB/HER family of proteins was detected by ELISA. The specific procedure was as follows, the HER4 recombinant protein and other members of the human ErbB/HER family (EGFR, HER2 and HER 3) were diluted to 1 μg/mL with PBS, then added to a 96-well ELISA plate at 100 μl/well and incubated overnight at 4 ℃. After washing the plate with PBS, 200. Mu.L of blocking solution (PBST [ PBS+0.1% Tween-20] containing 1% BSA) was added to each well, and the plate was left to stand at room temperature for incubation for 1 hour. After PBST plates were washed, antibody samples, diluted 4-fold from 5. Mu.g/mL in blocking solution, were added, 100. Mu.L/well, and incubated at room temperature for 2 hours. Plates were washed with PBST and 100. Mu.L of anti-human IgG (H+L) -HRP antibody (Jackson Immuno) diluted in blocking solution at a volume ratio of 1:5,000 was added to each well and incubated at room temperature for 1 hour. After PBST wash plates 100. Mu.L TMB chromogenic solution (Thermo) was added to each well, after incubation for 3-10 min at RT and light-proof 50. Mu.L stop solution (2M HCl) was added to each well, and then OD450 values were read by a multifunctional microplate reader (Varioskan, thermo). The raw data were analyzed using GRAPHPAD PRISM software and as shown in figure 3, antibody G202-K201 bound only to HER4 (EC 50 value of about 0.01827 μg/mL) and did not bind to other ErbB/HER family protein members.

4.2 Optimization of binding Activity of molecules of Ab4261 to HER4 of different species

The binding activity of the optimized molecule G202-K201 of Ab4261 to HER4 of different species was tested as described above, and the test antigens included HER4 recombinant proteins derived from humans, mice, rats and dogs. As shown in FIG. 4, the binding activity of the antibody G202-K201 to mouse and rat HER4 was comparable to that to human HER4, and the binding activity to canine HER4 was slightly lower than that to human HER 4.

Example 5 binding Activity of optimized molecules of Ab4261 to 293T-HER4 cells

The binding activity of optimized molecules G201-K203 and G202-K201 of Ab4261 to 293T-HER4 cells was detected by flow cytometry according to the method described in item 3.1 above.

As shown in FIG. 5 and Table 3, the cell binding activity of antibodies G201-K203 and G202-K201 was significantly higher than that of the parent antibody Ab4261, and the cell binding activity of G202-K201 was slightly better than that of G201-K203.

TABLE 3 binding Activity of optimized molecules of Ab4261 to 293T-HER4 cells

Antibody numbering	Cell binding Activity EC50 (μg/mL)
		G201-K203	0.02967
G202-K201	0.02398
		Ab4261	1.726

Example 6 Effect of optimized antibodies of Ab4261 on NRG-1 induced dimerization of HER2/HER4 receptor

Further validation was performed on whether Ab4261 optimized molecule G202-K201 would affect NRG-1 induced dimerization of the HER2/HER4 receptor using the NanoBiT structural complementary reporter system of Promega, respectively. The NanoBiT system consists of a LgBiT subunit and a SmBiT subunit, which can be respectively fused with the proteins to be detected, and if the two proteins to be detected interact to form a dimer, lgBiT and SmBiT structural complement form functional luciferase, so that bright luminous signals are generated by reaction with a substrate. In this example, the nucleotide sequences encoding the human HER2 and HER4 membrane ectodomains and transmembrane domains were first inserted into NanoBiT systems pbitt 1.3-C and pbitt 2.3-C vectors by molecular cloning techniques, then the two plasmids were co-transfected into U2OS cells, and the cells were harvested 24 hours after transfection and plated in 96 well white-wall plates and incubated overnight at 37 ℃. The parent antibody Ab4261 and its optimized antibody G202-K201, and the control antibody Pertuzumab were subjected to gradient dilution, and then mixed with NRG-1 in a 1:1 volume ratio for use (final concentration of NRG-1 is 1 nM). Nano-Glo LIVE CELL REAGENT (Promega) was added to the 96-well white plate and the chemiluminescent value at this time was read as background, followed by adding the prepared samples to each well and incubating at 37℃for 6 minutes. As shown in fig. 6 and table 4, both antibody G202-K201 and control antibody Pertuzumab were able to significantly inhibit NRG-1-induced HER2/HER4 receptor dimerization, while maternal antibody Ab4261 was significantly less inhibitory than G202-K201 and control antibody Pertuzumab, indicating that antibody G202-K201 was able to affect the normal biological function of HER4 receptor, e.g., inhibit the HER4 receptor downstream signaling pathway, by inhibiting NRG-1-induced HER4 receptor dimerization.

TABLE 4 inhibitory Activity of optimized molecules of Ab4261 on NRG-1 induced dimerization of HER4 receptor

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

While the invention has been described in terms of one or more embodiments, it will be understood that the invention is not limited to those embodiments, but is intended to cover all alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All references cited herein are incorporated herein by reference in their entirety.

Claims (21)

1. An isolated anti-HER 4 antibody or antigen-binding fragment thereof, wherein the anti-HER 4 antibody or antigen-binding fragment thereof comprises at least one of the following properties:

(1) The anti-HER 4 antibody or antigen-binding fragment thereof specifically binds to human HER4 extracellular domain III;

(2) The anti-HER 4 antibody or antigen-binding fragment thereof is capable of inhibiting ligand-induced HER4 receptor dimerization and downstream signal transduction pathways thereof;

(3) The anti-HER 4 antibody or antigen binding fragment thereof has no cross-reactivity with other members of the human ErbB/HER family (including EGFR, HER2 and HER 3), and

(4) The anti-HER 4 antibody or antigen-binding fragment thereof is capable of cross-reacting with HER4 of other species (e.g., mouse, rat, canine).

2. The anti-HER 4 antibody or antigen-binding fragment thereof of claim 1, comprising HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and LCDR1, LCDR2 and LCDR3 of the light chain variable region (1) the amino acid sequence of HCDR1 as shown in SEQ ID NO:1, (2) the amino acid sequence is X₁IX₂X₃X₄X₅GX₆TYYAX₇X₈VKG(X₁＝A,E,G,I,R,S,V,W or Y, X ₂ = N, Y, S, W, S, G or H;

X ₃ = N, a, F, H, P, Q, R, F, H, Y or G, X ₄ = E, H, I, R, Y, G or K, X ₅ = G, a, F or K, X ₆ = S, D, N, R, V

Or Y, X ₇ = D, Q or P, X ₈ = S or K, i.e. as shown in SEQ ID NO: 81), preferably X ₁ = a,

X ₂ = N or S, X ₃ = a, F, H, P, S, Y, W or G, X ₄ = I, R, Y or S, X ₅ = G or a, X3554 = S, N, R or Y, X ₇＝P,X₈ = S, (3) amino acid sequence GX ₉X₁₀AFDI(X₉ = G, a, D, E, F, H, I, K, Q, R, S, V, W or Y;

X ₁₀ = G, a, D, E, F, H, K, L, N, Q, R, T, V, W or Y; i.e. as shown in SEQ ID NO: 82), preferably X ₉ = G or Y, X ₁₀ = G, (4) amino acid sequence KSSQSLLX ₁₁SX₁₂X₁₃X₁₄X₁₅YLX₁₆(X₁₁ = H or Y, X ₁₂ = N, G, H, I, Y, L, S, T or V; X ₁₃ = G, a, I, N, P, S, T or V; X ₁₄ = Y, D, G, K, N or R;

X ₁₅ = H, D, K, N, Q, S, T or Y, X ₁₆ = D, a, G, H, N, Q, S or Y, i.e. as shown in SEQ ID NO: 83), preferably X ₁₁＝H,X₁₂＝N,X₁₃ = G, R, S or T, X ₁₄＝Y,X₁₅ = H, K or T, X ₁₆ = D, (5) an amino acid sequence of X ₁₇GSX₁₈RAS(X₁₇ = L, a, E, G, K, Q, R, S, V, W or Y, X ₁₈ = N, D, E, K, Q, R, S or T, i.e. as shown in SEQ ID NO: 84), LCDR2, preferably X ₁₇ = L, a, W or Y, X ₁₈ = N, (6) an amino acid sequence of X ₁₉QX₂₀LX₂₁TTRT(X₁₉ = M, Q, a, H, L or S, X ₂₀ = a, D, F, G, H, L, R, S, T, W or Y;

X ₂₁ = Q, D, G, H, I, R, S, T; i.e. as shown in SEQ ID NO: 85), preferably X ₁₉＝L,X₂₀＝A,X₂₁ = Q, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.

3. The anti-HER 4 antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising (1) an amino acid sequence of HCDR1 as shown in SEQ ID No. 1, (2) an amino acid sequence of HCDR2 as shown in any one of SEQ ID nos. 2-22, and (3) an amino acid sequence of HCDR3 as shown in SEQ ID No. 23 or 24, and (4) an amino acid sequence of LCDR1 as shown in any one of SEQ ID nos. 25-31, (5) an amino acid sequence of LCDR2 as shown in any one of SEQ ID nos. 32-35, and (6) an amino acid sequence of LCDR3 as shown in SEQ ID nos. 36 or 37.

4. The anti-HER 4 antibody or antigen-binding fragment thereof of any one of claims 1-3, comprising HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and LCDR1, LCDR2 and LCDR3 of the light chain variable region, each CDR comprising:

(1) An HCDR1 amino acid sequence as set forth in SEQ ID NO. 1, an HCDR2 amino acid sequence as set forth in any one of SEQ ID NO. 2-22, an HCDR3 amino acid sequence as set forth in SEQ ID NO. 23, and an LCDR1 amino acid sequence as set forth in SEQ ID NO. 25, an LCDR2 amino acid sequence as set forth in SEQ ID NO. 32, an LCDR3 amino acid sequence as set forth in SEQ ID NO. 36, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, or

(2) An HCDR1 amino acid sequence as set forth in SEQ ID NO. 1, an HCDR2 amino acid sequence as set forth in SEQ ID NO. 2, an HCDR3 amino acid sequence as set forth in SEQ ID NO. 24, and an LCDR1 amino acid sequence as set forth in SEQ ID NO. 25, an LCDR2 amino acid sequence as set forth in SEQ ID NO. 32, an LCDR3 amino acid sequence as set forth in SEQ ID NO. 36, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, or

(3) An HCDR1 amino acid sequence as set forth in SEQ ID NO. 1, an HCDR2 amino acid sequence as set forth in SEQ ID NO. 2, an HCDR3 amino acid sequence as set forth in SEQ ID NO. 23, and an LCDR1 amino acid sequence as set forth in any one of SEQ ID NO. 26-30, an LCDR2 amino acid sequence as set forth in SEQ ID NO. 32, an LCDR3 amino acid sequence as set forth in SEQ ID NO. 36, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, or

(4) An HCDR1 amino acid sequence as set forth in SEQ ID NO. 1, an HCDR2 amino acid sequence as set forth in SEQ ID NO. 2, an HCDR3 amino acid sequence as set forth in SEQ ID NO. 23, and an LCDR1 amino acid sequence as set forth in SEQ ID NO. 25, an LCDR2 amino acid sequence as set forth in SEQ ID NO. 33 or 35, an LCDR3 amino acid sequence as set forth in SEQ ID NO. 36, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, or

(5) An HCDR1 amino acid sequence as set forth in SEQ ID NO. 1, an HCDR2 amino acid sequence as set forth in SEQ ID NO. 2, an HCDR3 amino acid sequence as set forth in SEQ ID NO. 23, and an LCDR1 amino acid sequence as set forth in SEQ ID NO. 25, 28 or 31, an LCDR2 amino acid sequence as set forth in SEQ ID NO. 34, an LCDR3 amino acid sequence as set forth in SEQ ID NO. 36, or amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, or

(6) An HCDR1 amino acid sequence as set forth in SEQ ID NO. 1, an HCDR2 amino acid sequence as set forth in SEQ ID NO. 2, an HCDR3 amino acid sequence as set forth in SEQ ID NO. 23, and an LCDR1 amino acid sequence as set forth in SEQ ID NO. 25, an LCDR2 amino acid sequence as set forth in SEQ ID NO. 32, an LCDR3 amino acid sequence as set forth in SEQ ID NO. 37, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively, or

(7) An HCDR1 amino acid sequence as set forth in SEQ ID NO. 1, an HCDR2 amino acid sequence as set forth in SEQ ID NO. 21 or 22, an HCDR3 amino acid sequence as set forth in SEQ ID NO. 23, and an LCDR1 amino acid sequence as set forth in SEQ ID NO. 28 or 31, an LCDR2 amino acid sequence as set forth in SEQ ID NO. 34, an LCDR3 amino acid sequence as set forth in SEQ ID NO. 36, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.

5. The anti-HER 4 antibody or antigen binding fragment thereof of claim 1-4 comprising an HCDR1 amino acid sequence as set forth in SEQ ID NO. 1, an HCDR2 amino acid sequence as set forth in SEQ ID NO. 21 or 22, an HCDR3 amino acid sequence as set forth in SEQ ID NO. 21, and an LCDR1 amino acid sequence as set forth in SEQ ID NO. 28 or 31, an LCDR2 amino acid sequence as set forth in SEQ ID NO. 34, an LCDR3 amino acid sequence as set forth in SEQ ID NO. 36, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

6. The anti-HER 4 antibody or antigen-binding fragment thereof of any one of claims 1-5 comprising a heavy chain variable region VH having the HCDR1, HCDR2, and HCDR3 and a light chain variable region VL having the LCDR1, LCDR2, and LCDR3, comprising a VH having at least 80% identity to the amino acid sequence shown in any one of SEQ ID NOs 36-58 and a VL having at least 80% identity to the amino acid sequence shown in any one of SEQ ID NOs 59-76.

7. The anti-HER 4 antibody or antigen-binding fragment thereof of any one of claims 1-6, comprising a heavy chain variable region VH with the HCDR1, HCDR2, and HCDR3 and a light chain variable region VL with the LCDR1, LCDR2, and LCDR3, each comprising:

(1) Amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a VH shown in any one of SEQ ID NOS.38-62 and a VL shown in SEQ ID NO. 63, respectively, or

(2) Amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a VH shown in SEQ ID NO:38 and a VL shown in any one of SEQ ID NO:64-80, respectively, or

(3) Amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a VH represented by SEQ ID No. 61 or 62 and a VL represented by any one of SEQ ID nos. 77 to 80, respectively;

Preferably, the amino acid sequence 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. 61 and the VL shown in SEQ ID NO. 79, respectively, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL shown in SEQ ID NO. 62 and the VL shown in SEQ ID NO. 73, respectively.

8. A nucleic acid encoding the anti-HER 4 antibody or antigen-binding fragment thereof of any one of claims 1-7.

9. An expression vector capable of expressing the nucleic acid of claim 8.

10. A host cell comprising the nucleic acid of claim 8 or the expression vector of claim 9.

11. A method of making the anti-HER 4 antibody or antigen-binding fragment thereof of any one of claims 1-7 using the host cell of claim 10, comprising (i) expressing the anti-HER 4 antibody or antigen-binding fragment thereof in the host cell, and (ii) isolating the anti-HER 4 antibody or antigen-binding fragment thereof from the host cell or cell culture.

12. A bispecific or multispecific molecule comprising the anti-HER 4 antibody or antigen-binding fragment thereof of any one of claims 1-7, and having binding specificity for an Fc receptor, or an antigen associated with cancer selected from HER2, HER3, VEGF, VEGFR, c-Met, TFR (transferrin receptor), IGF-1R (human insulin-like growth factor I receptor), or an immune checkpoint protein antigen 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) moiety.

14. An immunoconjugate, chimeric antigen receptor, engineered T-cell receptor, or oncolytic virus, characterized in that it comprises the anti-HER 4 antibody or antigen binding fragment thereof according to any one of claims 1-7.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-7, or the bispecific 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 kit comprising the pharmaceutical composition of claim 15, together with one or more other therapeutic agents selected from the group consisting of signal pathway modulators, cell cycle inhibitors, and inflammatory response modulators.

17. Use of an anti-HER 4 antibody or antigen-binding fragment thereof according to any one of claims 1-7 for the preparation of a pharmaceutical composition or formulation for the treatment or prevention of a HER 4-related disease or disorder.

18. A method of treating or preventing a HER 4-related disease or disorder, the method comprising administering to a subject the anti-HER 4 antibody or antigen-binding fragment of any one of claims 1-7, or the pharmaceutical composition of claim 15, or the kit of claim 16.

19. The use according to claim 17 and the method according to claim 18, wherein the disease or condition comprises cancer and other HER4 related diseases, the cancer comprising glioma and the other HER4 related diseases comprising gliosis, e.g. stroke, alzheimer's disease, viral encephalitis, traumatic brain injury, multiple sclerosis, epilepsy, the subject being a human, non-human primate or other mammal (e.g. a dog), etc.

20. The method of claim 18 or 19, wherein the method is capable of inhibiting ligand-induced HER4 dimerization in the subject, the ligand comprising NRG-1-4, preferably NRG-1.

21. A method of detecting and/or measuring HER4 or HER4 expressing cells in a sample, and a method of screening a patient for a HER 4-related disorder responsive to treatment with an anti-HER 4 antibody or antigen-binding fragment thereof of any one of claims 1-7, comprising incubating the anti-HER 4 antibody or antigen-binding fragment thereof of 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.