erb-B2 bispecific antibody, pharmaceutical composition and its use

Abstract

This invention provides an anti-erb-B2 bispecific antibody. It also describes related polynucleotides encoding the provided anti-erb-B2 bispecific antibody or antigen-binding fragment, cells expressing the provided antibody or antigen-binding fragment, and methods for preparing, purifying, and identifying the bispecific antibody. Furthermore, this invention describes the excellent effects of the anti-erb-B2 bispecific antibody, including in vitro binding activity, cancer cell inhibition and killing, and antibody-mediated cytotoxicity. The above-described anti-erb-B2 bispecific antibody can be used to prepare drugs or compositions for treating conditions associated with high erb-B2 expression in individuals.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the field of antibody technology, and more specifically to the erb-B2 bispecific antibody, pharmaceutical compositions, and their uses. Background Technology

[0002] Monoclonal antibodies (mAbs) possess unique specificity, enabling them to bind to target proteins with high specificity and selectivity due to their broad binding surface. Monoclonal antibodies have made significant contributions to the development of novel diagnostic and therapeutic methods and have been widely applied. However, before truly effective monoclonal antibody molecules can be obtained, extensive antibody engineering and structural optimization are required. Since the approval of the first anti-CD20 monoclonal antibody, rituximab (Rituxan) (WO200002743), several monoclonal antibody molecules have been approved for clinical use in cancer treatment. These monoclonal antibody drugs have shown significant clinical efficacy and good safety profiles. Examples include rituximab in hematological malignancies and nivolumab (US8779105) in solid tumors.

[0003] Erb-B (also known as HER) receptors are expressed in a variety of tissues derived from epithelium, mesenchyme, and neurons, and are involved in controlling a range of biological processes, such as proliferation, differentiation, migration, and apoptosis. Furthermore, dysregulation of their expression is associated with multiple types of human cancers and poor clinical outcomes. Due to the importance of ErbB proteins in development and cellular transformation, significant attention has been paid to the intracellular signaling initiated by this receptor's tyrosine kinase family.

[0004] Ligand binding to the Erb-B receptor leads to dimerization and activation of its intrinsic kinase activity, followed by phosphorylation of receptor cytoplasmic tail-specific tyrosine residues. These phosphorylated tyrosine residues, in turn, provide recognition sites for intracellular signaling intermediates, thereby linking receptor tyrosine kinases (RTKs) to downstream transduction cascades. The selection and combination of activated pathways ultimately result in changes in gene expression, triggering appropriate biological responses to received extracellular signals.

[0005] Erb-B2 is a single receptor-like transmembrane protein with a molecular weight of 185 kDa. Structurally associated with the epidermal growth factor receptor, it is overexpressed in various cancers, including breast cancer, ovarian cancer, and gastric cancer. Erb-B2 mediates cell growth, differentiation, and survival, while also promoting the invasive spread of cancer cells. Overexpression of erb-B2 has been observed in 25%-30% of patients with primary breast cancer. Erb-B2 gene amplification results in increased expression of the erb-B2 protein on the surface of these tumor cells, leading to erb-B2 protein activation. Compared to erb-B2-negative cancers, erb-B2-positive tumors are more aggressive and associated with shorter survival times, poorer overall survival, a higher risk of recurrence, and central nervous system disorders. Approximately 30% to 50% of erb-B2-positive breast cancer patients develop brain metastases over time. Clinically, erb-B2 expression is closely related to patient prognosis; patients with high erb-B2 expression are more prone to tumor metastasis and have shorter survival times. Because of the significant differences in expression levels between normal cells and tumor cells, erb-B2 has become an ideal target for tumor immunotherapy and a hot topic in current tumor treatment research.

[0006] Trastuzumab is currently used as a first-line drug for the treatment of breast cancer. It is effective in treating metastatic breast cancer with erb-B2 overexpression, and the objective response rate of first-line monotherapy is 30-50%. However, it is not ideal in treating metastatic breast cancer with low erb-B2 expression, and resistance has developed in many patients who initially responded to the antibody within one year. Summary of the Invention

[0007] To address the aforementioned technical problems, the present invention aims to provide an erb-B2 bispecific antibody, a pharmaceutical composition, and its uses.

[0008] To achieve the objectives of this invention, the technical solution adopted is as follows:

[0009] The present invention provides an erb-B2 bispecific antibody comprising (a) a single-chain antibody fragment capable of binding to the juxtamembrane domain of erb-B2; and (b) a hapten capable of binding to the distal membrane domain of erb-B2; wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the single-chain antibody fragment comprise SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0010] In some embodiments, the single-chain antibody fragment is composed of GGGSGGGGSGGGGGGGG (SEQ ID NO:3) linking the variable region heavy chain and light chain together.

[0011] In some embodiments, the single-chain antibody fragment is directly connected to the hinge region at the end of the single-chain antibody; the connection order is scFV-hinge region-non-wild-type constant region.

[0012] In some embodiments, the amino acid sequence of the variable region of the single-chain antibody fragment includes: SEQ ID NO: 4, SEQ ID NO: 5.

[0013] In some embodiments, the amino acid sequence of the half-antibody heavy / light chain comprises: SEQ ID NO: 6 / SEQ ID NO: 7, SEQ ID NO: 8 / SEQ ID NO: 7.

[0014] In some embodiments, the amino acid sequence of the single-chain antibody / hemipeptide heavy chain / hemipeptide light chain includes:

[0015] SEQ ID NO:9 / SEQ ID NO:6 / SEQ ID NO:7, SEQ ID NO:10 / SEQ ID NO:8 / SEQ ID NO:7, SEQ ID NO:11 / SEQ ID NO:8 / SEQ ID NO:7.

[0016] In some embodiments, the nucleic acid sequences corresponding to the single-chain antibody / half-antibody heavy chain / half-antibody light chain include: SEQ ID NO: 12 / SEQ ID NO: 13 / SEQ ID NO: 14, SEQ ID NO: 15 / SEQ ID NO: 16 / SEQ ID NO: 14, SEQ ID NO: 17 / SEQ ID NO: 16 / SEQ ID NO: 14.

[0017] The present invention also provides a pharmaceutical composition comprising the above-mentioned erb-B2 bispecific antibody and a pharmaceutically optional carrier and composition.

[0018] The present invention also provides the use of the above-described erb-B2 bispecific antibody in the preparation of a medicament for treating erb-B2-related conditions in individuals, including tumors, blood disorders, infectious diseases, autoimmune diseases, or fibrotic diseases.

[0019] In some embodiments, the erb-B2-related condition is a metastatic tumor expressing erb-B2.

[0020] In some embodiments, the individual was detected as erb-B2 positive or as having elevated erb-B2 expression levels.

[0021] In some embodiments, the tumor disease is at least one of hematologic malignancies and solid tumor diseases. The hematologic malignancies are at least one of acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, multiple myeloma, mixed leukemia, and non-Hodgkin's lymphoma. The solid tumors are at least one of lymphoma, breast cancer, head and neck cancer, gastric cancer, lung cancer, esophageal cancer, colorectal cancer, ovarian cancer, cervical cancer, liver cancer, kidney cancer, pancreatic cancer, bladder cancer, colorectal cancer, glioma, and melanoma.

[0022] The beneficial effects of this invention are as follows:

[0023] Any erb-B2 bispecific antibody, compared to trastuzumab and pertuzumab, can simultaneously bind to both the proximal and distal membrane domains of erb-B2.

[0024] Any erb-B2 bispecific antibody, compared to trastuzumab, pertuzumab, and the erb-B2 bispecific reference antibody, exhibits significantly superior antibody-dependent cell-mediated cytotoxicity in in vitro cell experiments compared to these antibodies.

[0025] Any erb-B2 bispecific antibody showed significant cell-inhibiting activity compared to trastuzumab, pertuzumab, and the erb-B2 bispecific reference antibody.

[0026] Compared to trastuzumab, pertuzumab, and the erb-B2 bispecific antibody, any erb-B2 bispecific antibody exhibits less steric hindrance and higher affinity in its proximal membrane binding domain, while also demonstrating better affinity in its distal membrane domain. This allows for a more effective synergistic effect. Attached Figure Description

[0027] Figure 1 This is a three-dimensional structural diagram of the distal membrane segment and proximal membrane end of erb-B2 in Embodiment 1 of the present invention.

[0028] Figure 2 This is an SDS-PAGE image of the bispecific antibody after expression and purification in Example 3 of the present invention. From left to right, it shows 9203-3 non-reducing and reducing gels, 9203-2 non-reducing and reducing gels, 9203-1 non-reducing and reducing gels, and Marker.

[0029] Figure 3A and Figure 3B These are images from Example 4 of the present invention, showing the ELISA affinity identification of antibodies with antigens BPS-C-11 and BPS-C-12.

[0030] Figure 4A and Figure 4BThis is the result of the binding experiment of the bispecific antibody in Example 5 of the present invention with the human gastric cancer N87 cell line and the human ovarian cancer SKOV3 cell line. Figure 4A These are the results of a dose-response assay (FACS) of the binding of the bispecific antibody to human gastric cancer N87 cells. Figure 4B This is the result of a dose-response assay (FACS) of the combination of bispecific antibodies with the human ovarian cancer SKOV3 cell line.

[0031] Figure 5A and Figure 5B This is the result of antibody-mediated cytotoxicity in human gastric cancer cell line N87 and human ovarian cancer cell line SKOV3 in Example 6 of the present invention.

[0032] Figure 6 This is the result of the target inhibitor dose-effect experiment of the bispecific antibody in the human gastric cancer cell line N87 in Example 7 of the present invention.

[0033] Figure 7 is a diagram illustrating the Kd results of the bispecific antibody at both ends and the control antibody in Example 8 of the present invention. Figure 7A , Figure 7B , Figure 7C and Figure 7D The Kd plots are for mab1, trastuzumab, mab2, and pertuzumab, respectively. Detailed Implementation

[0034] To illustrate the present invention more clearly, the present invention will be further described in detail below with reference to the embodiments and the accompanying drawings.

[0035] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methodologies, schemes, and reagents described herein, as these can be varied and adapted. It should also be understood that the terminology used herein is for describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] Terminology definition:

[0037] The term "antibody" is used in the broadest sense herein and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity. "Antibody" (or "Abs") and "immunoglobulin" (or "Igs") are glycoproteins with the same structural features. Antibodies typically contain at least two full-length heavy chains and two full-length light chains, but in some cases may include fewer chains; for example, naturally occurring antibodies in camels may contain only heavy chains. Each light chain is connected to the heavy chain by a covalent disulfide bond (also known as a "VH / VL pair"), and each heavy and light chain also has regularly arranged intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain.

[0038] The term "monoclonal antibody" refers to a preparation of an antibody molecule consisting of a single amino acid, without specifying the method of its preparation. Monoclonal antibodies or their immunologically active fragments can be produced using hybridoma technology, recombinant technology, phage display technology, synthetic technology, or other production technologies known in the art. The methods for preparing monoclonal antibodies described in this invention include in vitro culture of hybridoma cells or preparation via DNA recombination technology. Monoclonal antibodies are highly specific, targeting a single antigenic site.

[0039] Antibodies can be classified into five classes based on the amino acid sequence of the constant region of the antibody heavy chain: IgA, IgD, IgE, IgG, and IgM. Several isotypes within these classes can be further subdivided into subclasses, such as IgG1, IgG2, IgG3 and IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different types of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. Antibodies can be classified into κ and λ regions based on the different constant regions (CL) of the antibody light chain. Within the full-length light and heavy chains, the variable and constant regions are typically linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also includes "D" regions of approximately 10 or more amino acids.

[0040] "Antigen-binding fragments" refer to molecules distinct from intact antibodies, containing a portion of the intact antibody capable of binding antigens bound by the intact antibody. This portion does not contain the constant heavy chain domains (i.e., CH2, CH3, and CH4, depending on the antibody isotype) of the intact antibody's Fc region. Examples of immunologically active antibody fragments include Fab, Fab′, Fab′-SH, F(ab′)2, ScFv, and Fv fragments. Fab fragments include a heavy / light chain variable region and a constant domain of the light chain, as well as a first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments in that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. F(ab′)2 antibody fragments are initially produced as Fab′ fragment pairs, with a hinge cysteine ​​residue between the Fab′ fragments. Fv fragments are the smallest fragments in the products of enzymatic analysis of IgG and IgM type antibodies. The antigen-binding region of Fv fragments consists of VH and VC regions, but they lack the CH1 and CL regions. VH and VL are linked together in the Fv fragment via non-covalent bonds. ScFv is an Fv-type fragment that includes VH and VL regions linked together by a flexible polypeptide.

[0041] The term "bispecific antibody" refers to an artificial antibody containing two specific antigen-binding sites. It bridges the gap between target cells and functional molecules (cells), stimulating a directed immune response, and is a type of genetically engineered antibody. Biantibodies can be bivalent or bispecific. Biantibodies are more fully described, for example, in EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 90:6444-6448 (1993).

[0042] The term "epitope" refers to any antigenic determinant on an antigen that binds to the complementary site of an antibody. Epitope determinants typically consist of chemically active surface groups of a molecule, such as amino acid or sugar side chains, and usually possess specific three-dimensional structural features as well as specific charge characteristics.

[0043] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it through transformation. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0044] In this article, the term "half-antibody" refers to an antibody that contains an Fc-modified heavy chain and a light chain at one end in order to obtain a bispecific antibody.

[0045] The term "host cell" refers to a cell to which foreign nucleic acids have been introduced, including the progeny of such cells. It is capable of expressing the foreign nucleic acids intracellularly or on the cell membrane, or releasing them extracellularly.

[0046] The term "cancer" refers to a growth or tumor resulting from the abnormal, uncontrolled growth of cells. As used herein, cancer explicitly includes leukemia and lymphoma. The term "cancer" also refers to a disease involving cells with the potential to metastasize to distant sites and exhibiting phenotypic characteristics distinct from non-cancer cells, such as the formation of colonies in a three-dimensional matrix like soft agar or the formation of tubular networks or reticular matrices in a three-dimensional basement membrane or extracellular matrix formulation. Non-cancer cells do not form colonies in soft agar but rather form distinct spherical structures in a three-dimensional basement membrane or extracellular matrix formulation.

[0047] The terms "immune," "immunological," or "immune" response refer to the development of beneficial humoral (antibody-mediated) and / or cellular (mediated by antigen-specific T cells or their secreted products) responses to peptides in a recipient patient. Such responses can be active responses induced by the administration of an immunogen or passive responses induced by the administration of antibodies or primitivic T cells. Cellular immune responses are induced by the presentation of polypeptide epitopes associated with class I or II MHC molecules to activate antigen-specific CD4+ helper T cells and / or CD8+ cytotoxic T cells. This response may also involve the activation or recruitment of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, neutrophils, or other components of innate immunity. The presence of a cell-mediated immune response can be determined by proliferation assays (CD4+ T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by isolating antibodies and T cells separately from immunized syngeneic animals and measuring the protective or therapeutic effect in a second subject.

[0048] Example 1: erb-B2 antigen design

[0049] A search of uniprot yielded the full-length erb-B2 sequence with the code P04626. Its extracellular terminal sequence was then extracted.

[0050] (SEQ ID NO:20).

[0051] The modeller program was used to retrieve the extracellular terminal template of ERB-B2 cells and construct its three-dimensional structural model, followed by energy optimization. PyMOL was used to visualize the results after energy minimization. Based on the structure, four three-dimensional structural regions were found at the ERB-B2 extracellular terminal, with the fourth region located close to the cell membrane. Figure 1As shown. The complete extracellular structure of erb-B2 revealed that its four structural regions are not completely independent; numerous non-bonded interactions exist between them. Designing antigens for different regions is challenging, as structural collapse can easily occur due to the lack of support from these non-bonded interactions. To address this, different regions were remodeled, energy minimized, and then molecular dynamics simulations were performed using NAMD2. By controlling the temperature and pressure during the simulation, an isothermal-barrier ensemble (NPT) was obtained to simulate the experimental conditions. Solvation was performed in a solvent chamber with periodic boundary conditions, followed by a 2ns molecular dynamics simulation. The final result was superimposed on the original extracellular full-field structure to calculate the RMSD value. Structures with an RMSD less than 1 Å were selected, yielding sequences containing the fourth region and the first and second regions. 6HIS tags were added to both sequences, resulting in SEQ ID NO:18 and SEQ ID NO:19.

[0052] Example 2: Preparation of erb-B2 antigen

[0053] The two expression vectors to be constructed in Example 2 were modified by adding an optimized signal peptide to the N-terminus for secretory expression. The amino acids of the recombinant protein were codon-optimized, and the Kozak sequence GCCGCCACC was added to the 5' end of the nucleotide. EcoRI / HindIII restriction enzyme sites of pcDNA3.4 were added to both ends of the nucleotide. The synthesized gene was then ligated into the pcDNA3.4 vector after enzyme digestion. The expression vectors were amplified and extracted from *E. coli* and detoxified. They were then transfected into Expi-293F cells at a plasmid:culture medium ratio of 1 μg / ml. The transfection reagent used was the ExpiFectamine 293 Transfection Kit (Theromfisher, Lot#: A14524), and the cell density at transfection was 25*102. 5 Cells / ml, expression enhancers Enhancer1 and Enhancer2 were added 16-18 h post-transfection, and cell supernatant was collected 5 days post-transfection. Purification was performed using a nickel column, with the column washed with 3-5 column volumes of deionized water. The column was equilibrated with 5 column volumes of Lysis Buffer. After loading the sample, the column was equilibrated with Wash Buffer, and eluted with 5-10 column volumes of Elution Buffer, collecting the protein solution in the eluent. The collected protein eluent was concentrated using a 4000G ultrafiltration tube (Millipore UFC903096) and the buffer was replaced with PBS (HyClone SH30256.01). After SDS-PAGE analysis, the eluent was stored at -20°C. Endotoxin removal, sterilization by filtration, and SDS-PAGE analysis were performed. The two successfully expressed and purified antigens were BPS-C-11 and BPS-C-12.

[0054] Example 3: Preparation of Bispecific Antibodies

[0055] Following the combinations SEQ ID NO: 12 / SEQ ID NO: 13 / SEQ ID NO: 14, SEQ ID NO: 15 / SEQ ID NO: 16 / SEQ ID NO: 14, SEQ ID NO: 17 / SEQ ID NO: 16 / SEQ ID NO: 14, the bispecific antibodies 9203-3, 9203-2, and 9203-1 were constructed, with corresponding amino acid sequences SEQ ID NO: 9 / SEQ ID NO: 6 / SEQ ID NO: 7, SEQ ID NO: 10 / SEQ ID NO: 8 / SEQ ID NO: 7, and SEQ ID NO: 11 / SEQ ID NO: 8 / SEQ ID NO: 7, respectively. Plasmids were constructed, and an optimized signal peptide was added to the N-terminus for secretory expression. The amino acids of the recombinant antibodies were codon-optimized, and the Kozak sequence GCCGCCACC was added to the 5' end of the nucleotides. EcoRI / HindIII restriction enzyme sites of pcDNA3.4 were added to both ends of the nucleotides. The synthesized gene was digested with enzymes and ligated into the pcDNA3.4 vector. The expression vector was amplified and extracted from *E. coli* and detoxified. It was then transfected into Expi-293F cells at a plasmid:culture medium ratio of 1 μg / ml. The transfection reagent used was the ExpiFectamine293 Transfection Kit (Theromfisher, Lot#: A14524), with a single-chain:heavy-chain:light-chain transfection ratio of 2:2:1. The cell density at transfection was 25*102. 5Cells / ml were transfected, and expression enhancers Enhancer1 and Enhancer2 were added 16-18 hours post-transfection. Cell supernatant was collected 5 days post-transfection. Simultaneously, ZW25 (US20210260210), trastuzumab, and pertuzumab (US20090285837A1) were constructed according to the above methods. The wild-type FC antibody mab1 was constructed using SEQ ID NO: 1 and SEQ ID NO: 2, and manb2 was constructed using the variable regions of SEQ ID NO: 6 and SEQ ID NO: 7. After purification with Protein A, the supernatant was centrifuged at 10,000 rpm for 30 min at 4°C to remove cell debris. The Protein A column was equilibrated with 10 column volumes of equilibration buffer (0.02 MPa, 0.15 M NaCl, pH 7.0). The supernatant was then passed through the column at a rate of 2 ml / min. After loading, the column was washed with equilibration buffer for 5 column volumes. Elution buffer (0.02 MPa, 0.15 M NaCl, pH 3.0) was added, and the eluent was collected in a collection tube containing neutralization buffer (1 M Tris, pH 9.0). The collected protein eluent was concentrated using a 4000G ultrafiltration tube (Millipore UFC903096) and the buffer was replaced with PBS (HyClone SH30256.01). After SDS-PAGE analysis, the eluent was stored at -20°C. Endotoxin removal, filtration and sterilization, and SDS-PAGE analysis to determine the purity of its reduced and non-reduced states.

[0056] SDS-PAGE images of the purified antibodies 9203-3, 9203-2, and 9203-1 are shown below. Figure 2 As can be seen, highly pure bispecific antibodies were obtained. From left to right, the images show 9203-3 non-reducing and reducing gels, 9203-2 non-reducing and reducing gels, 9203-1 non-reducing and reducing gels, and a marker. The non-reducing gel shows one band, while the reducing gel shows three clear bands. The molecular weights at the positions on the reducing gels represent the bispecific antibody, single-chain antibody, heavy chain antibody, and light chain antibody, respectively.

[0057] Example 4: Affinity Identification for the Preparation of Bispecific Antibodies

[0058] BPS-C-11-HIS and BPS-C-12-HIS were coated at 1 μg / ml using PBS (HyClone Lot: AC13298279) at 100 μl / well. Coating was carried out at 4°C for 16-18 h, followed by washing three times with TBST. Blocking was then performed with PBS + 2% BSA at 200 μl / well at room temperature (25°C) for 16-18 h, followed by washing once with TBST. The plates were then dried at 37°C for 2 h. The bispecific antibody and control antibody of this invention were prepared at 330 μl / 100 μg / ml. A 4-fold serial dilution was performed with 10 μg / ml as the first gradient. For example, the second gradient was achieved by adding 80 μl of the first gradient to 240 μl of PBS, and so on, for a total of 11 gradient concentrations. The plates were incubated at 37°C for 1 h. After washing the plate three times with PBST using an automated plate washer, add 100 μl of goat anti-human HRP secondary antibody (abcam Lot#: ab98624) diluted 1:20000 to each well and incubate at 37°C for 60 minutes. After incubation, wash the plate three times with the automated plate washer. After the final wash, remove the plate from the microplate and place it on absorbent paper to remove any remaining liquid. Add 100 μl of TMB chromogenic buffer to each well. Incubate in the dark for 3-5 minutes, then add 50 μl of 1% H2SO4 to each well to stop the reaction. Set the MD (I3X) microplate reader to read absorbance values ​​at 450 nm and 630 nm, and save the data after automatic reading.

[0059] Experiments revealed that the antibodies of this invention exhibit high affinity for both BPS-C-11 and BPS-C-12 antigens, while trastuzumab or pertuzumab failed to show high affinity for both antigens simultaneously. Figure 3A and Figure 3B .

[0060] Example 5: Bispecific antibody cell binding

[0061] The binding activity of the bispecific antibody and control antibody of this invention with human gastric cancer cell line N87 and human ovarian cancer cell line SKOV3 was detected by flow cytometry. The cells were digested (suspension cells did not require digestion), centrifuged at 1000 rpm for 5 min at room temperature, the supernatant was discarded, and the cells were washed with PBS and resuspended in flow cytometry tubes. The cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 6Cells / ml. Each tube contains 250 μL of PBS. Eight antibody concentrations were set, 100 μg / ml, serially diluted 3-fold. After adjusting the antibody concentration, 250 μL of the corresponding protein was added to each tube. After incubation at room temperature for 1 h, the cells were washed three times with PBS (1500 rpm, 5 mins). 100 μL of secondary antibody Goat Anti-human IgG / Alexa 647 (Bioss Lot: AE041526) was added to each sample tube. After incubation at room temperature in the dark for 30 mins, 1 mL of PBS was added and the cells were washed three times (1500 rpm, 5 mins). Cells were resuspended in 500 μL of PBS in each tube and analyzed using GraphPad. Examples demonstrate that the antibody of this invention exhibits high binding activity in both N87 and SKOV3 cell lines. Figure 4A and 4B As shown.

[0062] Example 6: Antibody-mediated cytotoxicity of bispecific antibodies

[0063] The antibody-mediated cytotoxicity of human gastric cancer cell line N87 and human ovarian cancer cell line SKOV3 was detected using Jurkat-FcγRIIIa-NFAT-Luciferase cells as effector cells, and the safety of the antibody-mediated killing of tumor cells and normal cells was evaluated. The procedure was as follows: Target cells were first observed under a microscope. Cells were collected when they were round, clear, and in normal condition. Cells were counted and centrifuged at 1500 rpm for 5 min. The supernatant was discarded, and the target cells were resuspended in 1640 + 0.5% FBS. After mixing, the cells were added to a white, flat-bottomed sterile plate at a depth of 1×10⁻⁶ cells / mL. 4 Cells / well, 25 μl per well. Eight gradients of the sample were prepared, 10-fold diluted, 25 μl per well, and incubated at 37°C in a 5% CO2 incubator for 30 min. After incubation, Jurkat-FcγRIIIa-NFAT-Luciferase cells were added at 6 × 10⁶ cells / well. 4 Cells / well, 50 μl per well. For control wells with a volume less than 100 μl, add 1640 + 0.5% FBS medium to a final volume of 100 μl, mix well, and incubate at 37°C in a 5% CO2 incubator for 6 h. Add 40 μl of Bio-glo to each well, incubate in the dark for 5 min, and read the chemiluminescence using a microplate reader. Examples show that the bispecific antibody of this invention exhibits significantly superior cytotoxicity in human gastric cancer cell line N87 and human ovarian cancer cell line SKOV3 compared to antibody-mediated cytotoxicity of trastuzumab, pertuzumab, a mixture of trastuzumab and pertuzumab, and ZW25 molecules. Figure 5A and 5B As shown.

[0064] Example 7: In vitro killing effect of bispecific antibodies

[0065] The in vitro inhibitory activity of the bispecific antibody of this invention was evaluated using the human gastric cancer cell line N87. The procedure was as follows: Target cells were collected and counted, centrifuged at 1500 rpm for 5 min, and collected at a concentration of 3000 cells / well / 100 μl. The cells were then seeded into a white, flat-bottomed 96-well sterile plate, using only the inner 60 wells. Cell-free wells were replaced with culture medium as background controls, and the outer wells were placed in an equal volume of culture medium to prevent edge effects. The plates were incubated overnight at 37°C with 5% CO2. 100 μl of the antibody at a final concentration (2-fold) was added to each well. The antibody was serially diluted with culture medium, typically by 3-fold (10 dilutions). An equal volume of culture medium was used as a blank control, and the outer wells were placed in an equal volume of culture medium to prevent edge effects. The plates were incubated at 37°C with 5% CO2 for 72 h. After the plates returned to room temperature, 40 μl of CTG was added to each well, and the plates were gently shaken at 60 rpm for 10 min to mix. The chemiluminescence was then read using a microplate reader. Examples have shown that the antibody of the present invention exhibits superior cell-inhibiting effects, with a measurable IC50 that is 1000 times more potent. Figure 6 As shown.

[0066] Example 8: Biacore determination of the affinity of the two end elements of a bispecific antibody

[0067] The affinity of the two end elements of bispecific antibodies was determined using a Biacore T200. A Protein A chip (GE, Cat# 29127556) was used. The Protein A chip can capture many mammalian antibodies, including human IgG1, IgG2, and IgG4. It binds only to the antibody Fc heavy chain, not the antibody Fab fragment, ensuring the directionality of antibody binding. mab1, mab2, trastuzumab, and pertuzumab were diluted to 1 μg / mL with running buffer 1*HBS-EP+ and captured onto the chip surface fc2, fc3, and fc24 at a flow rate of 10 μL / min for 30 s. Subsequently, a series of corresponding antigen concentrations were injected into channels fc1-2, fc1-3, and fc1-4 at a flow rate of 30 μL / min, with a binding period of 300 s followed by dissociation for 300 s. Statistical analysis showed that mab1 had a Kd (negative affinity) in the nM range and an affinity 4.26 times that of trastuzumab. mab2 also had a Kd in the nM range and an affinity 3.41 times that of pertuzumab. (See...) Figure 7A , Figure 7B , Figure 7C and Figure 7D The high affinity is likely a key reason for the excellent inhibitory and cell-mediated cytotoxic effects of the bispecific antibodies in this invention.

[0068] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. An erb-B2 bispecific antibody comprising (a) a single chain antibody fragment capable of binding to the juxtamembrane domain of erb-B2; and (b) a half antibody capable of binding to the distal membrane binding domain of erb-B2; the amino acid sequences of the heavy chain variable region and the light chain variable region of the single chain antibody fragment comprising SEQ ID NO: 1, SEQ ID NO: 2, respectively.

2. The erb-B2 bispecific antibody according to claim 1, characterized in that The single chain antibody fragment is linked together by GGGSGGGGSGGGGSGGGGSG of the variable region heavy chain and the light chain.

3. The erb-B2 bispecific antibody according to claim 1, characterized in that The amino acid sequences of the variable region of the single chain antibody fragment comprise SEQ ID NO: 4 or SEQ ID NO:

5.

4. The erb-B2 bispecific antibody according to claim 1, characterized in that, The amino acid sequences of the heavy chain / light chain of the half antibody comprise SEQ ID NO: 6 / SEQ ID NO: 7 or SEQ ID NO: 8 / SEQ ID NO:

7.

5. The erb-B2 bispecific antibody of claim 1, wherein the antibody is a humanized antibody. The amino acid sequences of the single chain antibody / half antibody heavy chain / half antibody light chain comprise SEQ ID NO: 9 / SEQ ID NO: 6 / SEQ ID NO: 7, SEQ ID NO: 10 / SEQ ID NO: 8 / SEQ ID NO: 7 or SEQ ID NO: 11 / SEQ ID NO: 8 / SEQ ID NO:

7.

6. The erb-B2 bispecific antibody according to claim 1, characterized in that The nucleic acid sequences corresponding to the single chain antibody / half antibody heavy chain / half antibody light chain comprise SEQ ID NO: 12 / SEQ ID NO: 13 / SEQ ID NO: 14, SEQ ID NO: 15 / SEQ ID NO: 16 / SEQ ID NO: 14 or SEQ ID NO: 17 / SEQ ID NO: 16 / SEQ ID NO:

14.

7. A pharmaceutical composition comprising the erb-B2 bispecific antibody of any one of claims 1-6 and a pharmaceutically acceptable carrier and composition.

8. Use of the erb-B2 bispecific antibody of any one of claims 1-6 for the manufacture of a medicament for treating a condition associated with erb-B2 in an individual, the condition associated with erb-B2 being at least one of breast cancer, gastric cancer, lung cancer, ovarian cancer, bladder cancer, colorectal cancer.

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