WO2022144025A1 - Anti-erbb3 receptor antibody or antigen-binding fragment thereof, and medical use thereof - Google Patents

Abstract

Provided are an anti-ERBB3 receptor antibody or an antigen-binding fragment thereof, and the medical use thereof. The present invention particularly relates to a fully humanized antibody for an anti-ERBB3 receptor or an antigen-binding fragment thereof, and the medical use thereof. Furthermore, provided are a pharmaceutical composition containing the fully humanized antibody for the anti-ERBB3 receptor or the antigen-binding fragment thereof, and the use thereof as an anti-cancer drug.

Description

Antibody against ERBB3 receptor or antigen-binding fragment thereof and medical use thereof technical field

The present invention relates to an anti-ERBB3 receptor antibody or an antigen-binding fragment thereof, further, the present invention relates to an ERBB3 antibody or an antigen-binding fragment thereof comprising a CDR region; the present invention also relates to a fully human antibody comprising the ERBB3 or an antigen thereof The pharmaceutical composition of the binding fragment and its use as a diagnostic agent and a therapeutic drug for ERBB3-related diseases.

Background technique

ERBB receptors are widely expressed in neuronal cells, epithelial cells, and mesenchymal cells, and are involved in the development of cardiovascular, neural, musculoskeletal and other organs, and are involved in cancer pathogenesis. ERBB receptors have four family proteins: EGFR, ERBB2, ERBB3 and ERBB4, all of which are membrane proteins with similar molecular structures, including an extracellular domain (ECD), a transmembrane domain, and an intracellular domain with tyrosine kinase activity. , and the C-terminus. The ECD can be subdivided into 4 subdomains: subdomains I, II, III, IV (BMC Bioinformatics 2001, 2:4.; Mol Cell 2003, 11:507-17.), where I and III are rich in leucine Acid, is the ligand-binding domain; II and IV are rich in cysteine and are dimer-forming domains. With the exception of ERBB2, the other three ERBB receptors bind II and IV in the inactive state and only open when bound and activated by ligands, exposing II to dimerization. At present, there are many studies on EGFR and ERBB2, and the development of corresponding targeted drugs is relatively intensive. In recent years, the attention of ERBB3 has been continuously increased. In EGFR and ERBB2-related drug resistance, the ERBB3 bypass plays a key role, and ERBB3 is involved in breast, lung, prostate, colorectal, ovarian, gastric, and bladder cancers. , melanoma and other tumors have high expression, so ERBB3 is another potential target for tumor therapy. 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 to promote Growth and proliferation of tumor cells. In addition, in cancer, ERBB3 can also transmit signals through "cross talk" with other RTKs, such as forming a complex with IGF1R, FGFR2 and HGFR(c-Met) (BioDrugs 2017, 31:63-73), it can be seen that ERBB3 plays an important role in tumors. important role in growth.

Currently, therapeutic strategies targeting ERBB3 follow several mechanisms: 1) locking ERBB3 in an inactive state (eg CDX-3379); 2) capturing ERBB3 ligand NRG (eg RB200); 3) blocking Binding of ERBB3 to ligands (such as U3-1287); 4) promoting the endocytosis of ERBB3 (antibody drug conjugates); 5) blocking the dimerization of ERBB3 with other EGFR family members; 6) recruiting immune cells to kill expression Cancer cells with endogenous ERBB3. For these mechanisms, ERBB3-targeted therapy can include: monoclonal antibodies, bispecific antibodies, vaccines against ERBB3, ligand traps, RNA inhibitors that lock ERBB3, and small molecules that inhibit ERBB3 kinase activity. Among them, the research and development of antibodies targeting ERBB3 is relatively high.

In clinical studies, a murine-derived ERBB3 antibody significantly inhibited the growth of patient tumors (citing Celldex data). The ERBB3 antibodies currently under development are humanized antibodies that have been humanized and modified from mouse-derived antibodies, and the immunogenicity of humanized antibodies during immunization is higher than that of fully human antibodies without mouse-derived antibody components. Human application is a disadvantage; some ERBB3 antibody drugs under development are fully human antibodies obtained by phage library display technology, which can solve the problem of immunogenicity.

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

Phage libraries generally include synthetic libraries, immune libraries, and natural libraries. The natural library is the most widely used. The natural library is usually a fully human antibody library constructed from immune cells of human peripheral blood. In vivo immunization, however, a highly diverse fully human antibody that has been produced by the human body can be obtained. In addition, the phage antibody library also has the following advantages: (1) The unification of genotype and phenotype is achieved. In addition, the experimental method is simple and fast, the traditional method of antibody production by hybridoma technology takes months, while the antibody library technology only takes a few weeks. ② It expresses fully human antibodies with small molecular weight, mainly in the form of active fragments Fab and scFV, which have obvious advantages in tissue penetration compared with complete antibodies. ③ The screening capacity is large. Hybridoma technology can screen thousands of clones, and antibody library technology can select millions or even hundreds of millions of molecules. There are more types of antibodies screened. ④It has a wide range of uses and adopts a prokaryotic expression system, which has more obvious advantages in large-scale production (Curr Opin Biotechnol. 2002 Dec; 13(6): 598-602; Immunotechnology, 2013, 48(13) 48(13): 63- 73).

At present, WO2007077028 and other patents have reported ERBB3 antibodies, most of which are murine or humanized antibodies, and some are fully human antibodies obtained from phage libraries. In the early stage and discovery stage, 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 the treatment research and application of related diseases.

SUMMARY OF THE INVENTION

The present invention provides an anti-ERBB3 antibody or an antigen-binding fragment thereof, in particular to a fully human anti-ERBB3 antibody or an antigen-binding fragment thereof, the anti-ERBB3 antibody or an antigen-binding fragment thereof comprising: an antibody heavy chain variable region and antibody light chain variable region; wherein, the antibody heavy chain variable region comprises at least one HCDR selected from the following sequences: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; The variable region of the antibody light chain comprises at least one LCDR selected from the following sequences: SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

In a preferred embodiment, the present invention also relates to an anti-ERBB3 antibody or an antigen-binding fragment thereof as described above, wherein the antibody heavy chain variable region comprises:

HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, and HCDR3 shown in SEQ ID NO:3.

In a preferred embodiment, the present invention also relates to an anti-ERBB3 antibody or an antigen-binding fragment thereof as described above, wherein the antibody light chain variable region comprises:

LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6.

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

The antibody heavy chain variable region comprises: HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2 and HCDR3 shown in SEQ ID NO:3;

The antibody light chain variable region comprises: LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:6.

In a preferred embodiment, the present invention also relates to 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:7 , or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology therewith.

In a preferred embodiment, the present invention also relates to 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 light chain variable region shown in SEQ ID NO: 8 , or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology therewith.

In a preferred embodiment, the present invention also relates to 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: 7, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology thereto, and a light chain variable region shown in SEQ ID NO: 8, or A light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology thereto.

In a preferred embodiment of the present invention, the anti-ERBB3 antibody or its antigen-binding fragment further comprises a heavy chain constant region derived from human IgG1, IgG2, IgG3 or IgG4 or a mutant thereof;

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

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

In a further preferred embodiment of the present invention, the anti-ERBB3 antibody or its antigen-binding fragment further comprises a heavy chain constant region as shown in SEQ ID NO: 11;

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

In a further preferred embodiment of the present invention, the light chain constant region derived from human λ chain is included;

In a further preferred embodiment of the present invention, the anti-ERBB3 antibody or its antigen-binding fragment further comprises a light chain constant region as shown in SEQ ID NO: 12.

In a preferred embodiment, the present invention also relates to 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: 9, or has at least the heavy chain shown in SEQ ID NO: 9. Full-length heavy chains of 80%, 85%, 90%, 95% or 99% homology.

In a preferred embodiment, the present invention also relates to 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 light chain shown in SEQ ID NO: 10, or has at least the light chain shown in SEQ ID NO: 10. Full-length light chains of 80%, 85%, 90%, 95% or 99% homology.

In a preferred embodiment, the present invention also relates to 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: 9, or its A full-length heavy chain having at least 80%, 85%, 90%, 95% or 99% homology, and the light chain shown in SEQ ID NO: 10, or at least 80%, 85%, 90%, Full-length light chain with 95% or 99% homology.

The present invention further provides polynucleotides encoding the above-mentioned anti-ERBB3 antibodies or antigen-binding fragments thereof.

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

The present invention further provides host cells into which the above-described expression vector is introduced or contained.

In a preferred embodiment of the present invention, the above-mentioned host cells are bacteria, preferably Escherichia coli.

In a preferred embodiment of the present invention, the above-mentioned host cell is a 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 present invention further provides a method for producing an anti-ERBB3 antibody or an antigen-binding fragment thereof, comprising the steps of:

a) culturing the above-mentioned host cells;

b) isolating the antibody from the culture; and

c) purifying the antibody.

The present invention further provides a pharmaceutical composition comprising 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 an antigen-binding fragment thereof, and an excipient, diluent or carrier that can be used for detection or diagnosis.

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

The present invention further provides use of the above-mentioned anti-ERBB3 antibody or antigen-binding fragment thereof or the above-mentioned detection or diagnosis reagent in the preparation of a reagent for detection, diagnosis and prognosis of ERBB3-mediated diseases or disorders.

In a preferred embodiment of the present invention,

The aforementioned disease or condition 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.

In a preferred embodiment of the present invention, the above-mentioned anti-ERBB3 antibody or its antigen-binding fragment or the above-mentioned composition is used for detecting, diagnosing and prognosing ERBB3-mediated diseases; 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.

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

A therapeutically effective amount or a prophylactically effective amount of the above-mentioned anti-ERBB3 antibody or an antigen-binding fragment thereof is provided to a subject; or the above-mentioned pharmaceutical composition of a therapeutically effective amount or a prophylactically effective amount is provided to the subject; wherein the ERBB3 mediates The disease is 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

For easier understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise 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 one-letter codes for amino acids used in the present invention are as described in J. Biol. Chem, 243, p3558 (1968).

The term "antibody" in the present invention refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain 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 classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, and γ chains, respectively. , alpha chains and epsilon chains. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into kappa chains or lambda chains by the difference in the constant region. Each of the five classes of Ig can have a kappa chain or a lambda chain.

In the present invention, the antibody light chain variable region described in this application may further comprise a light chain constant region, and the light chain constant region comprises human or murine κ, λ chains or variants thereof.

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

The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) are composed of 3 CDR regions and 4 FR regions. The sequence from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The number and position of CDR amino acid residues in the VL and VH regions of the antibodies or antigen-binding fragments of the present invention conform to 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 MHC. T cells recognize this complex using the T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DCs), topical 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, eg, 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, involving techniques and methods well known in the art, such as:

1. Antibodies isolated from human immunoglobulin gene transgenic, transchromosomal animals (eg mice) or hybridomas prepared therefrom;

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

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

4. Antibodies prepared, expressed, created or isolated by methods such as 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. Human antibodies of the invention may include amino acid residues 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 a mouse) have been grafted onto human backbone sequences (ie, "humanized antibodies") .

The term "antigen-binding fragment" refers to antigen-binding fragments and antibody analogs of antibodies, which typically include at least a portion of the antigen-binding or variable regions (eg, one or more CDRs) of the parental antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, antibody fragments retain at least 10% of the parent binding activity when the 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.

The term "Fab fragment" consists of the CH1 and variable regions of one light chain and one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

The term "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 hydrophobic interactions of the CH3 domains.

The term "Fab' fragment" contains a light chain and a portion of a heavy chain comprising the VH and CH1 domains and the region between the CH1 and CH2 domains, and thus can be located between the two heavy chains of two Fab' fragments. Interchain disulfide bonds are formed between them to form F(ab')2 molecules.

The term "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. Thus, an F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

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

The term "multispecific antibody" is used in its broadest sense to encompass antibodies with specificity for multiple epitopes. 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 has polyepitope specificity; having two or more VL and VH regions antibodies, each VH-VL unit binds to a different target or a different epitope of the same target; antibodies with two or more single variable domains, each single variable domain to a different target or different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments covalently or non-covalently linked together Wait.

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

The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment containing only the variable region of the heavy chain or the variable region of the light chain. In certain instances, 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" refers to the ability to interact with human ERBB3.

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

The term "epitope" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically include 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 assays, among others. Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.

The terms "specifically binds" and "selectively binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, when human ^ERBB3 is used as the analyte and the antibody is used as the ligand, the antibody exhibits an equilibrium dissociation constant ( K _D ) binds to a predetermined antigen with at least twice the affinity for binding to the predetermined antigen or non-specific antigens other than the predetermined antigen or closely related antigens (eg, BSA, etc.). 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 invention to bind to ERBB3 from different species. For example, an antibody of the invention that binds to human ERBB3 can also bind to ERBB3 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (eg SPR and ELISA), or binding or functional interaction with cells that physiologically express ERBB3. Methods to determine 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 are also intended to include any measurable reduction in ligand binding affinity when contacted with an anti-ERBB3 antibody compared to a ligand not contacted with an anti-ERBB3 antibody.

The term "inhibiting growth" (eg, in relation 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 the stimulation (ie, passive or adaptive) of an immune response to a specific antigen. The term "induction" with respect to inducing CDC or ADCC refers to stimulating a specific mechanism of direct cell killing.

The "ADCC" mentioned in the present invention, namely antibody-dependent cell-mediated cytotoxicity (antibody-dependent cell-mediated cytotoxicity), antibody-dependent cell-mediated cytotoxicity, refers to the cells expressing Fc receptors through the The Fc segment that recognizes the antibody directly kills the target cell coated by the antibody. The ADCC effector function of the antibody can be enhanced, reduced or eliminated by modification of the Fc region of IgG. The modification refers to mutation in the constant region of the heavy chain of the antibody.

The engineered antibodies or antigen-binding fragments of the invention can be prepared and purified using conventional methods. The cDNA sequences of the corresponding antibodies can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more preferred prior art, mammalian-like expression systems lead to glycosylation of the antibody, especially 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 for antibody production. The antibody-secreted culture medium can be purified and collected by conventional techniques. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The obtained product should be frozen immediately, eg -70°C, or lyophilized.

"Administering," "administering," and "treating," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions that interact with the animal. , contact of humans, subjects, cells, tissues, organs or biological fluids. "Administering," "administering," and "treating" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells. "Administering," "administering," and "treating" also mean in vitro and ex vivo treatment of, eg, cells by an agent, diagnostic, binding composition, or by another cell. "Treatment" when applied to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

"Treatment" means administering an internal or external therapeutic agent, such as comprising any of the antibodies of the invention, 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 in an amount effective to alleviate one or more symptoms of a disease in a patient or population to be treated, either by inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinically measured degree. The amount of a therapeutic agent effective to relieve symptoms of any particular disease (also referred to as a "therapeutically effective amount") can vary depending on factors such as the patient's disease state, age and weight, and the ability of the drug to produce the desired effect in the patient. Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms. Although embodiments of the present invention (eg, methods of treatment or articles of manufacture) may be ineffective in alleviating symptoms of the target disease that each patient has, according to any statistical test 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 determine that it should reduce target disease symptoms in a statistically significant number of patients.

The term "consisting essentially of," or variations thereof, as used throughout the specification and claims, is meant to include all of the stated elements or groups of elements, and optionally other elements of similar or different nature to the stated elements, said other elements An element does not significantly alter the basic or new properties of a given dosing regimen, method or composition.

The term "naturally occurring" applied to an object as used herein refers to the fact that the object can be found in nature. For example, a polypeptide sequence or a polynucleotide sequence that exists in an organism (including a virus) that can be isolated from natural sources and has not been intentionally modified artificially in the laboratory is naturally occurring.

An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of the medical condition. 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 side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

"Exogenous" refers to a substance that is to be produced outside of an organism, cell or human in context.

"Endogenous" refers to a substance produced in a cell, organism or human body depending on the context.

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

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably and all such designations include progeny thereof. Thus, the words "transformants" and "transformed cells" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that, due to deliberate or unintentional mutations, all progeny may not be exactly the same in terms of DNA content. Mutant progeny that have the same function or biological activity as screened in the original transformed cell are included. Where a different name is meant, it 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 occurs or instances where it does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a particular sequence may, but need not, be present.

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

2. Example

The following examples are used to further describe the present invention, but these examples do not limit the scope of the present invention. The experimental methods that do not specify specific conditions in the examples of the present invention generally follow conventional conditions, such as Cold Spring Harbor Antibody Technology Experiment Manual, Molecular Cloning Manual; or conditions suggested by raw material or commodity manufacturers. Reagents with no specific source 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 human ERBB3 protein shown in SEQ ID NO: 13 was used as the template for designing the antigen of the present invention. The following ERBB3 antigens without special description refer to human ERBB3.

ERBB3 full-length protein: ERBB3 (SEQ ID NO: 13) sequence is as follows:

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

The monkey ERBB3 antigen (SEQ ID NO: 15) used for detection is the commercial product rhesus monkey ERBB3 Protein-His tag (Sino biological, cat#90043-K08H), which is the ECD region sequence of the monkey ERBB3 antigen with a His tag at the C-terminus , the sequence is as follows:

Multiple human PBMCs were collected, B cells were isolated, RNA was extracted, reverse transcribed into cDNA, and then a natural phage surface Fab display library (library capacity 1.6×10 ¹¹ ) was constructed using the cDNA as a template. The constructed natural Fab phage library was packaged to form phage particles, which were panned by liquid phase method. The phage was combined with biotinylated ERBB3 in liquid phase, and then separated by streptavidin magnetic beads. In order to obtain a positive sequence that binds to human ERBB3, biotinylated human ERBB3 was used for 2-3 rounds of panning, and 384 monoclonal colonies were picked and packaged into phage-displayed Fab fragments for ELISA test. Test the binding activity of monoclonal phage to human ERBB3: ELISA plates were coated with 1 μg/mL ERBB3 respectively, phage supernatant was added, and finally detected with anti-human IgG Fab HRP; 86 positive OD ₄₅₀ values detected by ELISA were greater than 0.2 The clones were sequenced, and the sequences were analyzed to obtain unique sequences: 45 VH (variable heavy chain) and 51 VL (variable light chain). The obtained heavy chain and light chain variable region sequences are respectively connected with human IgG1 heavy chain constant region and light chain constant region sequences to obtain full-length antibodies, and these full-length antibodies are subjected to experimental evaluation to finally determine a binding force and Antibody H01 with better functions; wherein, the connected human IgG1 heavy chain constant region and light chain constant region sequences are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

Table 1. Heavy and light chain variable region CDR sequences of antibodies

name	sequence	Numbering
HCDR1	TYSMN	SEQ ID NO: 1
HCDR2	SISSGSSYIYYADSLKG	SEQ ID NO: 2
HCDR3	EGGYFDL	SEQ ID NO: 3
LCDR1	GGNNIGSKSVH	SEQ ID NO: 4
LCDR2	YDTDRPS	SEQ ID NO: 5
LCDR3	QVWGTSSEV	SEQ ID NO: 6

Table 2. Heavy and light chain variable region sequences of antibodies

Note: CDR regions are marked by underline.

Table 3. Heavy and light chain sequences of antibodies

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

Antibody number	HCVR	HC	LCVR	LC
H01	SEQ ID NO: 7	SEQ ID NO: 9	SEQ ID NO: 8	SEQ ID NO: 10

The IgG1 heavy chain constant region sequence is as follows:

The light chain constant region sequence of the human lambda chain is as follows:

Example 2: Preparation of fully human antibodies

cDNA fragments were synthesized according to the amino acid sequences of the light and heavy chains of the H01 antibody and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). HEK293 cells (Life Technologies Cat. No. 11625019) were transfected with the expression vector and transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) at a ratio of 1: ₂ , and incubated in a CO incubator for 4- 5 days. The cell culture medium was collected, and after centrifugal filtration, the sample was applied to the antibody purification affinity column, and the column was washed with phosphate buffer, eluted with glycine hydrochloric acid buffer (pH2.7 0.1M Gly-HCl), neutralized with 1M Tris hydrochloric acid pH 9.0, and Phosphate buffer was dialyzed to obtain the antibody protein of the present invention, and its concentration and purity are as shown in Table 5:

Table 5. Concentration and Purity of Antibodies

Antibody	Concentration (mg/mL)	purity(%)
H01	0.85	96%

Example 3: In vitro binding activity of fully human antibodies

3.1 In vitro indirect ELISA binding experiment:

Human ERBB3 His protein (Sino biological, cat#10201-H08H), monkey ERBB3 His protein (Sino biological, cat#90043-K08H), murine ERBB3 His protein (Sino biological, cat#51003-M08H) were mixed with PBS at pH 7.4. ), human EGFR His protein (Sino biological, cat#10001-H08H), human ERBB2 His protein (Sino biological, cat#10004-H08H), human ERBB4 His protein (Sino biological, cat#10363-H08H) were diluted to 0.5 The concentration of μg/mL was added to a 96-well high-affinity ELISA plate at a volume of 100 μL/well, and incubated overnight (16-20 hours) in a refrigerator at 4°C. After washing the plate 3 times with PBST (pH 7.4 PBS containing 0.05% Tween-20), 200 μL/well of 1% bovine serum albumin (BSA) blocking solution diluted with PBST was added and incubated at 37°C for 2 hours for blocking. After blocking, the blocking solution was discarded, and the plate was washed once with PBST buffer.

The antibody to be tested was diluted with PBST containing 1% BSA, starting at 10 nM, 3-fold serial dilution, 11 doses, added to the ELISA plate at 100 μL/well, and incubated at 37° C. for 1 hour. After the incubation, the plate was washed three times with PBST, and 200 μL/well of secondary antibody anti-human HRP (Abeam, cat#ab97225) diluted with PBST containing 1% BSA was added, and incubated at 37° C. for 0.5 hour. After washing the plate 5 times with PBST, add 100 μL/well of TMB chromogenic substrate (Suzhou Yake Chemical Reagent Co., Ltd., cat#S0025), incubate at 25°C for 8-15 minutes in the dark, and add 50 μL/well of 1M HCl to stop After the reaction, the absorbance was read at 450 nm with a microplate reader (Thermo, Lux), and the data were analyzed.

Concentration signal value curve analysis was performed. The results are shown in the table below. H01 antibody has good affinity for human, monkey and murine ERBB3, and does not bind to other ERBB receptors (EGFR, ERBB2, ERBB3).

Table 6. Affinity of antibodies to antigenic proteins

Note: "-" means not combined

3.2 In vitro cell binding experiments:

(1) Binding experiment with stably transfected cell line 293T human ERBB3

HEK293T cells overexpressing human ERBB3 (293T-human ERBB3) were digested with trypsin, collected by centrifugation, adjusted to cell density with FACS buffer (1×PBS containing 2% FBS), and plated in 96-well U bottom plates. Wells 1 x 10 ⁵ to 2 x 10 ⁵ cells. Centrifugation: 1200g, 5 minutes, discard the supernatant, add 100 μL of antibody solution that has been serially diluted with FACS buffer (200nM initial, 5-fold serial dilution, 8 doses), incubate at 4°C for 1 hour. Centrifuge at 1200 g for 5 minutes, discard the supernatant, wash the cells twice with FACS buffer, add the fluorescently labeled secondary antibody working solution PE anti-human IgG Fc Antibody (Biolegend, Cat#409304) prepared in FACS buffer, and resuspend in 100 μL per well Cells were incubated at 4°C for 1 hour. Centrifuge at 1200g for 5 minutes and discard the supernatant. After washing the cells twice with FACS buffer, the cells were resuspended in PBS, and the fluorescence signal was detected by a flow cytometer Bio-Rad (ZE5), and the EC ₅₀ concentration of the antibody-binding cells was analyzed by curve.

Table 7. Antibody binding ability (EC ₅₀ value) to 293T-hERBB3 cells

The results show that the antibody of the present invention can specifically bind to the stably transfected cell line overexpressing human ERBB3.

(2) Binding experiment with tumor cell lines

Through the FACS experiment, the binding ability of the antibody to the following tumor cells expressing human ERBB3 was detected: human breast cancer cells SK-BR-3 (Nanjing Kebai, Cat#CBP60413) and MX-1 (Nanjing Kebai, Cat#CBP60640), Human colon adenocarcinoma cell SW620 (Nanjing Kebai, Cat#CBP60036), human gastric cancer cell NCI-N87 (Chinese Academy of Sciences Cell Bank, Cat#TCHu130), human head and neck cancer cell FaDu (Chinese Academy of Sciences Cell Bank, Cat#TCHu132), human non-small cell Cell lung cancer cell HCC827 (Cell Bank of Chinese Academy of Sciences, Cat#TCHu153). After the tumor cells were trypsinized, the cells were collected by centrifugation, the cell density was adjusted with FACS buffer (1×PBS containing 2% FBS), and then plated in 96-well U-bottom plates, ¹ ×105 to ² ×105 cells per well cell. Centrifugation: 1200g, 5 minutes, discard the supernatant, add 100 μL of antibody solution that has been serially diluted with FACS buffer (200nM initial, 5-fold serial dilution, 8 doses), and incubate at 4°C for 1 hour. Centrifuge at 1200 g for 5 minutes, discard the supernatant, wash the cells twice with FACS buffer, add the fluorescently labeled secondary antibody working solution PE anti-human IgG Fc Antibody (Biolegend, Cat#409304) prepared in FACS buffer, and resuspend in 100 μL per well Cells were incubated at 4°C for 1 hour. Centrifuge at 1200g for 5 minutes and discard the supernatant. After washing the cells twice with FACS buffer, the cells were resuspended in PBS, and the fluorescence signal was detected by a flow cytometer Bio-Rad (ZE5), and the EC ₅₀ concentration of the antibody-binding cells was analyzed by curve.

Table 8. Antibody binding ability to tumor cells (EC ₅₀ value)

The results show that the antibody of the present invention can specifically bind to tumor cell lines expressing human ERBB3.

Example 4: Endocytosis of fully human antibodies

To detect whether the antibody of the present invention can be endocytosed with human ERBB3 after binding to ERBB3, the human breast cancer cells SK-BR-3 expressing ERBB3 and HEK293T cells overexpressing human ERBB3 (293T-human ERBB3) were used for evaluation.

4.1 Endocytosis of fully human antibody in tumor cells SK-BR-3

The cells were trypsinized, collected and resuspended in pre-chilled FACS buffer to adjust the cell concentration 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, and add 1 mL of the prepared antibody to be tested to resuspend the cells. The supernatant was discarded by centrifugation (4°C, 1500 rpm × 5 minutes), washed twice with FACS buffer, and the supernatant was discarded. 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 at 4°C for 30 minutes on a shaker, and discard the supernatant by centrifugation (4°C, 1500 rpm × 5 minutes), Wash twice with FACS buffer and discard the supernatant. Add 1 mL of pre-warmed cell culture medium to each tube to resuspend the cells and mix them evenly. Divide them into 5 tubes, each tube of 200 μL, for the 0-minute group, blank group, 15-minute group, 30-minute group and 60-minute group, respectively. 10 minutes and blanks were placed on ice, and the rest were placed in a 37°C incubator for 15 minutes, 30 minutes, and 60 minutes, respectively. The EP tubes were taken out at the corresponding time points and placed on ice for 5 minutes to pre-cool. All treatment groups were centrifuged and discarded. The supernatant (4° C., 1500 rpm×5 minutes) was washed once with FACS buffer, and the supernatant was discarded. Add 100 μL strip buffer to the EP tubes of all treatment groups except the 0-minute group, 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 . All treatment groups were added with 100 μL of PBS to resuspend the cells and detected by 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 The table shows:

Table 9. Endocytosis of fully human antibody in tumor cells SK-BR-3

The results show that the fully human antibody H01 of the present invention has obvious endocytosis in human breast adenocarcinoma cell SK-BR-3.

4.2 Endocytosis of fully human antibody in stable cell line 293T-human ERBB3

The cells were trypsinized, collected and resuspended in pre-chilled FACS buffer to adjust the cell concentration 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, and add 1 mL of the prepared antibody to be tested to resuspend the cells. The supernatant was discarded by centrifugation (4°C, 1500 rpm × 5 minutes), washed twice with FACS buffer, and the supernatant was discarded. 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 at 4°C for 30 minutes on a shaker, and discard the supernatant by centrifugation (4°C, 1500 rpm × 5 minutes), Wash twice with FACS buffer and discard the supernatant. Add 1 mL of pre-warmed cell culture medium to each tube to resuspend the cells and mix well, divide into 4 tubes, each tube is 250 μL, which are divided into 0 minutes group, blank group, 30 minutes group, 60 minutes group and 120 minutes group. 10 minutes and blanks were placed on ice, and the rest were placed in a 37°C incubator for 30 minutes, 60 minutes, and 120 minutes, respectively, and the EP tubes were taken out at the corresponding time points and placed on ice to pre-cool for 5 minutes. All treatment groups were centrifuged and discarded. The supernatant (4° C., 1500 rpm×5 minutes) was washed once with FACS buffer, and the supernatant was discarded. Add 250 μL of strip buffer to the EP tubes of all treatment groups except the 0-minute group, 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. All treatment groups were added with 100 μL of PBS to resuspend the cells and detected by 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 The table shows:

Table 10. Endocytosis of fully human antibody in 293T-human ERBB3

The results show that the fully human antibody H01 of the present invention has obvious endocytosis in HEK293T cells (293T-human ERBB3) overexpressing human ERBB3.

Example 5: Ligand Blocking Experiment of Fully Human Antibody

The blocking effect of the antibody on the binding between the ligand and tumor cells expressing human ERBB3 was tested by cell-base receptor blocking assay (cRBA). The human breast cancer cells SK-BR-3 expressing ERBB3 (Nanjing Kebai, Cat#CBP60413) were trypsinized and counted, resuspended in FACS buffer to 1×10 ⁶ /mL, and plated with 100 μL per well to 96 In the well plate, centrifuge: 1500rpm, 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 in 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 1.2 μg/mL ligand bio-HRG1 (Sino Biological, Cat#11609-HNCH-B), mix well, incubate on ice for 10 minutes, centrifuge: 1500rpm, 4°C, 5 minutes, discard The supernatant was washed twice with FACS buffer. Add 100 μL of prepared 100-fold dilution SA-PE (R&D, Cat#F0040), incubate at 4°C for 45 minutes, wash twice with FACS buffer, resuspend cells in 100 μL, and use flow cytometer Bio-Rad (ZE5) Assays were performed, blocking ratios were calculated and the _IC50 concentrations of the antibodies were plotted.

Blocking rate (%)=(control well MFI-sample well MFI)/control well MFI×100, control wells are wells without fully human antibody, and sample wells are fully human antibody wells added with different concentration gradients.

Table 11. Ligand blocking effect of fully human antibodies

The results showed that the fully human antibody H01 could block the binding between the ligand and ERBB3 expressed by tumor cells.

Example 6: Dimerization Blocking Experiment

In this experiment, the cell line EGFR/ERBB3 dimerized cells containing the reporter system (

Cat#93-1125C3) and ERBB2/ERBB3 dimerized cells (

Cat#93-1042C3), to determine the blocking effect of fully human antibodies on EGFR/ERBB3 and ERBB2/ERBB3 heterodimer formation. The cultured cells were prepared at 1×10 ⁵ /mL, 20 μL per well was added to a white 384-well plate, and incubated overnight. Add 5 μL of the prepared antibody solution (initial working concentration of 30 μg/mL, 3-fold gradient dilution, 10 concentration points), and incubate at room temperature for 1 hour. Then add 5 μL of ligand (the working concentration of EGFR/ERBB3 cell line is 64ng/mL, and the working concentration of ERBB2/ERBB3 cell line is 36ng/mL), and set the well with ligand but no antibody as dimerization positive control The wells, the wells without antibodies and ligands are the background wells, incubate in a 37°C incubator for 6 to 16 hours, and add 15 μL (50% v/v) of PathHunter detection reagent mixture (

Cat#93-0247) for 1 hour at room temperature. Chemiluminescence readings were performed on a PerkinElmer Envision ^™ . The inhibition rate of the sample wells was calculated by the following formula and plotted to obtain the _IC50 value.

Inhibition rate (%) = 100% x (1-(sample well-background well)/(positive control well-background well)

Table 12. Dimerization blocking effect of fully human antibodies

The results showed that the fully human antibody H01 could block the heterodimerization of EGFR/ERBB3 and ERBB2/ERBB3.

Example 7: Phosphorylation blocking experiment

In the ligand-dependent ERBB3 pathway, the ligand HRG binds to the ERBB3 protein expressed by tumor cells to phosphorylate and activate it, and then forms a heterodimer with EGFR or ERBB2, thereby activating the downstream AKT and ERK pathways. In this experiment, the phosphorylation detection kits of pHer3 (Cisbio, Cat#64HR3PEG), pAKT (Cisbio, Cat#64AKSPEG) and pERK (Cisbio, Cat#64ERKPPEH) were used to test the ERBB3-positive human breast cancer cells MCF-7 in the ligand. The 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, the supernatant was discarded by centrifugation, and the cells were pelleted with reaction medium (RPMI 1640 medium + 10% heat-inactivated FBS + 1% penicillin/streptomycin) ) was resuspended to 1×10 ⁶ /mL, and 100uL of cell suspension was added to each well of a flat-bottom 96-well plate (low adsorption plate). The supernatant was discarded by suction, and 100 uL of starvation medium (RPMI 1640 medium + 1% penicillin/streptomycin) was added to each well, and incubated overnight in a 37-degree incubator. Add 50uL of fully human antibody prepared in starvation medium (4 times the working concentration, ie 800nM), set the well without antibody as a positive control well for phosphorylation activation, and incubate at 37°C for 60 minutes after mixing. Take out the reaction plate, add 50ul of ligand HRG1-β (200ng/mL) with 4 times working concentration prepared in Starved medium, set the well without ligand as the negative control well for phosphorylation, and set the well without antibody and ligand as background Control wells. After mixing, incubate in a 37°C incubator for 10 minutes. Since the cells are adherent, the supernatant can be discarded directly, and the phosphorylation effect can be detected by the kits of pHer3, pAKT and pERK. The cell pellet was added to 70uL of 1* supplemental lysis buffer in the kit, and the reaction was shaken at room temperature for 30 minutes. After the gun was mixed, 16ul of the lysis supernatant was transferred to a 384-well plate, and then 4uL of the two labeled antibody mixtures in the kit were added. (20-fold dilution). Seal with tin foil and incubate at room temperature for 4 h. Read HTRF (665nm and 620nm) on a microplate reader (Thermo, Lux). Calculate the Ratio value of each well according to 665nm/620nm×10000, and use this value to calculate the blocking rate.

Blocking rate (%)=(sample well-negative control well)/(positive control well-background control well).

Table 13. Phosphorylation blocking effect of fully human antibodies

The results showed that the fully human antibody H01 could block the phosphorylation of Her3, AKT and ERK.

Claims (21)

1. An anti-ERBB3 antibody or an antigen-binding fragment thereof, comprising: an antibody heavy chain variable region and an antibody light chain variable region; wherein, the antibody heavy chain variable region comprises at least one selected from the following sequence HCDR: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; the antibody light chain variable region comprises at least one LCDR selected from the following sequences: SEQ ID NO: 4, SEQ ID NO:5 and SEQ ID NO:6.
2. The anti-ERBB3 antibody or antigen-binding fragment thereof of claim 1, wherein the antibody heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2 and SEQ ID NO : HCDR3 shown in 3.
3. The anti-ERBB3 antibody or antigen-binding fragment thereof of claim 1, wherein the antibody light chain variable region comprises LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and SEQ ID NO : LCDR3 shown in 6.
4. The anti-ERBB3 antibody or antigen-binding fragment thereof of claim 1, comprising an antibody heavy chain variable region and an antibody light chain variable region, wherein,

   The antibody heavy chain variable region comprises: HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2 and HCDR3 shown in SEQ ID NO:3; and

   The antibody light chain variable region comprises: LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:6.
5. The anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the anti-ERBB3 antibody or antigen-binding fragment thereof comprises the heavy chain variable region shown in SEQ ID NO: 7, or a heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology therewith.
6. The anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the anti-ERBB3 antibody or antigen-binding fragment thereof comprises the light chain variable region shown in SEQ ID NO: 8, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology therewith.
7. The anti-ERBB3 antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the anti-ERBB3 antibody or antigen-binding fragment thereof comprises the heavy chain variable region shown in SEQ ID NO: 7, or A heavy chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology thereto, and a light chain variable region shown in SEQ ID NO: 8, or with A light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology.
8. The anti-ERBB3 antibody or antigen-binding fragment thereof of any one of claims 5-7, further comprising a heavy chain constant region derived from human IgG1, IgG2, IgG3 or IgG4 or a variant thereof;

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

   More preferably, it comprises a human IgG1 heavy chain constant region;

   Most preferably comprise the heavy chain constant region shown in SEQ ID NO: 11;

   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;

   It preferably comprises a light chain constant region derived from a human lambda chain;

   Most preferably it comprises a light chain constant region as set forth in SEQ ID NO:12.
9. The anti-ERBB3 antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the anti-ERBB3 antibody or antigen-binding fragment thereof comprises the heavy chain shown in SEQ ID NO: 9, or has at least 80 %, 85%, 90%, 95% or 99% homology of the full length heavy chain.
10. The anti-ERBB3 antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the anti-ERBB3 antibody or antigen-binding fragment thereof comprises, or has at least 80% of the light chain shown in SEQ ID NO: 10 %, 85%, 90%, 95% or 99% homology of the full length light chain.
11. The anti-ERBB3 antibody or antigen-binding fragment thereof according to any one of claims 1-8, wherein the anti-ERBB3 antibody or antigen-binding fragment thereof comprises the heavy chain shown in SEQ ID NO: 9, or has A full-length heavy chain of at least 80%, 85%, 90%, 95% or 99% homology, and the light chain shown in SEQ ID NO: 10, or at least 80%, 85%, 90%, 95% therewith % or 99% homology to the full-length light chain.
12. A polynucleotide encoding the anti-ERBB3 antibody or antigen-binding fragment thereof of any one of claims 1-11.
13. An expression vector containing the polynucleotide of claim 12.
14. A host cell, which introduces or contains the expression vector of claim 13 .
15. The host cell according to claim 14, wherein the host cell is bacteria, yeast or mammalian cells; preferably Escherichia coli, Pichia pastoris, CHO cells or HEK293 cells.
16. A method for producing an anti-ERBB3 antibody or an antigen-binding fragment thereof, comprising the steps of:

    a) culturing the host cell of any one of claims 14-15;

    b) isolating the antibody from the culture; and,

    c) purifying the antibody.
17. A pharmaceutical composition comprising the anti-ERBB3 antibody or antigen-binding fragment thereof of any one of claims 1-11, and a pharmaceutically acceptable excipient, diluent or carrier.
18. A detection or diagnostic reagent, which contains the anti-ERBB3 antibody or its antigen-binding fragment according to any one of claims 1-11, and one or more detectable markers that bind the ERBB3 antibody or its antigen-binding fragment to ERBB3 reagents.
19. Use of the anti-ERBB3 antibody or antigen-binding fragment thereof of any one of claims 1-11 or the pharmaceutical composition of claim 17 in the manufacture of a medicament for the treatment or prevention of ERBB3-mediated diseases or disorders .
20. The anti-ERBB3 antibody or antigen-binding fragment thereof of any one of claims 1-11 or the detection or diagnostic reagent of claim 18 is used in the preparation of a reagent for detecting, diagnosing, and prognosing ERBB3-mediated diseases or conditions Use in the box.
21. The use of claim 19 or 20, wherein:

    the disease or condition 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.