CN119013307A - Antibodies or antigen binding fragments thereof and medical uses thereof - Google Patents

Abstract

Provided are anti-ERBB3 antibodies or antigen-binding fragments thereof and medical uses thereof, and provided are fully human antibodies or antigen-binding fragments thereof against ERBB3 receptors and medical uses thereof. Provided are pharmaceutical compositions comprising fully human anti-ERBB3 antibodies or antigen-binding fragments thereof, and uses thereof as anticancer drugs.

Description

Antibodies or antigen binding fragments thereof and medical uses thereof Technical Field

The present invention relates to ERBB3 antibodies or antigen-binding fragments thereof, and further, the present invention relates to ERBB3 fully human antibodies or antigen-binding fragments thereof comprising CDR regions; the invention also relates to pharmaceutical compositions comprising said ERBB3 fully human antibodies or antigen-binding fragments thereof, and their use as diagnostic and therapeutic agents for ERBB3 related diseases.

Background

ERBB receptors are widely expressed in neuronal, epithelial, and mesenchymal cells, are involved in the development of cardiovascular, neural, musculoskeletal and other organs, and are involved in cancer pathogenesis. ERBB receptor has 4 family proteins: EGFR, ERBB2, ERBB3 and ERBB4 are membrane proteins, have similar molecular structures, and comprise an extracellular domain (ECD), a transmembrane domain, an intracellular domain with tyrosine kinase activity, and a C-terminal end. ECD can be subdivided into 4 subdomains: subdomain I, II, III, IV (BMC Bioinformatics 2001, 2:4; mol Cell 2003, 11:507-17.) wherein I and III are leucine-rich and are ligand binding domains; II and IV are cysteine-rich and are dimer-forming domains. In addition to ERBB2, the other three ERBB receptors bind II and IV in the inactive state and open when bound and activated by the ligand, exposing II to form dimers. EGFR and ERBB2 are more studied at present, and corresponding targeted drugs are more intensively researched and developed. In recent years, the attention of ERBB3 is continuously increased, in EGFR and ERBB2 related drug resistance, ERBB3 bypass plays a key role, and ERBB3 has higher expression in tumors such as breast cancer, lung cancer, prostate cancer, colorectal cancer, ovarian cancer, gastric cancer, bladder cancer, melanoma and the like, so ERBB3 is another potential target for tumor treatment. ERBB3 has 2 ligands NRG1 and NRG2, which activate the kinase activity of ERBB3, and activated ERBB3 forms a heterodimer with EGFR or ERBB2 and phosphorylates the latter, which in turn transmits signals to downstream pathways that promote tumor cell growth and proliferation. In addition, ERBB3 can also signal by "cross talk" with other RTKs in cancer, such as the formation of complexes with IGF1R, FGFR2 and HGFR (c-Met) (BioDrugs 2017, 31:63-73), and it is seen that ERBB3 plays an important role in tumor growth.

Currently, therapeutic strategies for ERBB3 targets follow several mechanisms: 1) Locking ERBB3 in an inactive state (e.g., CDX-3379); 2) Capture ERBB3 ligand NRG (e.g., RB 200); 3) Blocking ERBB3 binding to the ligand (e.g., U3-1287); 4) Promoting endocytosis of ERBB3 (antibody drug conjugate); 5) Blocking dimerization of ERBB3 with other EGFR family members; 6) Recruiting immune cells kills cancer cells expressing endogenous ERBB 3. . Targeted therapies for ERBB3 for these mechanisms may be: monoclonal antibodies, bispecific antibodies, vaccines against ERBB3, LIGAND TRAP, RNA inhibitors that lock ERBB3, small molecules that inhibit ERBB3 kinase activity, and the like. Wherein, the research and development heat of the targeted ERBB3 antibody is higher.

In clinical studies, an ERBB3 antibody from murine origin significantly inhibited the growth of cytoma in patients (data referencing Celldex). The humanized antibody of the murine antibody of the ERBB3 antibody which is currently being researched is subjected to humanized modification, and the immunogenicity of the humanized antibody is higher than that of the fully-humanized antibody without the murine antibody component when the humanized antibody is immunized, so that the humanized antibody is a disadvantageous factor when the humanized antibody is applied to human bodies; some ERBB3 antibody medicines are fully human antibodies obtained by phage display technology, and can solve the problem of immunogenicity.

Phage display technology (PHAGE DISPLAY technology) is to express exogenous proteins or polypeptides fused to phage coat proteins, thereby expressing the exogenous proteins on the surface of phage. The phage antibody library is an antibody library established by combining phage display technology, PCR amplification technology and protein expression technology and using comprehensive technical means.

The phage library generally comprises a synthetic library, an immune library and a natural library, the natural library is a fully human antibody library constructed by immune cells of human peripheral blood, and the biggest advantage of the phage library is that fully human antibodies with high diversity produced by a human body can be obtained without additional in-vivo immunization. In addition, phage antibody libraries have the following advantages: ① The unification of genotype and phenotype is realized. In addition, the experimental method is simple and rapid, and the conventional antibody production method by the hybridoma technique needs months, while the antibody library technique needs only a few weeks. ② The expression is fully human antibody, has small molecular weight, is mainly expressed in the form of active fragments Fab and scFV, and has obvious advantages in the aspect of tissue penetration compared with the whole antibody. ③ The screening capacity is large, the hybridoma technology is screened in thousands of clones, and the antibody library technology can select millions or even hundreds of millions of molecules. The screened antibodies were more abundant. ④ The use is wide, a prokaryotic expression system is adopted, and the advantage is more obvious when the prokaryotic expression system is produced in a large scale (Curr Opin Biotechnol.2002 Dec;13 (6): 598-602; immunotechnology,2013,48 (13) 48 (13): 63-73).

At present, WO2007077028 et al reports that antibodies of ERBB3, mostly murine or humanized antibodies, and also partially fully human antibodies obtained from phage libraries, are mostly in early clinical stages and in discovery stages at home and abroad, and no antibody drugs targeting ERBB3 are marketed, so that further development of ERBB3 fully human antibodies with higher activity, high affinity and high stability is necessary for therapeutic research and application of related diseases.

Disclosure of Invention

The present invention provides an anti-ERBB 3 antibody or antigen-binding fragment thereof comprising: heavy chain variable region and light chain variable region; wherein the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 1. 60 and 61, HCDR1, HCDR2 and HCDR3, the light chain variable region comprising the amino acid sequences as set forth in SEQ ID NOs: 62. LCDR1, LCDR2 and LCDR3 as shown in figures 6 and 63 wherein said SEQ ID NO: 60. SEQ ID NO: 61. SEQ ID NO:62 and SEQ ID NO:63 is an amino acid sequence having the general formula:

Wherein: x ₁ is selected from A or G; x ₂ is selected from T or K; x ₃ is selected from L, F, Y or Q; x ₄ is selected from G or S; x ₅ is selected from S or D; x ₆ is selected from S or E; x ₇ is selected from D, G or E; x ₈ is selected from G, F, K, L, V, R or Q; x ₉ is selected from S, W or F; x ₁₀ is selected from V, S or T; x ₁₁ is selected from T, S or N.

The invention also relates to a preferred embodiment, an anti-ERBB 3 antibody, or antigen-binding fragment thereof, as defined above, comprising a heavy chain variable region and an antibody light chain variable region, wherein,

The heavy chain variable region comprises HCDR1 shown as SEQ ID NO. 1, HCDR2 shown as SEQ ID NO. 3 or 30, and HCDR3 shown as any one of amino acid sequences of SEQ ID NO. 4, 28, 31, 32, 33 or 34;

The light chain variable region comprises LCDR1 shown in any one of the amino acid sequences of SEQ ID NO. 5, 19, 21, 23, 25, 27, 29 or 35, LCDR2 shown in SEQ ID NO. 6 and LCDR3 shown in any one of the amino acid sequences of SEQ ID NO. 7, 20, 22, 24 or 26.

The present invention also relates to a preferred embodiment, an anti-ERBB 3 antibody or antigen-binding fragment thereof as described above comprising a heavy chain variable region and a light chain variable region, wherein:

The heavy chain variable region comprises HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3 and HCDR3 shown in SEQ ID NO. 4; the light chain variable region comprises LCDR1 shown in SEQ ID NO. 5, LCDR2 shown in SEQ ID NO. 6 and LCDR3 shown in SEQ ID NO. 7; or alternatively, the first and second heat exchangers may be,

The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 4; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 19, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 20; or alternatively, the first and second heat exchangers may be,

The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 4; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 21, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 22; or alternatively, the first and second heat exchangers may be,

The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 4; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 23, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 24; or alternatively, the first and second heat exchangers may be,

The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 4; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 25, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 26; or alternatively, the first and second heat exchangers may be,

The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 4; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 27, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 22; or alternatively, the first and second heat exchangers may be,

The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 28; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 29, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 7; or alternatively, the first and second heat exchangers may be,

The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 30, and HCDR3 shown in SEQ ID NO. 31; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 5, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 7; or alternatively, the first and second heat exchangers may be,

The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 32; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 5, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 7; or alternatively, the first and second heat exchangers may be,

The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 33; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 29, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 7; or alternatively, the first and second heat exchangers may be,

The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 34; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 35, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 7.

The present invention also relates to a preferred embodiment, an anti-ERBB 3 antibody or antigen-binding fragment thereof as described above, wherein said heavy chain variable region is at least one heavy chain variable region selected from the group consisting of: SEQ ID NO. 9, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45 and SEQ ID NO. 46, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology thereto.

The invention also relates to a preferred embodiment, an anti-ERBB 3 antibody or antigen-binding fragment thereof as described above, wherein said light chain variable region is at least one light chain variable region selected from the group consisting of light chain variable region ：SEQ ID NO:10、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:47, shown in the following sequence or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology thereto.

The present invention also relates to a preferred embodiment, an anti-ERBB 3 antibody or antigen-binding fragment thereof as described above, wherein said anti-ERBB 3 antibody or antigen-binding fragment thereof comprises the heavy chain variable region shown in SEQ ID No. 9 and the light chain variable region shown in SEQ ID No. 10; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO. 9, and a light chain variable region shown in SEQ ID NO. 36; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO. 9, and a light chain variable region shown in SEQ ID NO. 37; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO. 9, and a light chain variable region shown in SEQ ID NO. 38; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO. 9, and a light chain variable region shown in SEQ ID NO. 39; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO. 9, and a light chain variable region shown in SEQ ID NO. 40; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO. 41, and a light chain variable region shown in SEQ ID NO. 42; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO. 43, and a light chain variable region shown in SEQ ID NO. 10; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown as SEQ ID NO. 44, and a light chain variable region shown as SEQ ID NO. 10; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises the heavy chain variable region shown in SEQ ID NO. 45 and the light chain variable region shown in SEQ ID NO. 42 or,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises the heavy chain variable region shown in SEQ ID NO. 46, and the light chain variable region shown in SEQ ID NO. 47.

In a preferred embodiment of the invention, the anti-ERBB 3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region comprising a polypeptide derived from human IgG1, igG2, igG3 or IgG4 or a mutant thereof;

In a further preferred embodiment of the invention, the anti-ERBB 3 antibody or antigen-binding fragment thereof further comprises the heavy chain constant region of human IgG1 or variant thereof;

In a further preferred embodiment of the invention, the anti-ERBB 3 antibody or antigen-binding fragment thereof further comprises a human IgG1 heavy chain constant region;

In a further preferred embodiment of the invention, the anti-ERBB 3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO. 17;

In a further preferred embodiment of the invention, the anti-ERBB 3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain, lambda chain or mutant thereof;

In a further preferred embodiment of the invention, the anti-ERBB 3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human lambda chain.

In a further preferred embodiment of the invention, the anti-ERBB 3 antibody or antigen-binding fragment thereof further comprises the light chain constant region shown as SEQ ID NO. 18.

In a preferred embodiment of the invention, the anti-ERBB 3 antibody or antigen-binding fragment thereof, wherein the anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain selected from the group consisting of: SEQ ID NO. 12, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 57 or SEQ ID NO. 58, or a heavy chain having at least 80%, 85%, 90%, 95% or 99% homology thereto.

In a preferred embodiment of the invention, the anti-ERBB 3 antibody or antigen-binding fragment thereof, wherein the anti-ERBB 3 antibody or antigen-binding fragment thereof comprises light chain ：SEQ ID NO:13、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:59, or a light chain having at least 80%, 85%, 90%, 95% or 99% homology thereto.

The present invention also relates to a preferred embodiment, an anti-ERBB 3 antibody or antigen-binding fragment thereof as described above, wherein said anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID No. 12 and a light chain as shown in SEQ ID No. 13; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown as SEQ ID NO. 12, and a light chain shown as SEQ ID NO. 48; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain represented by SEQ ID NO. 12, and a light chain represented by SEQ ID NO. 49; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown as SEQ ID NO. 12, and a light chain shown as SEQ ID NO. 50; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown in SEQ ID NO. 12, and a light chain shown in SEQ ID NO. 51; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown in SEQ ID NO. 12, and a light chain shown in SEQ ID NO. 52; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown as SEQ ID NO. 53, and a light chain shown as SEQ ID NO. 54; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown as SEQ ID NO. 55, and a light chain shown as SEQ ID NO. 13; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown as SEQ ID NO. 56, and a light chain shown as SEQ ID NO. 13; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown as SEQ ID No. 57 and a light chain shown as SEQ ID No. 54; or alternatively, the first and second heat exchangers may be,

The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown as SEQ ID NO. 58, and a light chain shown as SEQ ID NO. 59.

The invention further provides a polynucleotide encoding the anti-ERBB 3 antibody or antigen-binding fragment thereof described above.

The present invention further provides an expression vector comprising the polynucleotide described above.

The present invention further provides a host cell into which or containing the above expression vector is introduced.

In a preferred embodiment of the invention, the host cell is a bacterium, preferably E.coli.

In a preferred embodiment of the invention, the host cell is a yeast, preferably pichia pastoris.

In a preferred embodiment of the invention, the host cell is a mammalian cell, preferably a CHO cell or a HEK293 cell.

The invention further provides a method of producing an anti-ERBB 3 antibody or antigen-binding fragment thereof, comprising the steps of:

a) Culturing the host cell described above;

b) Isolating fully human antibodies from the culture; and

C) Purifying the antibody.

The invention further provides a pharmaceutical composition comprising an anti-ERBB 3 antibody or antigen-binding fragment thereof as described above, and a pharmaceutically acceptable excipient, diluent or carrier.

The invention further provides a detection or diagnostic reagent comprising an anti-ERBB 3 antibody or antigen-binding fragment thereof as described above and an excipient, diluent or carrier useful for detection or diagnosis.

The invention further provides the use of an anti-ERBB 3 antibody as described above, or an antigen-binding fragment thereof, or a composition as described above, in the manufacture of a medicament for the treatment or prophylaxis of an ERBB3 mediated disease or condition.

The invention further provides the use of an anti-ERBB 3 antibody or antigen-binding fragment thereof as described above or a detection or diagnostic reagent as described above in the preparation of a kit for the detection, diagnosis, prognosis of an ERBB3 mediated disease or disorder.

In a preferred embodiment of the present invention,

The disease or disorder is cancer;

Preferably ERBB3 expressed or overexpressed cancers;

More preferably breast cancer, ovarian cancer, prostate cancer, endometrial cancer, thyroid cancer, kidney cancer, lung cancer, stomach cancer, colon cancer, bladder cancer, cervical cancer, gall bladder cancer, pancreatic cancer, testicular cancer, soft tissue sarcoma, head and neck cancer, glioma or melanoma.

The invention further provides a method of treating or preventing an ERBB3 mediated disease comprising the steps of:

Providing a therapeutically effective amount or a prophylactically effective amount of an anti-ERBB 3 antibody or antigen-binding fragment thereof as described above to a subject; or providing a therapeutically effective amount or a prophylactically effective amount of the above-described pharmaceutical composition to a subject; wherein the ERBB3 mediated disease is selected from the group consisting of: breast cancer, ovarian cancer, prostate cancer, endometrial cancer, thyroid cancer, kidney cancer, lung cancer, gastric cancer, colon cancer, bladder cancer, cervical cancer, gall bladder cancer, pancreatic cancer, testicular cancer, soft tissue sarcoma, head and neck cancer, glioma or melanoma.

Detailed Description

Detailed Description

1. Terminology

In order that the application may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The three-letter and one-letter codes for amino acids used in the present application are as described in J.biol. Chem,243, p3558 (1968).

The term "antibody" as used herein refers to an immunoglobulin that is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains joined by interchain disulfide bonds. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins, igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α and ε chains, respectively. The same class of Ig can be further classified into different subclasses according to the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chain, e.g., igG can be classified into IgG1, igG2, igG3, and IgG4. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each class Ig of the five classes of Igs may have either a kappa chain or a lambda chain.

In the present application, the antibody light chain variable region of the present application may further comprise a light chain constant region comprising a kappa, lambda chain of human or murine origin or variants thereof.

In the present application, the antibody heavy chain variable region of the present application may further comprise a heavy chain constant region comprising IgG1, igG2, igG 3, igG 4 or variants thereof of human or murine origin.

The sequences of the heavy and light chains of the antibody near the N-terminus vary widely, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions, arranged in the order from amino-to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3. The CDR amino acid residues of the VL and VH regions of the antibodies or antigen-binding fragments of the application conform in number and position to known Kabat or Chothia or ABM definition rules (http:// bioinf org. Uk/abs /).

The term "antigen presenting cell" or "APC" is a cell displaying on its surface a foreign antigen complexed with MHC. T cells recognize this complex using the T Cell Receptor (TCR). Examples of APCs include, but are not limited to, dendritic Cells (DCs), peripheral Blood Mononuclear Cells (PBMCs), monocytes, B lymphoblastic cells, and monocyte derived dendritic cells.

The term "antigen presentation" refers to the process by which APCs capture antigens and enable them to be recognized by T cells, for example as a component of MHC-I/MHC-II conjugates.

The term "recombinant human antibody" includes human antibodies prepared, expressed, created, or isolated by recombinant methods, the techniques and methods involved are well known in the art, such as:

1. An antibody isolated from a transgene of a human immunoglobulin gene, a transchromosomal animal (e.g., mouse) or a hybridoma prepared therefrom;

2. An antibody isolated from a host cell transformed to express the antibody, such as a transfectoma;

3. an antibody isolated from a recombinant combinatorial human antibody library; and

4. Antibodies are prepared, expressed, created or isolated by splicing human immunoglobulin gene sequences to other DNA sequences, and the like.

Such recombinant human antibodies comprise variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include rearrangements and mutations that occur later, such as during antibody maturation.

The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the application may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (i.e., a "humanized antibody").

The term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which generally include at least a portion of the antigen-binding or variable regions (e.g., one or more CDRs) of the parent antibody (parental antibody). The antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, an antibody fragment retains at least 10% of the parent binding activity when expressed on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target. Examples of antigen binding fragments include, but are not limited to: fab, fab ', F (ab') 2, fv fragments, linear antibodies, single chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson,2005, nat. Biotechnol.23: 1126-1136.

"Fab fragment" consists of a light chain and a heavy chain CH1 and variable domains. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

The "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic effect of the CH3 domain.

"Fab ' fragments" contain portions of one light chain and one heavy chain comprising the VH domain and CH1 domain and the region between the CH1 and CH2 domains, whereby an inter-chain disulfide bond can be formed between the two heavy chains of two Fab ' fragments to form a F (ab ') 2 molecule.

"F (ab') 2 fragments" contain two light chains and two heavy chains comprising portions of the constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Thus, a F (ab ') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

The "Fv region" comprises variable regions from both the heavy and light chains, but lacks constant regions.

The term "multispecific antibody" is used in its broadest sense to encompass antibodies with multiple epitope specificities. Such multispecific antibodies include, but are not limited to: an antibody comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL units have polyepitopic specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies (triabodies), antibody fragments that have been covalently or noncovalently linked together, and the like.

The term "single chain antibody" is a single chain recombinant protein formed by joining the heavy chain variable region VH and the light chain variable region VL of an antibody through a connecting peptide, which is the smallest antibody fragment with a complete antigen binding site.

The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment that contains only heavy chain variable region or light chain variable region chains. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment. The two VH regions of a bivalent domain antibody fragment may target the same or different antigens.

The term "binds to ERBB 3" in the present application means that it is capable of interacting with human ERBB 3.

The term "antigen binding site" according to the application refers to a three-dimensional spatial site recognized by an antibody or antigen binding fragment of the application.

The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed by contiguous amino acids, or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically maintained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation detection assays, among others. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography, two-dimensional nuclear magnetic resonance, and the like.

The terms "specifically bind", "selectively bind" and "selectively bind" as used herein refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, when human ERBB3 is used as the analyte and an antibody is used as the ligand, the antibody binds to the predetermined antigen with an equilibrium dissociation constant (K _D) of about less than 10 ^-7 M or even less, and with an affinity that is at least twice that of its binding to a predetermined antigen or to a non-specific antigen other than a closely related antigen (e.g., BSA, etc.), as determined by Surface Plasmon Resonance (SPR) techniques in an instrument. The term "antibody that recognizes an antigen" may be used interchangeably herein with the term "antibody that specifically binds".

The term "cross-reactive" refers to the ability of an antibody of the application to bind to ERBB3 from a different species. For example, an antibody of the application that binds human ERBB3 may also bind ERBB3 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen, or binding or functional interaction with cells physiologically expressing ERBB3, in binding assays (e.g., SPR and ELISA). Methods of determining cross-reactivity include standard binding assays as described herein, such as Surface Plasmon Resonance (SPR) analysis, or flow cytometry.

The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of the ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking is also intended to include any measurable decrease in ligand binding affinity upon contact with an anti-ERBB 3 antibody as compared to a ligand not contacted with an anti-ERBB 3 antibody.

The term "inhibit growth" (e.g., involving a cell) is intended to include any measurable decrease in cell growth.

The terms "inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to stimulation (i.e., passive or adaptive) of an immune response to a particular antigen. The term "induce" with respect to inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.

The term "ADCC", namely, anti-body-DEPENDENT CELL-mediated cytotoxicity, as used herein, refers to antibody-dependent cell-mediated cytotoxicity, which means that cells expressing Fc receptors directly kill target cells coated with antibodies by recognizing the Fc segment of the antibodies. The ADCC effector function of an antibody may be reduced or decreased by modification of the Fc fragment on IgG. The modification refers to mutation in the heavy chain constant region of the antibody.

The engineered antibodies or antigen binding fragments of the application can be prepared and purified by conventional methods. The cDNA sequences of the corresponding antibodies can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of the antibody, particularly at the highly conserved N-terminus of the FC region. Stable clones were obtained by expressing antibodies that specifically bound to human antigens. Positive clones were expanded in serum-free medium of the bioreactor to produce antibodies. The antibody-secreting culture may be purified and collected using conventional techniques. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

"Administering," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administration," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a reagent with the cell, and contacting the reagent with a fluid, wherein the fluid is in contact with the cell. "administration," "administration," and "treatment" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

"Treatment" means administration of a therapeutic agent, such as an antibody comprising any of the present application, for internal or external use to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") effective to alleviate any particular disease symptom can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test method that a physician or other healthcare professional typically uses to assess the severity or progression of the symptom. While embodiments of the present application (e.g., therapeutic methods or articles of manufacture) may be ineffective in alleviating the symptoms of each patient suffering from the disease of interest, they should alleviate the symptoms of the disease of interest in a statistically significant number of patients as determined by any statistical test methods known in the art, such as Student t test, chi-square test, U test according to Mann and Whitney, kruskal-Wallis test (H test), jonckheere-Terpstra test, and Wilcoxon test.

The term "consisting essentially of … …" or variations thereof as used throughout the specification and claims is meant to include all such elements or groups of elements, and optionally other elements of similar or different nature to those described, which do not significantly alter the basic or novel nature of the prescribed dosing regimen, method or composition.

The term "naturally occurring" as applied to an object in the present application refers to the fact that the object may be found in nature. For example, polypeptide sequences or polynucleotide sequences that are present in organisms (including viruses) that can be isolated from natural sources and that have not been intentionally modified by man in the laboratory are naturally occurring.

An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the route of administration and the dosage and severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.

"Exogenous" refers to a substance that is to be produced by background outside an organism, cell or human.

"Endogenous" refers to substances produced in cells, organisms or humans according to background.

"Homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both comparison sequences is occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, in the optimal alignment of sequences, if there are 6 matches or homologies at 10 positions in the two sequences, then the two sequences are 60% homologous. In general, a comparison is made when two sequences are aligned to give the greatest percent homology.

The expressions "cell", "cell line" and "cell culture" are used interchangeably herein and all such designations include their progeny. Thus, the words "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to deliberate or unintentional mutations. Including mutant progeny having the same function or biological activity as screened in the original transformed cell. Where different names are meant, they are clearly visible from the context.

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may be, but need not be, present.

"Pharmaceutical composition" means a composition comprising one or more antibodies or antigen-binding fragments thereof as described herein, as well as other components such as physiological/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

2. Examples

The application will be further described with reference to the following examples, which are not intended to limit the scope of the application. The experimental method without specific conditions being noted in the embodiment of the application is generally according to conventional conditions, such as an antibody technical laboratory manual and a molecular cloning manual of cold spring harbor; or according to the conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.

Example 1: screening and construction of fully human antibodies

ERBB3 antigen design:

The amino acid sequence of human ERBB3 protein shown in SEQ ID NO. 14 is used as the template for designing the antigen of the present invention. Hereinafter, the ERBB3 antigen is not specifically described and refers to human ERBB3.

ERBB3 full-length protein: ERBB3 (SEQ ID NO: 14) sequence is as follows:

screening ERBB3 antigen as commercial product Biotinylated human ERBB-His tag (Sino biological, cat# 10201-H08H-B); the ERBB3 antigen for detection is a commercial product human ERBB3-His tag (Sino biological, cat# 10201-H08H). The ERBB3 antigen for detection is ECD region sequence of human ERBB3, its C end is provided with His tag, and the ERBB3 antigen for screening is biotinylated. The ECD sequence of human ERBB3 is as follows:

The monkey ERBB3 antigen (SEQ ID NO: 16) used for detection is commercial product Rhesus ERBB3 Protein-His Tag (Sino biological, cat# 90043-K08H), and is characterized in that the ECD region sequence of the monkey ERBB3 antigen is labeled with His at the C terminal, and the sequence is as follows:

PBMC of a plurality of persons are collected, B cells are separated, RNA is extracted, reverse transcription is carried out to cDNA, and then the cDNA is used as a template to construct a natural phage surface Fab display library (the library capacity is 1.6X10 ¹¹). Packaging the constructed natural Fab phage library to form phage particles, then panning by a liquid phase method, combining phage with biotinylated ERBB3 liquid phase, and separating by using streptavidin magnetic beads. To obtain positive sequences binding to human ERBB3, 2-3 rounds of panning were performed using biotinylated human ERBB3, 384 monoclonal colonies were picked and packaged as phage display Fab fragments for ELISA testing. Testing the binding activity of monoclonal phages to human ERBB 3: coating ERBB3 with 1 μg/mL on ELISA plate, adding phage supernatant, and detecting with anti-human IgG Fab HRP; 86 positive clones with OD ₄₅₀ value greater than 0.2 tested by ELISA were sequenced, and the sequences were analyzed to obtain unique sequences: 45 VH (heavy chain variable region) and 51 VL (light chain variable region). The obtained heavy chain variable region sequences and the light chain variable region sequences are respectively connected with the human IgG1 heavy chain constant region sequences and the human light chain constant region sequences to obtain full-length antibodies, and experimental evaluation is carried out on the full-length antibodies to finally determine 1 antibody Ab1 with better binding force and function; wherein the sequences of the heavy chain constant region and the light chain constant region of the connected human IgG1 are respectively shown as SEQ ID NO. 17 and SEQ ID NO. 18.

S57E (Kabat numbering) site-directed mutagenesis was performed on HCDR2 of Ab1 antibody to form novel HCDR2 (SEQ ID NO: 3), thereby obtaining antibody Ab2.

TABLE 1 heavy and light chain variable region CDR sequences of antibodies

TABLE 2 heavy and light chain variable region sequences of antibodiesNote that: CDR regions are underlined.

TABLE 3 heavy and light chain sequences of antibodies Note that: CDR regions are underlined.

TABLE 4 sequence numbering of antibodies and their heavy, light and variable regions

The IgG1 heavy chain constant region and light chain constant region sequences are as follows:

Example 2: engineering of fully human antibodies

Site mutation was performed on the basis of the sequence of Ab2, random saturation mutation was performed on heavy and light chain CDR regions, phage libraries of single CDR or double CDR combination mutations were constructed, and the libraries were displayed in the form of scFv (VH-VL), and library capacities were evaluated and above 10 ⁷ as shown in Table 5. 5 rounds of alternate enrichment with ERBB3 antigen protein and HEK293T cells overexpressing human ERBB3 (293T-human ERBB 3) were performed, the primary screening was performed by ELISA to obtain 1056 clones, then FACS re-screening was performed and sample sequencing was performed, finally 103 unique candidate sequences were obtained, and 10 antibodies binding to TOP10 were selected for production purification.

TABLE 5 fully humanized antibody scFv mutant phage library

Clones selected from the mutant library based on the Ab2 antibody sequence were different from the Ab2 antibodies in HCDR2, HCDR3, LCDR1, LCDR3 on the light and heavy chains. The relevant CDR (amino acid residues of the CDR are determined and annotated by the Kabat numbering system) sequences or formulae, the corresponding light/heavy chain variable regions and their light/heavy chains are as follows:

TABLE 6 heavy and light chain variable region CDR sequences of mutant antibodies

TABLE 7 heavy and light chain variable region sequences of antibodies Note that: CDR regions are underlined.

TABLE 8 heavy and light chain sequences of mutant antibodies Note that: CDR regions are underlined

Example 3: preparation of fully human antibodies and mutants thereof

CDNA fragments were synthesized from the amino acid sequences of the light and heavy chains of Ab1 and Ab2 antibodies and inserted into pcDNA3.1 expression vectors (Life Technologies Cat. No. V790-20). The expression vector and transfection reagent PEI (Polysciences, inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a 1:2 ratio and incubated in a CO ₂ incubator for 4-5 days. Collecting cell culture solution, centrifugally filtering, loading the cell culture solution to an antibody purification affinity column, eluting by a phosphate buffer solution, eluting by glycine-hydrochloric acid buffer solution (pH2.7.1M Gly-HCl), neutralizing by 1M Tris-hydrochloric acid pH 9.0, and dialyzing by the phosphate buffer solution to obtain the antibody protein, wherein the concentration and the purity of the antibody protein are shown in Table 9:

TABLE 9 concentration and purity of fully human antibodies

The 10 antibodies Ab2_M1-M10 combined with TOP10 are produced and purified by the same method to obtain the fully human antibody mutant protein.

Example 4: in vitro binding experiments of fully human antibody mutants

4.1 Stable cell-binding assays

HEK293T cells overexpressing human ERBB3 (293T-human ERBB 3), after pancreatin digestion, cells were collected by centrifugation, cell density was adjusted with FACS buffer (1 XPBS with 2% FBS) and plated on 96-well U-bottom plates at 1X 10 ⁵ to 2X 10 ⁵ cells per well. And (3) centrifuging: 1200g, 5 min, the supernatant was discarded, 100. Mu.L of antibody solution which had been diluted with FACS buffer gradient was added and incubated for 1 hour at4 ℃. And (3) centrifuging: 1200g, 5 min, the supernatant was discarded, and after 2 times of FACS buffer washing, the fluorescent-labeled secondary antibody working solution PE anti-human IgG Fc Antibody (Biolegend, cat#409304) prepared by FACS buffer was added, and 100. Mu.L of cells were resuspended per well and incubated at4℃for 1 hour. And (3) centrifuging: 1200g, 5 min, the supernatant was discarded. After 2 times FACS buffer washing, the cells were resuspended in PBS, fluorescence signals were detected using a flow cytometer Bio-Rad (ZE 5) and the EC ₅₀ concentration of antibody-bound cells was plotted.

The results show that: EC ₅₀ of the engineered mutants ab2_m6, ab2_m7, ab2_m8, ab2_m9 and ab2_m10 showed enhanced binding compared to ab2, and the binding of the other 5 mutants was similar to ab2.

TABLE 10 binding force of fully human antibody mutants to 293T-hERBB3 cells

4.2 Tumor cell binding experiments

Binding force of 10 purified fully human antibody mutants to human breast cancer cells MX-1 expressing ERBB3 antigen was examined by the detection method of example 4.1, curve fitting was performed according to the Log (agonist) vs. response-Variable slope (four parameters) formula with MFI as abscissa and concentration logarithmic values as abscissa, and EC ₅₀ was calculated, and the results are shown in the following table.

The results show that: the EC50 of the engineered fully human antibody mutant was stronger for MX-1 cells than for its parent Ab2.

TABLE 11 binding of fully human antibody mutants to MX-1 expressing cells

Example 5: endocytic assay of fully human antibody mutants

The antibodies of the invention were tested for their ability to bind ERBB3 and then to be co-engulfed with human ERBB3 and evaluated against human breast adenocarcinoma MX-1 expressing ERBB 3. Cells were digested with pancreatin, collected and resuspended with pre-chilled FACS buffer to adjust the cell concentration to 2x10 ⁶/mL. EP tube was taken, 1mL of cell suspension was added, centrifuged at 1500rpm for 5 minutes, the supernatant was discarded, 1mL of the prepared antibody to be assayed was added to re-suspend the cells, the final concentration of the antibody was 20. Mu.g/mL, the incubation was carried out for 1 hour at 4℃in a shaker, the supernatant was discarded by centrifugation (4℃at 1500 rpm. Times.5 minutes), FACS buffer was washed twice, and the supernatant was discarded. 1mL of a fluorescent-labeled secondary antibody working solution PE anti-human IgG Fc Antibody (Biolegend, cat#409304) was added to each tube, the cells were resuspended, incubated for 30min at 4℃with a shaker, the supernatant was discarded by centrifugation (4 ℃,1500 rpm. Times.5 min), and the supernatant was discarded by FACS buffer washing twice. 1mL of preheated cell culture medium was added to each tube to resuspend the cells and mix them uniformly, each tube was divided into 4 tubes of 250. Mu.L, each of which was 0 minute group, blank group, 15 minute group and 30 minute group, and the rest was placed on ice for 0 minute and blank, and the rest was placed in a37℃incubator for 15 minutes and 30 minutes of endocytosis, respectively, EP tubes were taken out at the corresponding time points, placed on ice for 5 minutes of precooling, and the supernatant was discarded by centrifugation (4℃for 1500rpm X5 minutes) for all treatment groups, washed once with FACS buffer, and discarded. 250. Mu.L of strip buffer was added to all treatment EP tubes outside the 0-minute group, incubated at room temperature for 8 minutes, the supernatant was centrifuged off (4 ℃ C., 1500 rpm. Times.5 minutes), FACS buffer washed twice, and the supernatant was discarded. All treatment groups were resuspended in 100. Mu.L PBS and examined by flow cytometry Bio-Rad (ZE 5).

Percentage of endocytosis (%) = (fluorescence intensity value at each time point-average fluorescence intensity value of Blank group)/(average light fluorescence value of 0 minutes-average fluorescence intensity value of Blank group) ×100, and specific results are shown in table 12:

TABLE 12 fully human antibody mutants endocytose in tumor cells MX-1

The result shows that the fully human antibody mutant has obvious endocytosis in human breast adenocarcinoma cells MX-1, the endocytosis efficiency of the cells on the antibody is gradually improved along with the time extension, the peak is reached for 30min, the saturation is reached for 60min, and the endocytosis efficiency of the mutant antibody cells is integrally improved.

Example 6: cell proliferation inhibition assay

The present experiment aims at evaluating that candidate antibodies block the binding of ligand HRG and ERBB3 by binding to ERBB3 receptor on the surface of tumor cell membrane, thereby blocking the formation of heterodimer of ERBB3 and other EGFR family members, blocking the cell signal transduction of phosphorylation transduction, and further inhibiting proliferation of tumor cells. ERBB 3-positive tumor cells MCF-7 (ATCC, cat#CRL-3435) were treated by pancreatin digestion, the supernatant was discarded by centrifugation, the cell pellet was resuspended in complete medium (RPMI 1640 medium+10% heat-inactivated FBS), the cell density was adjusted to 1X 10 ⁴ cells/mL, 100uL of cell suspension was added to each well in the center 60 wells of a 96-well plate (Corning, cat#3610), 200uL of PBS solution was added to the surrounding wells, and the incubator was incubated overnight at 37 ℃. The next day, 400nM of maximum working concentration was prepared with complete medium, 3-fold diluted, 9 concentration gradient candidate antibodies were added to 50 ul/well of candidate antibody solution in the cell plate, the blank was added with equal amounts of complete medium, each concentration was multiplexed, mixed well, incubated for 1 hour in 37 degrees incubator, then 50 ul/well was added with 80ng/mL of beta-HRG ligand solution prepared with complete medium, the negative control (cells only) was added with equal amounts of complete medium, and the plate was incubated in 37℃incubator for 5 days. The cell plates were removed at room temperature equilibrium temperature for 10min, after which 75 ul/well CELL TITER Glo (Promega, cat#: G7573) solution was added, and the reaction was allowed to stand for 15min, using chemiluminescence parameters reading at 490nm for 500 ms. The cell proliferation inhibition rate was calculated as follows:

inhibition% = (positive control signal value-experimental signal value)/positive control signal value × 100

Performing curve fitting by GRAPHPAD PRISM 8.0.0 with the inhibition rate corresponding to each concentration gradient of the fully human antibody mutant as an ordinate and the concentration logarithmic value as an abscissa, and calculating the IC ₅₀ of the candidate antibody on tumor cell proliferation inhibition, wherein the result is as follows:

TABLE 13 proliferation inhibition of MC-7 cells by fully human antibody mutants Note that: "-" means no inhibition

Experimental results show that the antibody mutants can inhibit the cell proliferation induced by ERBB3 ligand beta-HRG compared to their parent.

Example 9: ligand blocking assay

The blocking effect of the antibody mutants on binding between the ligand and human ERBB3 expressed by tumor cells was tested by cRBA (cell-base receptor blocking assay) experiments. Human breast cancer cells SK-BR-3 expressing ERBB3 (Nanjac Bai, cat#CBP 60413) were pancreatin digested and counted, resuspended to 1X 10 ⁶/mL with FACS buffer, plated into 96-well plates at 100. Mu.L per well, and centrifuged: 1500rpm, 4 ℃,5 minutes, discard the supernatant. A 2-fold working concentration of fully human antibody solution (working concentration 30 μg/mL) was prepared, cell pellet was resuspended at 50 μl per well, wells without antibody were set as control wells, and incubated on ice for 30 min. Then 50. Mu.L of ligand bio-HRG1 (Sino Biological, cat# 11609-HNCH-B) at a concentration of 1.2. Mu.g/mL was added, mixed well and incubated on ice for 10 min, centrifuged: the supernatant was discarded at 1500rpm at 4℃for 5 minutes and washed twice with FACS buffer. 100. Mu.L of 100-fold dilutions of SA-PE (R & D, cat#F0040) were added, incubated at 4℃for 45 min, FACS buffer washed twice, and the cells were resuspended in 100. Mu.L, detected with a flow cytometer Bio-Rad (ZE 5), blocking rates were calculated and the IC ₅₀ concentration of the antibodies was analyzed as a curve.

Blocking rate (%) = (control wells MFI-sample wells MFI)/control wells MFI x 100, control wells were wells without fully human antibody, and sample wells were wells with different concentration gradients of fully human antibody.

TABLE 14 ligand blocking effect of fully human antibody mutantsNote that: "-" means no break

The results show that the fully human antibody mutant has significantly improved ligand HRG blocking effect compared to the parent Ab 2.

Example 10: phosphorylation blocking assay

In the ligand-dependent ERBB3 pathway, ligand HRG binds to ERBB3 protein expressed by tumor cells, phosphorylating and activating it, thereby forming a heterodimer with EGFR or ERBB2, activating the downstream AKT and ERK pathways. This experiment tested the blocking effect of fully human antibodies on Her3, AKT and ERK phosphorylation under ligand activation conditions by human breast cancer cells MCF-7 positive for ERBB3 by means of a phosphorylation assay kit of pHer (Cisbio, cat#64hr3 PEG), pAKT (Cisbio, cat# AKSPEG) and perrk (Cisbio, cat# ERKPPEH).

Cells MCF-7 (ATCC, cat#CRL-3435) were treated with pancreatin, the supernatant was discarded by centrifugation, the cell pellet was resuspended to 1X 10 ⁶/mL with Reaction medium (RPMI 1640 medium+10% heat-inactivated FBS+1% penicillin/streptomycin), 100uL of cell suspension was added to each well of a flat bottom 96-well plate (low adsorption plate), four-sided wells were blocked with 200uL PBS, and incubated for 6 hours in a 37℃incubator. The supernatant was pipetted off with a row gun, 100uL Starved medium (RPMI 1640 medium+1% penicillin/streptomycin) was added per well and incubated overnight in a 37℃incubator. 50uL of fully human antibody (4-fold working concentration, i.e., 800 nM) formulated in Starved medium was added, wells without antibody were set as positive control wells for phosphorylation activation, and incubated at 37℃for 60 min after homogenization. The reaction plate was removed, 50ul of ligand HRG 1-beta (200 ng/mL) at 4-fold working concentration formulated with Starved medium was added, no ligand wells were set as negative control wells for phosphorylation, no antibody and ligand wells were set as background control wells. After mixing, incubation was carried out in a 37℃incubator for 10 minutes. Since the cells are adherent, the supernatant can be discarded directly and the phosphorylation detected using the pHer, pAKT and pERK kit. The cell pellet was added to 1*supplemented lysis buffer in 70uL of the kit, subjected to shaking reaction at room temperature for 30 minutes, and after mixing in a row gun, 16uL of the lysed supernatant was taken to 384 well plates, and then added to a mixture of two labeled antibodies in 4uL of the kit (20-fold dilution). And (3) attaching a tinfoil film to seal, incubating for 4 hours at room temperature, reading HTRF (665 nm and 620 nm) on an enzyme-labeled instrument (Thermo, lux), calculating the Ratio value of each hole according to 665nm/620nm multiplied by 10000, and calculating the blocking rate by using the Ratio value, wherein the formula is as follows: blocking rate (%) = (sample well-negative control well)/(positive control well-background control well).

TABLE 15 phosphorylation blocking of fully human antibody mutantsNote that: "-" means not measured

The results show that the fully human antibody mutants have significantly improved phosphorylation blocking effects on pHer, pAKT, and pERK compared to the parent Ab2.

Claims (21)

1. An anti-ERBB 3 antibody or antigen-binding fragment thereof, comprising: heavy chain variable region and light chain variable region; wherein the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 1. 60 and 61, HCDR1, HCDR2 and HCDR3, the light chain variable region comprising the amino acid sequences as set forth in SEQ ID NOs: 62. LCDR1, LCDR2 and LCDR3 as shown in figures 6 and 63 wherein said SEQ ID NO: 60. SEQ ID NO: 61. SEQ ID NO:62 and SEQ ID NO:63 is an amino acid sequence having the general formula:

   HCDR2：X₁ISDTGGEX₂YYADSVKG SEQ ID NO：60；

   HCDR3：DX₃X₄X₅X₆X₇VDYFD SEQ ID NO：61；

   LCDR1：X₈GDNIGIKX₉VH SEQ ID NO：62；

   LCDR3：QX₁₀WDDX₁₁SDHPV SEQ ID NO：63；

   Wherein: x ₁ is selected from A or G; x ₂ is selected from T or K; x ₃ is selected from L, F, Y or Q; x ₄ is selected from G or S; x ₅ is selected from S or D; x ₆ is selected from S or E; x ₇ is selected from D, G or E; x ₈ is selected from G, F, K, L, V, R or Q; x ₉ is selected from S, W or F; x ₁₀ is selected from V, S or T; x ₁₁ is selected from T, S or N.
2. The anti-ERBB 3 antibody or antigen-binding fragment thereof of claim 1, which comprises a heavy chain variable region and an antibody light chain variable region, wherein,

   The heavy chain variable region comprises HCDR1 shown as SEQ ID NO. 1, HCDR2 shown as SEQ ID NO. 3 or 30, and HCDR3 shown as any one of amino acid sequences of SEQ ID NO. 4, 28, 31, 32, 33 or 34;

   The light chain variable region comprises LCDR1 shown in any one of the amino acid sequences of SEQ ID NO. 5, 19, 21, 23, 25, 27, 29 or 35, LCDR2 shown in SEQ ID NO. 6 and LCDR3 shown in any one of the amino acid sequences of SEQ ID NO. 7, 20, 22, 24 or 26.
3. The anti-ERBB 3 antibody, or antigen-binding fragment thereof, of claim 1-2, which comprises a heavy chain variable region and a light chain variable region, wherein,

   The heavy chain variable region comprises HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3 and HCDR3 shown in SEQ ID NO. 4; the light chain variable region comprises LCDR1 shown in SEQ ID NO. 5, LCDR2 shown in SEQ ID NO. 6 and LCDR3 shown in SEQ ID NO. 7; or alternatively, the first and second heat exchangers may be,

   The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 4; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 19, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 20; or alternatively, the first and second heat exchangers may be,

   The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 4; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 21, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 22; or alternatively, the first and second heat exchangers may be,

   The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 4; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 23, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 24; or alternatively, the first and second heat exchangers may be,

   The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 4; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 25, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 26; or alternatively, the first and second heat exchangers may be,

   The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 4; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 27, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 22; or alternatively, the first and second heat exchangers may be,

   The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 28; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 29, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 7; or alternatively, the first and second heat exchangers may be,

   The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 30, and HCDR3 shown in SEQ ID NO. 31; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 5, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 7; or alternatively, the first and second heat exchangers may be,

   The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 32; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 5, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 7; or alternatively, the first and second heat exchangers may be,

   The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 33; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 29, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 7; or alternatively, the first and second heat exchangers may be,

   The heavy chain variable region comprises: HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 3, and HCDR3 shown in SEQ ID NO. 34; the light chain variable region comprises: LCDR1 shown in SEQ ID NO. 35, LCDR2 shown in SEQ ID NO. 6, and LCDR3 shown in SEQ ID NO. 7.
4. The anti-ERBB 3 antibody or antigen-binding fragment thereof according to any one of claims 1-3 wherein the heavy chain variable region is at least one heavy chain variable region selected from the group consisting of: SEQ ID NO. 9, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45 and SEQ ID NO. 46, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology thereto.
5. The anti-ERBB 3 antibody or antigen-binding fragment thereof according to any one of claims 1-4 wherein the light chain variable region is at least one light chain variable region selected from the group consisting of light chain variable region ：SEQ ID NO:10、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:47, shown in the following sequence or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology thereto.
6. The anti-ERBB 3 antibody or antigen-binding fragment thereof according to any one of claims 1-5 wherein the anti-ERBB 3 antibody or antigen-binding fragment thereof comprises the heavy chain variable region shown in SEQ ID No. 9 and the light chain variable region shown in SEQ ID No. 10; or alternatively, the first and second heat exchangers may be,

   The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO. 9, and a light chain variable region shown in SEQ ID NO. 36; or alternatively, the first and second heat exchangers may be,

   The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO. 9, and a light chain variable region shown in SEQ ID NO. 37; or alternatively, the first and second heat exchangers may be,

   The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO. 9, and a light chain variable region shown in SEQ ID NO. 38; or alternatively, the first and second heat exchangers may be,

   The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO. 9, and a light chain variable region shown in SEQ ID NO. 39; or alternatively, the first and second heat exchangers may be,

   The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO. 9, and a light chain variable region shown in SEQ ID NO. 40; or alternatively, the first and second heat exchangers may be,

   The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO. 41, and a light chain variable region shown in SEQ ID NO. 42; or alternatively, the first and second heat exchangers may be,

   The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO. 43, and a light chain variable region shown in SEQ ID NO. 10; or alternatively, the first and second heat exchangers may be,

   The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown as SEQ ID NO. 44, and a light chain variable region shown as SEQ ID NO. 10; or alternatively, the first and second heat exchangers may be,

   The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises the heavy chain variable region shown in SEQ ID NO. 45 and the light chain variable region shown in SEQ ID NO. 42 or,

   The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises the heavy chain variable region shown in SEQ ID NO. 46, and the light chain variable region shown in SEQ ID NO. 47.
7. The anti-ERBB 3 antibody or antigen-binding fragment thereof according to any one of claims 1-6 further comprising a heavy chain constant region derived from human IgG1, igG2, igG3 or IgG4 or a mutant thereof;

   Preferably, the anti-ERBB 3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1 or variant thereof;

   more preferably comprising a human IgG1 heavy chain constant region;

   most preferably comprises the heavy chain constant region as shown in SEQ ID NO. 17;

   Optionally, the anti-ERBB 3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain, lambda chain, or mutant thereof;

   Preferably comprising a light chain constant region derived from a human lambda chain;

   Most preferably comprises the light chain constant region as set forth in SEQ ID NO. 18.
8. The anti-ERBB 3 antibody or antigen-binding fragment thereof of any one of claims 1-7 wherein the heavy chain is selected from the group consisting of heavy chains comprising the sequence: SEQ ID NO. 12, SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 57 or SEQ ID NO. 58, or a heavy chain having at least 80%, 85%, 90%, 95% or 99% homology thereto.
9. The anti-ERBB 3 antibody or antigen-binding fragment thereof of any one of claims 1-7 wherein the light chain is selected from the group consisting of light chain ：SEQ ID NO:13、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:59, comprising the following sequences or a light chain having at least 80%, 85%, 90%, 95% or 99% homology thereto.
10. The anti-ERBB 3 antibody or antigen-binding fragment thereof of claim 1-9, wherein,

    The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown as SEQ ID NO. 12, and a light chain shown as SEQ ID NO. 13; or alternatively, the first and second heat exchangers may be,

    The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown as SEQ ID NO. 12, and a light chain shown as SEQ ID NO. 48; or alternatively, the first and second heat exchangers may be,

    The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain represented by SEQ ID NO. 12, and a light chain represented by SEQ ID NO. 49; or alternatively, the first and second heat exchangers may be,

    The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown as SEQ ID NO. 12, and a light chain shown as SEQ ID NO. 50; or alternatively, the first and second heat exchangers may be,

    The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown in SEQ ID NO. 12, and a light chain shown in SEQ ID NO. 51; or alternatively, the first and second heat exchangers may be,

    The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown in SEQ ID NO. 12, and a light chain shown in SEQ ID NO. 52; or alternatively, the first and second heat exchangers may be,

    The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown as SEQ ID NO. 53, and a light chain shown as SEQ ID NO. 54; or alternatively, the first and second heat exchangers may be,

    The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown as SEQ ID NO. 55, and a light chain shown as SEQ ID NO. 13; or alternatively, the first and second heat exchangers may be,

    The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown as SEQ ID NO. 56, and a light chain shown as SEQ ID NO. 13; or alternatively, the first and second heat exchangers may be,

    The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown as SEQ ID No. 57 and a light chain shown as SEQ ID No. 54; or alternatively, the first and second heat exchangers may be,

    The anti-ERBB 3 antibody or antigen-binding fragment thereof comprises a heavy chain shown as SEQ ID NO. 58, and a light chain shown as SEQ ID NO. 59.
11. A polynucleotide encoding the anti-ERBB 3 antibody or antigen-binding fragment thereof of any one of claims 1-10.
12. An expression vector comprising the polynucleotide of claim 11.
13. A host cell into which or containing the expression vector of claim 12 has been introduced.
14. The host cell of claim 13, wherein the host cell is a bacterial, yeast or mammalian cell; preferably E.coli, pichia pastoris, CHO cells or HEK293 cells.
15. A method of producing an anti-ERBB 3 antibody or antigen-binding fragment thereof, comprising the steps of:

    a) Culturing the host cell of any one of claims 13-14;

    b) Isolating antibodies from the culture; and

    C) Purifying the antibody.
16. A pharmaceutical composition comprising the anti-ERBB 3 antibody or antigen-binding fragment thereof of any one of claims 1-10, and a pharmaceutically acceptable excipient, diluent or carrier.
17. A detection or diagnostic reagent comprising an anti-ERBB 3 antibody or antigen-binding fragment thereof according to any one of claims 1-10 and an excipient, diluent or carrier useful in detection or diagnosis.
18. Use of an anti-ERBB 3 antibody or antigen-binding fragment thereof according to any one of claims 1-10, or a composition according to claim 16, in the manufacture of a medicament for the treatment or prophylaxis of an ERBB3 mediated disease or condition.
19. Use of an anti-ERBB 3 antibody or antigen-binding fragment thereof according to any one of claims 1-10 or a detection or diagnostic reagent according to claim 17 in the manufacture of a kit for the detection, diagnosis, prognosis of an ERBB3 mediated disease or disorder.
20. The use according to any one of claims 18-19, wherein:

    The disease or disorder is cancer;

    Preferably ERBB3 expressed or overexpressed cancers;

    More preferably breast cancer, ovarian cancer, prostate cancer, endometrial cancer, thyroid cancer, kidney cancer, lung cancer, stomach cancer, colon cancer, bladder cancer, cervical cancer, gall bladder cancer, pancreatic cancer, testicular cancer, soft tissue sarcoma, head and neck cancer, glioma or melanoma.
21. A method of treating or preventing ERBB3 mediated diseases comprising the steps of:

    Providing a therapeutically effective amount or a prophylactically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-10 to a subject against ERBB 3; or providing a therapeutically effective amount or a prophylactically effective amount of the pharmaceutical composition of claim 16 to a subject; wherein the ERBB3 mediated disease is selected from the group consisting of: breast cancer, ovarian cancer, prostate cancer, endometrial cancer, thyroid cancer, kidney cancer, lung cancer, gastric cancer, colon cancer, bladder cancer, cervical cancer, gall bladder cancer, pancreatic cancer, testicular cancer, soft tissue sarcoma, head and neck cancer, glioma or melanoma.