WO2023198138A1

WO2023198138A1 - Antibody or antigen-binding fragment thereof and medical use thereof

Info

Publication number: WO2023198138A1
Application number: PCT/CN2023/087967
Authority: WO
Inventors: 毛东杰; 谢岳峻
Original assignee: 上海翰森生物医药科技有限公司; 常州恒邦药业有限公司; 江苏豪森药业集团有限公司
Priority date: 2022-04-13
Filing date: 2023-04-13
Publication date: 2023-10-19

Abstract

Provided are an anti-ERBB3 antibody or an antigen-binding fragment thereof, and the medical use thereof. Provided are an anti-ERBB3 receptor fully human antibody or an antigen-binding fragment thereof, and the medical use thereof. Provided are a pharmaceutical composition comprising the anti-ERBB3 fully human antibody or the antigen-binding fragment thereof, and the use of the pharmaceutical composition as an anti-cancer drug.

Description

Antibodies or antigen-binding fragments thereof and their medicinal uses

Technical field

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

Background technique

ERBB receptor is widely expressed in neuronal cells, epithelial cells, and mesenchymal cells. It is related to the development of cardiovascular, neural, musculoskeletal and other organs, and is involved in the pathogenesis of cancer. ERBB receptor has four family proteins: EGFR, ERBB2, ERBB3 and ERBB4. They are all membrane proteins with similar molecular structures, including extracellular domain (ECD), transmembrane domain, and intracellular domain with tyrosine kinase activity. and the C terminus. ECD can be subdivided into 4 subdomains: subdomain I, II, III, IV (BMC Bioinformatics 2001,2:4.; Mol Cell 2003,11:507-17.), among which I and III are rich in leucine, It is the ligand binding functional domain; II and IV are rich in cysteine and are the functional domains that form dimers. In addition to ERBB2, the other three ERBB receptors bind II and IV in the inactive state, and are opened when bound and activated by ligands, exposing II to form dimers. Currently, there are many studies on EGFR and ERBB2, and the research and development of corresponding targeted drugs is intensive. In recent years, ERBB3 has received increasing attention. ERBB3 bypass plays a key role in EGFR and ERBB2-related drug resistance, and ERBB3 plays a key role in breast cancer, lung cancer, prostate cancer, colorectal cancer, ovarian cancer, gastric cancer, and bladder cancer. , melanoma and other tumors, so ERBB3 is another potential target for tumor treatment. ERBB3 has two ligands, NRG1 and NRG2. The ligands can activate the kinase activity of ERBB3. The activated ERBB3 can form a heterodimer with EGFR or ERBB2 and phosphorylate the latter, thereby transmitting signals to downstream pathways and promoting Growth and proliferation of tumor cells. In addition, in cancer, ERBB3 can also transmit signals through "cross talk" with other RTKs, such as forming complexes with IGF1R, FGFR2 and HGFR (c-Met) (BioDrugs 2017, 31:63-73). It can be seen that ERBB3 plays an important role in tumors. plays an important role in growth.

Currently, therapeutic strategies targeting ERBB3 targets follow the following mechanisms: 1) locking ERBB3 in an inactive state (such as CDX-3379); 2) capturing the ERBB3 ligand NRG (such as RB200); 3) blocking Binding of ERBB3 to ligands (such as U3-1287); 4) Promoting the endocytosis of ERBB3 (antibody drug conjugate); 5) Blocking the dimerization of ERBB3 with other EGFR family members; 6) Recruiting immune cell killing expression Cancer cells with endogenous ERBB3. . For these mechanisms, ERBB3-targeted treatments can include: monoclonal antibodies, bispecific antibodies, anti-ERBB3 vaccines, ligand traps, RNA inhibitors that lock ERBB3, small molecules that inhibit ERBB3 kinase activity, etc. Among them, the research and development of antibodies targeting ERBB3 is relatively hot.

In clinical studies, an ERBB3 antibody derived from mice can significantly inhibit the growth of patients' cell tumors (citing Celldex data). The ERBB3 antibodies currently under development include humanized antibodies that are mouse-derived antibodies that have been humanized. The immunogenicity of humanized antibodies during immunization is higher than that of fully human antibodies that do not contain mouse-derived antibody components. This is a disadvantage when applied to humans; there are also some ERBB3 antibody drugs under development that are fully developed through phage library display technology. Humanized antibodies can solve the problem of immunogenicity.

Phage display technology is the fusion expression of foreign proteins or polypeptides with the phage coat protein, thereby expressing the foreign protein on the surface of the phage. The phage antibody library is an antibody library established using comprehensive technical means that combines phage display technology, PCR amplification technology, and protein expression technology.

Phage libraries generally include synthetic libraries, immune libraries and natural libraries. The most commonly used phage library is the natural library. The natural library is usually a fully human antibody library constructed from immune cells from human peripheral blood. Its biggest advantage is that it does not require additional In vivo immunity can obtain highly diverse fully human antibodies produced by the human body. In addition, the phage antibody library also has the following advantages: ① It achieves the unification of genotype and phenotype. In addition, the experimental method is simple and fast. The traditional antibody production method through hybridoma technology takes several months, while the antibody library technology only takes a few weeks. ② What is expressed is a fully human antibody with a small molecular weight. It is mainly expressed in the form of active fragment Fab and scFV. Compared with intact antibodies, it has obvious advantages in tissue penetration. ③The screening capacity is large. Hybridoma technology screens thousands of clones, and antibody library technology can select millions or even billions of molecules. More types of antibodies were screened. ④ It has a wide range of uses and uses a prokaryotic expression system. The advantages are more obvious when mass-produced (Curr Opin Biotechnol. 2002 Dec; 13(6):598-602; Immunotechnology, 2013, 48(13) 48(13): 63- 73).

At present, ERBB3 antibodies have been reported in WO2007077028 and other patents. Most of them are mouse antibodies or humanized antibodies, and some are fully human antibodies obtained from phage libraries. Most of them are in clinical use at home and abroad. In the early and discovery stages, there are no antibody drugs targeting ERBB3 on the market. Therefore, it is necessary to further develop fully human ERBB3 antibodies with higher activity, high affinity and high stability for treatment research and application of related diseases.

Contents of the invention

The invention provides an anti-ERBB3 antibody or an antigen-binding fragment thereof, which includes: a heavy chain variable region and a light chain variable region; wherein, the heavy chain variable region includes SEQ ID NO: 1, 60 respectively. and HCDR1, HCDR2 and HCDR3 shown in 61 and 61, the light chain variable region includes LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 62, 6 and 63 respectively, wherein said SEQ ID NO: 60, SEQ ID NO: 61. SEQ ID NO: 62 and SEQ ID NO: 63 respectively have the amino acid sequences represented by the following general formulas:

Among them: 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; 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 present invention also relates to a preferred embodiment, an anti-ERBB3 antibody or an antigen-binding fragment thereof as described above, which contains a heavy chain variable region and an antibody light chain variable region, wherein,

The heavy chain variable region includes HCDR1 as shown in SEQ ID NO: 1, as shown in SEQ ID NO: 3 or 30 The HCDR2 shown is the HCDR3 shown in any amino acid sequence of SEQ ID NO: 4, 28, 31, 32, 33 or 34;

The light chain variable region includes LCDR1 as shown in any amino acid sequence of SEQ ID NO: 5, 19, 21, 23, 25, 27, 29 or 35, LCDR2 as shown in SEQ ID NO: 6, as SEQ ID NO: LCDR3 shown in any amino acid sequence of 7, 20, 22, 24 or 26.

The present invention also relates to a preferred embodiment, an anti-ERBB3 antibody or an antigen-binding fragment thereof as described above, which contains a heavy chain variable region and a light chain variable region, wherein:

The heavy chain variable region includes 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 includes SEQ ID NO:1 LCDR1 shown in ID NO:5, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:7; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:20; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:21, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:22; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:23, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:24; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:25, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:26; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:27, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:22; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:29, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:7; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:5, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:7; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:5, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:7; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:29, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:7; or,

The heavy chain variable region includes: 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 includes : 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-ERBB3 antibody or an antigen-binding fragment thereof as described above, wherein the heavy chain variable region is at least one selected from the heavy chain variable region shown in the following sequence: 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 at least 70%, 75%, 80%, 85% thereof , a heavy chain variable region with 90%, 95% or 99% homology.

The present invention also relates to a preferred embodiment, an anti-ERBB3 antibody or an antigen-binding fragment thereof as described above, wherein the light chain variable region is at least one light chain variable region selected from the following sequence: 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, or related to A light chain variable region of at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology.

The present invention also relates to a preferred embodiment, an anti-ERBB3 antibody or an antigen-binding fragment thereof as described above, wherein the anti-ERBB3 antibody or an 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,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain variable region shown in SEQ ID NO: 9, and the light chain variable region shown in SEQ ID NO: 36; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain variable region shown in SEQ ID NO: 9, and the light chain variable region shown in SEQ ID NO: 37; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain variable region shown in SEQ ID NO: 9, and the light chain variable region shown in SEQ ID NO: 38; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain variable region shown in SEQ ID NO: 9, and the light chain variable region shown in SEQ ID NO: 39; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain variable region shown in SEQ ID NO: 9, and the light chain variable region shown in SEQ ID NO: 40; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain variable region shown in SEQ ID NO: 41, and the light chain variable region shown in SEQ ID NO: 42; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain variable region shown in SEQ ID NO: 43, and the light chain variable region shown in SEQ ID NO: 10; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof comprises the heavy chain variable region shown in SEQ ID NO:44, and the light chain variable region shown in SEQ ID NO:10; or,

The anti-ERBB3 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-ERBB3 antibody or antigen-binding fragment thereof includes 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 present invention, the anti-ERBB3 antibody or antigen-binding fragment thereof further comprises Heavy chain constant regions derived from human IgG1, IgG2, IgG3 or IgG4 or mutants thereof;

In a further preferred embodiment of the present invention, the anti-ERBB3 antibody or antigen-binding fragment thereof further comprises the heavy chain constant region of human IgG1 or a variant thereof;

In a further preferred embodiment of the present invention, the anti-ERBB3 antibody or antigen-binding fragment thereof further comprises a human IgG1 heavy chain constant region;

In a further preferred embodiment of the present invention, the anti-ERBB3 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 present invention, the anti-ERBB3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from human kappa chain, lambda chain or mutants thereof;

In a further preferred embodiment of the present invention, the anti-ERBB3 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 present invention, the anti-ERBB3 antibody or antigen-binding fragment thereof further comprises a light chain constant region as shown in SEQ ID NO: 18.

In a preferred embodiment of the present invention, the anti-ERBB3 antibody or antigen-binding fragment thereof, wherein the anti-ERBB3 antibody or antigen-binding fragment thereof comprises a heavy chain selected from the following: 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 present invention, the anti-ERBB3 antibody or antigen-binding fragment thereof, wherein the anti-ERBB3 antibody or antigen-binding fragment thereof includes 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 is at least 80%, 85%, 90%, 95% or 99% identical to source of light chains.

The present invention also relates to a preferred embodiment, an anti-ERBB3 antibody or an antigen-binding fragment thereof as described above, wherein the anti-ERBB3 antibody or an antigen-binding fragment thereof comprises the heavy chain shown in SEQ ID NO: 12, and SEQ ID NO. The light chain shown in NO:13; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 12, and the light chain shown in SEQ ID NO: 48; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 12, and the light chain shown in SEQ ID NO: 49; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 12, and the light chain shown in SEQ ID NO: 50; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 12, and the light chain shown in SEQ ID NO: 51; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 12, and the light chain shown in SEQ ID NO: 52; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof comprises the heavy chain shown in SEQ ID NO: 53, and the light chain shown in SEQ ID NO: 54; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 55, and the light chain shown in SEQ ID NO: 13; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 56, and the light chain shown in SEQ ID NO: 13; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 57, and the light chain shown in SEQ ID NO: 54; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 58, and the light chain shown in SEQ ID NO: 59.

The present invention further provides a polynucleotide encoding the above-mentioned anti-ERBB3 antibody or antigen-binding fragment thereof.

The present invention further provides an expression vector containing the above-mentioned polynucleotide.

The present invention further provides a host cell, which is introduced or contains the above-mentioned expression vector.

In a preferred embodiment of the present invention, the above-mentioned host cell is a bacterium, preferably Escherichia coli.

In a preferred embodiment of the present invention, the above-mentioned host cell is yeast, preferably Pichia pastoris.

In a preferred embodiment of the present invention, the above-mentioned host cells are mammalian cells, preferably CHO cells or HEK293 cells.

The invention further provides a method for producing anti-ERBB3 antibodies or antigen-binding fragments thereof, comprising the steps:

a) Cultivate the above host cells;

b) isolating fully human antibodies from culture; and

c) Purify the antibody.

The present invention further provides a pharmaceutical composition, which contains the above-mentioned anti-ERBB3 antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable excipient, diluent or carrier.

The present invention further provides a detection or diagnostic reagent, which contains the above-mentioned anti-ERBB3 antibody or antigen-binding fragment thereof and an excipient, diluent or carrier that can be used for detection or diagnosis.

The present invention further provides the use of the above-mentioned anti-ERBB3 antibody or antigen-binding fragment thereof or the above-mentioned composition in the preparation of a medicament for treating or preventing ERBB3-mediated diseases or disorders.

The present invention further provides the use of the above-mentioned anti-ERBB3 antibody or antigen-binding fragment thereof or the above-mentioned detection or diagnostic reagent in preparing a kit for detecting, diagnosing, and prognosticating ERBB3-mediated diseases or disorders.

In a preferred embodiment of the invention,

The disease or condition mentioned above is cancer;

Cancers that express or overexpress ERBB3 are preferred;

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

The present invention further provides a method for treating or preventing ERBB3-mediated diseases, including the steps:

Provide a therapeutically effective amount or a prophylactically effective amount of the above-mentioned anti-ERBB3 antibody or antigen-binding fragment thereof to a subject; or provide a therapeutically effective amount or a prophylactically effective amount of the above-mentioned pharmaceutical composition to a subject; wherein the ERBB3 mediates The diseases are selected from: breast cancer, ovarian cancer, prostate cancer, endometrial cancer, thyroid cancer, kidney cancer, lung cancer, stomach cancer, colon cancer, bladder cancer, cervical cancer, gallbladder cancer, pancreatic cancer, testicular cancer, soft tissue sarcoma, Head and neck cancer, glioma, or melanoma.

Detailed ways

Detailed description of the invention

1. Terminology

In order to make this application easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this application belongs.

The three-letter and single-letter codes for amino acids used in this application are as described in J. Biol. Chem, 243, p3558 (1968).

The term "antibody" used in this application refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by inter-chain disulfide bonds. The amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain and γ chain respectively. , α chain and ε chain. The same type of Ig can be divided into different subclasses based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into kappa or lambda chains through differences in constant regions. Each of the five types of Ig can have a kappa chain or a lambda chain.

In this application, the antibody light chain variable region described in this application may further comprise a light chain constant region, and the light chain constant region includes human or murine kappa, lambda chains or variants thereof.

In this application, the antibody heavy chain variable region described in this application may further comprise a heavy chain constant region, which includes human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof. body.

The sequence of about 110 amino acids near the N-terminus of antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and is the constant region (C region). The variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions. The order from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The number and position of the CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment described in this application comply with the known Kabat or Chothia or ABM definition rules (http://bioinf.org.uk/abs/) .

The term "antigen presenting cell" or "APC" is a cell that displays on its surface a foreign antigen complexed with an 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 lymphoblasts, and monocyte-derived dendritic cells.

The term "antigen presentation" refers to the process by which APCs capture antigens and enable their recognition by T cells, for example as components of MHC-I/MHC-II conjugates.

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

1. Antibodies isolated from transgenic or transchromosomal animals (such as mice) of human immunoglobulin genes or hybridomas prepared therefrom;

2. Antibodies isolated from host cells transformed to express antibodies, such as transfectomas;

3. Antibodies isolated from recombinant combinatorial human antibody libraries; and

4. Antibodies prepared, expressed, created or isolated by splicing human immunoglobulin gene sequences into other DNA sequences.

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

The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the present application may include amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, 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 mouse) have been grafted onto human backbone sequences (i.e., "humanized antibodies") .

The term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which generally include at least part of the antigen-binding region or variable region (such as one or more CDRs) of the parent antibody. 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 activity is 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 for 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.

A "Fab fragment" consists of the CH1 and variable regions of a light chain and a heavy chain. 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 the antibody. The two heavy chain segments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

A "Fab' fragment" contains a light chain and a portion of a heavy chain including the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby between the two heavy chains of two Fab' fragments Interchain disulfide bonds are formed to form F(ab')2 molecules.

An "F(ab')2 fragment" contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

An "Fv region" contains variable regions from both 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. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL unit Antibodies with multi-epitope specificity; antibodies with two or more VL and VH regions, each VH-VL unit binds to different targets or different epitopes of the same target; antibodies with two or more single Antibodies with variable regions, each single variable region binds to different targets or different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and tribodies (triabodies), antibody fragments that have been covalently or non-covalently linked together, etc.

The term "single-chain antibody" is a single-chain recombinant protein composed of an antibody's heavy chain variable region VH and light chain variable region VL connected through a connecting peptide. It is the smallest antibody fragment with a complete antigen-binding site.

The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment containing only a heavy chain variable region or a light chain variable region chain. 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 can target the same or different antigens.

The term "binding to ERBB3" in this application refers to the ability to interact with human ERBB3.

The term "antigen binding site" in this application refers to the three-dimensional space recognized by the antibody or antigen-binding fragment of this application.

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

The terms "specific binding" and "selective binding" used in this application 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, when measured in the instrument by surface plasmon resonance (SPR) technology, the antibody dissociates with an equilibrium dissociation constant of approximately less than 10 ^-7 M or even less ( _KD ) binds to the predetermined antigen, and its affinity for binding to the predetermined antigen is at least twice the affinity for binding to the predetermined antigen or a non-specific antigen (such as BSA, etc.) other than the predetermined antigen or a closely related antigen. The term "antibody that recognizes an antigen" is used interchangeably herein with the term "antibody that specifically binds."

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

The terms "inhibition" or "blocking" are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of a ligand preferably reduces or alters the normal level or type of activity that would occur if ligand binding occurred without inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in binding affinity of the ligand when contacted with the anti-ERBB3 antibody compared to the ligand not contacted with the anti-ERBB3 antibody.

The term "inhibition of growth" (eg, referring to a cell) is intended to include any measurable reduction in cell growth.

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

The "ADCC" described in this application, that is, antibody-dependent cell-mediated cytotoxicity, refers to cells expressing Fc receptors that are directly killed by recognizing the Fc segment of the antibody and are coated with the antibody. target cells. The ADCC effector function of the antibody can be enhanced or reduced, reduced or eliminated by modifying the Fc segment of IgG. The modification refers to mutation in the heavy chain constant region of the antibody.

The engineered antibodies or antigen-binding fragments of the present application can be prepared and purified by conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into the GS expression vector. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems result in glycosylation of the antibody, particularly at the highly conserved N-terminus of the FC region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in bioreactors to produce antibodies. The culture medium secreting antibodies can be purified and collected using conventional techniques. Antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The obtained product needs to be frozen immediately, such as -70°C, or freeze-dried.

"Administration," "administration," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, mean the administration of an exogenous drug, therapeutic, diagnostic, or composition to the animal , contact with persons, subjects, cells, tissues, organs or biological fluids. "Administration," "administration," and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of cells includes contact of reagents with the cells, and contact of the reagents with a fluid, wherein the fluid is in contact with the cells. "Administer," "administer," and "treat" also mean in vitro and ex vivo treatment of, for example, a cell by a reagent, a diagnostic, a binding composition or by another cell. "Treatment" when applied to human, veterinary medicine or research subjects means therapeutic treatment, prophylactic or prophylactic measures, research and diagnostic applications.

"Treat" means administering an internal or external therapeutic agent, such as one comprising any of the antibodies of the present application, to a patient who has one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Generally, a therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the subject or population, either by inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinically measurable extent. The amount of therapeutic agent effective to alleviate the symptoms of any particular disease (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether disease symptoms have been alleviated may be assessed by any of the clinical tests commonly used by physicians or other health care professionals to assess the severity or progression of symptoms. Although embodiments of the present application (e.g., treatments or articles of manufacture) may not be effective in alleviating the symptoms of the target disease in each patient, according to any statistical test method known in the art, such as Student's t test, Chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test determined that it should alleviate the target disease symptoms in a statistically significant number of patients.

As used throughout the specification and claims, the term "consisting essentially of" or variations thereof is meant to include all stated elements or groups of elements, and optionally other elements of similar or different nature to the stated elements, which other Elements that do not significantly alter the basic or novel properties of a given dosage regimen, method, or composition.

The term "naturally occurring" as applied to an object herein refers to the fact that the object is found in nature. For example, organisms (including viruses) that can be isolated from natural sources and have not been artificially A naturally occurring polypeptide sequence or polynucleotide sequence is one that is intentionally modified in the laboratory.

An "effective amount" includes an amount sufficient to ameliorate or prevent symptoms or symptoms of a medical disorder. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the method, route and dosage of administration, and the severity of the side effects. An effective amount may be the maximum dosage or dosage regimen that avoids significant side effects or toxic effects.

"Exogenous" refers to substances that are background produced outside an organism, cell or human body.

"Endogenous" refers to a substance that is produced in a cell, organism, or human body based on its background.

"Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example if each position in 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 matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. In general, comparisons are made when aligning two sequences yields the greatest percent homology.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include their progeny. Thus, the words "transformant" and "transformed cell" include primary subject cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring are unlikely to be exactly the same in terms of DNA content due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as screened in the originally transformed cells are included. Where different names are intended, this is clear from the context.

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

"Pharmaceutical composition" means containing one or more antibodies or antigen-binding fragments thereof as described herein, together with other ingredients such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of pharmaceutical compositions is to facilitate administration to living organisms and facilitate the absorption of active ingredients to exert biological activity.

2. Embodiments

The following examples are used to further describe the present application, but these examples do not limit the scope of the present application. Experimental methods without specifying specific conditions in the examples of this application usually follow conventional conditions, such as Cold Spring Harbor's Antibody Technology Experiment Manual, Molecular Cloning Manual; or conditions recommended by raw material or product manufacturers. Reagents whose specific sources are not indicated are conventional reagents purchased in the market.

Example 1: Screening and construction of fully human antibodies

ERBB3 antigen design:

The amino acid sequence of the human ERBB3 protein shown in SEQ ID NO: 14 is used as a template for the antigen design of the present invention. The following ERBB3 antigens unless otherwise specified refer to human ERBB3.

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

The ERBB3 antigen used for screening is the commercial product Biotinylated human ERBB3-His tag (Sino biological, cat#10201-H08H-B); the ERBB3 antigen used for detection is the commercial product human ERBB3-His tag (Sino biological, cat#10201-H08H) . The ERBB3 antigen for detection is the ECD region sequence of human ERBB3, with a His tag at the C terminus. The ERBB3 antigen for screening is biotinylated on this basis. The ECD sequence of human ERBB3 is as follows:

The monkey ERBB3 antigen (SEQ ID NO: 16) used for detection is the commercial product Rhesus ERBB3 Protein-His Tag (Sino biological, cat#90043-K08H). It is the ECD region sequence of the monkey ERBB3 antigen with a His tag added to the C terminus. The sequence as follows:

PBMC from multiple individuals were collected, B cells were isolated, RNA was extracted, reverse transcribed into cDNA, and then the cDNA was used as a template to construct a natural phage surface Fab display library (library capacity: 1.6×10 ¹¹ ). After the constructed natural Fab phage library is packaged to form phage particles, the liquid phase method is used for panning. The phages are combined with biotinylated ERBB3 liquid phase and then separated using streptavidin magnetic beads. In order to obtain positive sequences that bind to human ERBB3, biotinylated human ERBB3 was used for 2 to 3 rounds of screening, and 384 single clone colonies were selected and packaged into phage display Fab fragments for ELISA testing. Test the binding activity of monoclonal phage to human ERBB3: ELISA plates were coated with 1 μg/mL ERBB3, phage supernatant was added, and finally detected with anti-human IgG Fab HRP; 86 ELISA plates were tested with OD ₄₅₀ values greater than 0.2. The positive clones were sequenced and the sequences were analyzed. The unique sequences obtained were: 45 VH (heavy chain variable region) and 51 VL (light chain variable region). The obtained heavy chain and light chain variable region sequences were connected to the human IgG1 heavy chain constant region and light chain constant region sequences respectively to obtain full-length antibodies. These full-length antibodies were experimentally evaluated to finally determine a binding capacity and Antibody Abl with good functions; wherein, the connected human IgG1 heavy chain constant region and light chain constant region sequences are shown in SEQ ID NO: 17 and SEQ ID NO: 18 respectively.

S57E (Kabat numbering) site-directed mutation was performed on the HCDR2 of the Ab1 antibody to form a new HCDR2 (SEQ ID NO: 3), and then the antibody Ab2 was obtained.

Table 1. Antibody heavy chain and light chain variable region CDR sequences

Table 2. Antibody heavy chain and light chain variable region sequences

Note: CDR area is marked by underline.

Table 3. Heavy and light chain sequences of antibodies

Note: CDR area is marked by underline.

Table 4. Sequence numbers of antibodies and their heavy chains, light chains, and variable regions

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

Example 2: Transformation of fully human antibodies

Based on the sequence of Ab2, carry out site mutations, perform random saturation mutations on the heavy chain and light chain CDR regions, construct a phage library with single CDR or double CDR combination mutations, display it in the form of scFv (VH-VL), and evaluate the library capacity. The libraries above 10 ⁷ are shown in Table 5. ERBB3 antigen protein and HEK293T cells overexpressing human ERBB3 (293T-human ERBB3) were used for 5 rounds of alternating enrichment, and 1056 clones were obtained through preliminary screening by ELISA. Then FACS re-screening was performed and samples were sent for sequencing. Finally, 103 unique candidate sequences were obtained, and 10 antibodies with top 10 binding capabilities were selected for production and purification.

Table 5. Fully human antibody scFv mutant phage library

The clones selected based on the mutation library of the Ab2 antibody sequence are different from the Ab2 antibody in HCDR2, HCDR3, LCDR1, and LCDR3 on the light and heavy chains. The relevant CDR (the amino acid residues of the CDR are determined and annotated by the Kabat numbering system) sequence or general formula, the corresponding light/heavy chain variable region and its light and heavy chains are as follows:

Table 6. Heavy chain and light chain variable region CDR sequences of mutant antibodies

Table 7. Heavy and light chain variable region sequences of antibodies

Note: CDR area is marked by underline.

Table 8. Heavy and light chain sequences of mutant antibodies

Note: CDR area is marked by underline

Example 3: Preparation of fully human antibodies and mutants thereof

cDNA fragments were synthesized based on the amino acid sequences of the light and heavy chains of Ab1 and Ab2 antibodies and inserted into the pcDNA3.1 expression vector (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 ratio of 1:2 and placed in a CO ₂ incubator for 4- 5 days. Collect the cell culture fluid, centrifuge it, load it onto the antibody purification affinity column, wash the column with phosphate buffer, elute with glycine hydrochloride buffer (pH2.7 0.1M Gly-HCl), neutralize with 1M Tris hydrochloric acid pH 9.0, and After dialysis against phosphate buffer, the antibody protein of the present application was obtained. Its concentration and purity are shown in Table 9:

Table 9. Concentration and purity of fully human antibodies

The 10 antibodies Ab2_M1-M10 with TOP10 binding capacity were produced and purified using the same method as above to obtain fully human antibody mutant proteins.

Example 4: In vitro binding experiment of fully human antibody mutants

4.1 Stable transfection cell binding experiment

HEK293T cells overexpressing human ERBB3 (293T-human ERBB3) were digested with trypsin, collected by centrifugation, and the cell density was adjusted with FACS buffer (1×PBS containing 2% FBS) before being spread on a 96-well U-bottom plate. Wells contain 1 × 10 ⁵ to 2 × 10 ⁵ cells. Centrifuge: 1200g, 5 minutes, discard the supernatant, add 100 μL of antibody solution that has been gradient diluted with FACS buffer, and incubate at 4°C for 1 hour. Centrifuge: 1200g, 5 minutes, discard the supernatant, wash the cells twice with FACS buffer, add fluorescently labeled secondary antibody working solution PE anti-human IgG Fc Antibody (Biolegend, Cat#409304) prepared in FACS buffer, and resuspend 100 μL per well. Cells were incubated at 4°C for 1 hour. Centrifuge: 1200g, 5 minutes, discard the supernatant. After washing the cells twice with FACS buffer, they were resuspended in PBS. The fluorescence signal was detected using a flow cytometer Bio-Rad (ZE5), and a curve was drawn to analyze the EC ₅₀ concentration of the antibody-bound cells.

The results showed that the EC ₅₀ of the modified mutants Ab2_M6, Ab2_M7, Ab2_M8, Ab2_M9 and Ab2_M10 showed enhanced binding capacity compared to Ab2, and the binding capacity of the other five mutants was similar to Ab2.

Table 10. Binding ability of fully human antibody mutants to 293T-hERBB3 cells

4.2 Tumor cell binding experiment

Through the detection method of Example 4.1, the binding ability of 10 purified fully human antibody mutants to human breast cancer cells MX-1 expressing ERBB3 antigen was detected. MFI was used as the abscissa, and the concentration logarithm value was used as the abscissa. Log(agonist)vs.response--Variable slope(four parameters) formula is used for curve fitting and EC ₅₀ is calculated. The results are shown in the table below.

The results showed that the EC50 of the modified fully human antibody mutant was higher than that of its parent Ab2, and its affinity for MX-1 cells was stronger than that of its parent Ab2.

Table 11. Binding of fully human antibody mutants to cells expressing MX-1

Example 5: Endocytosis experiment of fully human antibody mutants

To detect whether the antibody of the present invention can be internalized into cells together with human ERBB3 after binding to ERBB3, human breast adenocarcinoma cell MX-1 expressing ERBB3 was used for evaluation. The cells were digested with trypsin, collected and resuspended in pre-chilled FACS buffer, and the cell concentration was adjusted to 2×10 ⁶ /mL. Take the EP tube, add 1 mL of cell suspension, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add 1 mL of the prepared antibody to be tested, resuspend the cells, the final concentration of the antibody is 20 μg/ml, and incubate on a shaker at 4°C for 1 hour. Centrifuge and discard the supernatant (4°C, 1500 rpm × 5 minutes), wash twice with FACS buffer, and discard the supernatant. Add 1 mL of fluorescently labeled secondary antibody working solution PE anti-human IgG Fc Antibody (Biolegend, Cat#409304) to each tube, resuspend the cells, incubate on a shaker at 4°C for 30 minutes, and centrifuge (4°C, 1500rpm × 5 minutes) , washed twice with FACS buffer, and discarded the supernatant. Add 1 mL of preheated cell culture medium to each tube to resuspend the cells and mix well. Divide into 4 tubes, 250 μL each, which are divided into 0-minute group, blank group, 15-minute group and 30-minute group. Take out the 0-minute and blank groups. on ice, and the rest were placed in a 37°C incubator for endocytosis for 15 minutes and 30 minutes respectively. At the corresponding time points, take out the EP tubes and place them on ice to precool for 5 minutes. Centrifuge all treatment groups and discard the supernatant (4°C, 1500rpm). ×5 minutes), wash once with FACS buffer, and discard the supernatant. Add 250 μL strip buffer to the EP tubes of all treatment groups except the 0-minute group, and incubate at room temperature for 8 minutes. Centrifuge and discard the supernatant (4°C, 1500 rpm × 5 minutes). Wash twice with FACS buffer and discard the supernatant. Add 100 μL PBS to resuspend cells in all treatment groups, and detect using flow cytometer Bio-Rad (ZE5).

Antibody endocytosis percentage (%) = (fluorescence intensity value at each time point - average fluorescence intensity value of Blank group) / (average fluorescence intensity value of 0 minute group - average fluorescence intensity value of Blank group) * 100, the specific results are as follows Table 12 shows:

Table 12. Endocytosis of fully human antibody mutants in tumor cells MX-1

The results show that the fully human antibody mutant of the present invention has obvious endocytosis in human breast adenocarcinoma cells MX-1. As time goes by, the cell's endocytosis efficiency of the antibody gradually increases, reaching a peak at 30 minutes and reaching a peak at 60 minutes. Due to saturation, the overall endocytosis efficiency of mutant antibody cells was improved.

Example 6: Cell proliferation inhibition experiment

The purpose of this experiment is to evaluate how candidate antibodies block the binding of ligand HRG to ERBB3 by binding to the ERBB3 receptor on the surface of tumor cell membranes, thereby blocking the formation of heterodimers between ERBB3 and other EGFR family members, and blocking phosphorylation-transduced cells. signal transduction, thereby inhibiting the proliferation of tumor cells. ERBB3-positive tumor cells MCF-7 (ATCC, Cat#CRL-3435) were trypsinized, centrifuged and the supernatant discarded, and the cell pellet was resuspended in complete medium (RPMI 1640 medium + 10% heat-inactivated FBS) for counting. , adjust the cell density to 1×10 ⁴ cells/mL, add 100uL cell suspension to each well in the center 60 wells of a 96-well plate (Corning, Cat# 3610), add 200uL/well PBS solution to the surrounding holes, and incubate in a 37°C incubator overnight. The next day, use complete culture medium to prepare the highest working concentration of 400nM, 3-fold dilution, and 9 concentration gradient candidate antibodies. Add 50ul/well of the candidate antibody solution to the cell plate. Add an equal amount of complete culture medium to the blank control. Each concentration Repeat the wells, mix well, and incubate in a 37-degree incubator for 1 hour. Then add 50ul/well of complete culture medium to prepare a β-HRG ligand solution with a concentration of 80ng/mL. For the negative control (cells only), add an equal amount of complete culture medium. , place the plate in a 37°C incubator and incubate for 5 days. Take out the cell plate and allow it to equilibrate at room temperature for 10 minutes, then add 75ul/well Cell Titer Glo (Promega, Cat#: G7573) solution, let it stand for 15 minutes, and read the chemiluminescence parameters at 490nm for 500ms. Calculate the cell proliferation inhibition rate according to the following formula:

Inhibition rate % = (positive control group signal value - experimental group signal value) / positive control group signal value * 100

Taking the inhibition rate corresponding to each concentration gradient of the fully human antibody mutant as the ordinate and the logarithm of concentration as the abscissa, curve fitting was performed using GraphPad Prism 8.0 to calculate the IC ₅₀ of the candidate antibody for inhibiting tumor cell proliferation. The results are as follows:

Table 13. Inhibition of proliferation of MC-7 cells by fully human antibody mutants

Note: "-" means no inhibitory effect

Experimental results showed that the antibody mutant could inhibit cell proliferation induced by the ERBB3 ligand β-HRG compared with its parent.

Example 9: Ligand blocking experiment

The cRBA (cell-base receptor blocking assay) experiment was used to test the blocking effect of the antibody mutants on the binding between the ligand and human ERBB3 expressed on tumor cells. Human breast cancer cells SK-BR-3 (Nanjing Kebai, Cat#CBP60413) expressing ERBB3 were trypsinized and counted, resuspended in FACS buffer to 1×10 ⁶ /mL, and spread into 96 wells at 100 μL per well. plate, centrifuge: 1500 rpm, 4°C, 5 minutes, discard the supernatant. Prepare a fully human antibody solution with 2 times the working concentration (working concentration 30 μg/mL), resuspend the cell pellet at 50 μL per well, set the well without antibody as a control well, and incubate on ice for 30 minutes. Then add 50 μL of ligand bio-HRG1 (Sino Biological, Cat#11609-HNCH-B) with a concentration of 1.2 μg/mL, mix and incubate on ice for 10 minutes, centrifuge: 1500 rpm, 4°C, 5 minutes, discard The supernatant was washed twice with FACS buffer. Add 100 μL of the prepared 100-fold dilution SA-PE (R&D, Cat#F0040), incubate at 4°C for 45 minutes, wash twice with FACS buffer, resuspend the cells in 100 μL, and perform flow cytometry using Bio-Rad (ZE5). Detect, calculate the blocking rate and draw a curve to analyze the IC ₅₀ concentration of the antibody.

Blocking rate (%) = (control well MFI - sample well MFI)/control well MFI × 100. The control well is a well without fully human antibody, and the sample well is a well with fully human antibody in different concentration gradients.

Table 14. Ligand blocking effect of fully human antibody mutants

Note: "-" means no blocking effect

The results showed that compared with the parent Ab2, the fully human antibody mutant had a significantly improved blocking effect on the ligand HRG.

Example 10: Phosphorylation blocking experiment

In the ligand-dependent ERBB3 pathway, the ligand HRG binds to the ERBB3 protein expressed in tumor cells to phosphorylate and activate it, and then forms a heterodimer with EGFR or ERBB2, thereby activating the downstream AKT and ERK pathways. This experiment uses the phosphorylation detection kits of pHer3 (Cisbio, Cat#64HR3PEG), pAKT (Cisbio, Cat#64AKSPEG) and pERK (Cisbio, Cat#64ERKPPEH) to test the ligand expression of ERBB3-positive human breast cancer cells MCF-7. Blocking effect of fully human antibodies on the phosphorylation of Her3, AKT and ERK under activation conditions.

Cells MCF-7 (ATCC, Cat#CRL-3435) were digested with trypsin, centrifuged and the supernatant was discarded. Cell pellet was precipitated using Reaction medium (RPMI 1640 medium + 10% heat-inactivated FBS + 1% penicillin/streptomycin). Resuspend to 1×10 ⁶ /mL, add 100uL cell suspension to each well of a flat-bottom 96-well plate (low adsorption plate), seal the empty wells on all sides with 200ul PBS, and incubate in a 37-degree incubator for 6 hours. Aspirate and discard the supernatant, add 100uL Starved medium (RPMI 1640 medium + 1% penicillin/streptomycin) to each well, and incubate overnight in a 37-degree incubator. Add 50uL of fully human antibody prepared in Starved medium (4 times the working concentration, i.e. 800nM), set the well without antibody as a positive control well for phosphorylation activation, mix well and incubate at 37 degrees for 60 minutes. Take out the reaction plate, add 50ul of 4 times the working concentration of ligand HRG1-β (200ng/mL) prepared in Starved medium, set the well without ligand as the phosphorylation negative control well, and set the well without antibody and ligand as the background Control wells. Mix well and incubate in a 37°C incubator for 10 minutes. Since the cells are adherent, you can directly discard the supernatant and use pHer3, pAKT and pERK kits to detect phosphorylation. Add 1*supplemented lysis buffer in the 70uL kit to the cell pellet, shake at room temperature for 30 minutes, mix evenly, take 16ul of the lysis supernatant into a 384-well plate, and then add 4uL of the two labeled antibody mixtures in the kit ( 20 times dilution). Seal with tin foil film, incubate at room temperature for 4 hours, read HTRF (665nm and 620nm) on a microplate reader (Thermo, Lux), calculate the Ratio value of each well based on 665nm/620nm×10000, and use the Ratio value to calculate the blocking rate. The formula is as follows:
Blocking rate (%) = (sample well-negative control well)/(positive control well-background control well).

Table 15. Phosphorylation blocking effect of fully human antibody mutants

Note: "-" means not measured

The results showed that compared with the parent Ab2, the fully human antibody mutant had a significantly higher blocking effect on the phosphorylation of pHer3, pAKT and pERK.

Claims

An anti-ERBB3 antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region and a light chain variable region; wherein, the heavy chain variable region includes as shown in SEQ ID NO: 1, 60 and 61 respectively HCDR1, HCDR2 and HCDR3, the light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 62, 6 and 63 respectively, wherein said SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63 are amino acid sequences represented by the following general formulas:

HCDR2:X 1 ISDTGGEX 2 YYADSVKG SEQ ID NO: 60;

HCDR3: DX 3 X 4 X 5 X 6 X 7 VDYFD SEQ ID NO: 61;

LCDR1:X 8 GDNIGIKX 9 VH SEQ ID NO: 62;

LCDR3: QX 10 WDDX 11 SDHPV SEQ ID NO: 63;

Among them: 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; 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 anti-ERBB3 antibody or antigen-binding fragment thereof according to claim 1, which comprises a heavy chain variable region and an antibody light chain variable region, wherein,

The heavy chain variable region includes HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3 or 30, or any of SEQ ID NO:4, 28, 31, 32, 33 or 34. HCDR3 represented by an amino acid sequence;

The light chain variable region includes LCDR1 as shown in any amino acid sequence of SEQ ID NO: 5, 19, 21, 23, 25, 27, 29 or 35, LCDR2 as shown in SEQ ID NO: 6, as SEQ ID NO: LCDR3 shown in any amino acid sequence of 7, 20, 22, 24 or 26.
The anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1-2, which comprises a heavy chain variable region and a light chain variable region, wherein,

The heavy chain variable region includes 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 includes SEQ ID NO:1 LCDR1 shown in ID NO:5, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:7; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:19, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:20; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:21, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:22; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:23, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:24; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:25, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:26; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:27, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:22; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:29, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:7; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:5, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:7; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:5, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:7; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:29, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:7; or,

The heavy chain variable region includes: 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 includes : LCDR1 shown in SEQ ID NO:35, LCDR2 shown in SEQ ID NO:6, and LCDR3 shown in SEQ ID NO:7.
The anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the heavy chain variable region is at least one selected from the heavy chain variable region shown in the following sequence: 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 at least 70%, 75%, 80%, 85%, 90% , 95% or 99% homologous heavy chain variable region.
The anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the light chain variable region is at least one selected from the light chain variable region shown in the following sequence: 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, or at least 70% of the same , 75%, 80%, 85%, 90%, 95% or 99% homology to the light chain variable region.
The anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the anti-ERBB3 antibody or antigen-binding fragment thereof comprises the heavy chain variable region shown in SEQ ID NO:9, and SEQ ID The light chain variable region shown in NO:10; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain variable region shown in SEQ ID NO: 9, and the light chain variable region shown in SEQ ID NO: 36; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain variable region shown in SEQ ID NO: 9, and the light chain variable region shown in SEQ ID NO: 37; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain variable region shown in SEQ ID NO: 9, and the light chain variable region shown in SEQ ID NO: 38; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain variable region shown in SEQ ID NO: 9, and the light chain variable region shown in SEQ ID NO: 39; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain variable region shown in SEQ ID NO: 9, and the light chain variable region shown in SEQ ID NO: 40; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain variable region shown in SEQ ID NO: 41, and the light chain variable region shown in SEQ ID NO: 42; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain variable region shown in SEQ ID NO: 43, and the light chain variable region shown in SEQ ID NO: 10; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof comprises the heavy chain variable region shown in SEQ ID NO:44, and the light chain variable region shown in SEQ ID NO:10; or,

The anti-ERBB3 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-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain variable region shown in SEQ ID NO: 46, and the light chain variable region shown in SEQ ID NO: 47.
The anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, further comprising a heavy chain constant region derived from human IgG1, IgG2, IgG3 or IgG4 or a mutant thereof;

Preferably, the anti-ERBB3 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1 or a variant thereof;

More preferably, it contains human IgG1 heavy chain constant region;

Most preferably, it contains the heavy chain constant region shown in SEQ ID NO:17;

Optionally, the anti-ERBB3 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human kappa chain, a lambda chain, or a mutant thereof;

Preferably it contains a light chain constant region derived from a human lambda chain;

Most preferably it contains a light chain constant region as shown in SEQ ID NO:18.
The anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the heavy chain is selected from the group consisting of heavy chains containing the following sequences: 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.
The anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the light chain is selected from the group consisting of light chains containing the following sequences: 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 at least 80%, 85%, 90%, 95% or 99 thereof % homology to the light chain.
The anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 12, and the light chain shown in SEQ ID NO: 13; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 12, and the light chain shown in SEQ ID NO: 48; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 12, and the light chain shown in SEQ ID NO: 49; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 12, and the light chain shown in SEQ ID NO: 50; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 12, and the light chain shown in SEQ ID NO: 51; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 12, and the light chain shown in SEQ ID NO: 52; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 53, and the light chain shown in SEQ ID NO: 54; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 55, and the light chain shown in SEQ ID NO: 13; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 56, and the light chain shown in SEQ ID NO: 13; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 57, and the light chain shown in SEQ ID NO: 54; or,

The anti-ERBB3 antibody or antigen-binding fragment thereof includes the heavy chain shown in SEQ ID NO: 58, and the light chain shown in SEQ ID NO: 59.
A polynucleotide encoding the anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1-10.
An expression vector containing the polynucleotide of claim 11.
A host cell introduced into or containing the expression vector of claim 12.
The host cell according to claim 13, wherein the host cell is a bacterium, yeast or mammalian cell; preferably Escherichia coli, Pichia pastoris, CHO cells or HEK293 cells.
A method for producing anti-ERBB3 antibodies or antigen-binding fragments thereof, comprising the steps:

a) Cultivate the host cell described in any one of claims 13-14;

b) isolating the antibodies from the culture; and,

c) Purify the antibody.
A pharmaceutical composition containing the anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and a pharmaceutically acceptable excipient, diluent or carrier.
A detection or diagnostic reagent, which contains the anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 and an excipient, diluent or carrier that can be used for detection or diagnosis.
The anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or the use of the composition according to claim 16 in the preparation of a medicament for the treatment or prevention of ERBB3-mediated diseases or disorders .
The anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 or the detection or diagnostic reagent according to claim 17 is used in the preparation of reagents for detecting, diagnosing, and prognosing ERBB3-mediated diseases or disorders. Purpose in the box.
The use according to any one of claims 18-19, wherein:

The disease or condition described is cancer;

Cancers that express or overexpress ERBB3 are preferred;

More preferably, breast cancer, ovarian cancer, prostate cancer, endometrial cancer, thyroid cancer, kidney cancer, lung cancer, stomach cancer, colon cancer, bladder cancer, cervical cancer, gallbladder cancer, pancreatic cancer, testicular cancer, soft tissue sarcoma, head and neck cancer cancer, glioma, or melanoma.
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 anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 to a subject; or providing a therapeutically effective amount or a prophylactically effective amount to a subject. The pharmaceutical composition described in 16; wherein the ERBB3-mediated disease is selected from: breast cancer, ovarian cancer, prostate cancer, endometrial cancer, thyroid cancer, kidney cancer, lung cancer, gastric cancer, colon cancer, bladder cancer, cervical cancer cancer, gallbladder cancer, pancreatic cancer, testicular cancer, soft tissue sarcoma, head and neck cancer, glioma, or melanoma.