AI-based de novo design, screening, and synthesis of HER2 protein-specific binding antibodies and their applications

Abstract

This invention discloses a HER2 protein-specific binding antibody synthesized through de novo design and screening based on AI and its applications. This invention utilizes AI prediction and phage screening technology to obtain the monoclonal antibody DY23HER202, which specifically binds to HER2. The amino acid sequence of its heavy chain variable region CDRH3 is SEQ ID NO.1. ELISA, flow cytometry, immunofluorescence, and immunohistochemical experiments validated the antibody's binding to HER2, demonstrating that DY23HER202 exhibits higher sensitivity and specificity compared to other screened monoclonal antibodies. BLI assays showed that DY23HER202 has a high affinity for HER2. The DY23HER202 monoclonal antibody provided by this invention can be used to prepare reagents or kits for detecting HER2 or HER2 protein-positive tumors or to develop drugs for treating HER2 protein-positive tumors.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to HER2 protein-specific binding antibodies designed, screened, and synthesized from scratch using AI and their applications. Background Technology

[0002] Human epidermal growth factor receptor 2 (HER2) is almost not expressed in normal cells but is highly expressed in various tumor cells, such as breast cancer, gastric cancer, prostate cancer, and ovarian cancer. HER2 has been considered a potential therapeutic target for these tumors. Trastuzumab was the first monoclonal antibody targeting highly aggressive HER2-positive breast cancer, paving the way for antibody therapy for HER2-positive tumors. However, despite significant success in treating HER2-positive tumors, several serious challenges remain, including the generation of anti-antibody antibodies, limited recognition of antibody HER2 epitopes, and heterogeneous expression of HER2 in tumors. This indicates the need to continue developing more novel antibodies targeting HER2. Furthermore, this novel antibody can be used for effective identification of HER2-positive tumors and biochemical studies of HER2, contributing to the diagnosis and research of HER2-positive tumors. Therefore, developing novel HER2-targeting antibodies has become an important task.

[0003] Classic antibody discovery is primarily based on animal immune responses, involving multiple processes such as animal immunization, molecular cloning, immunological detection, and antibody function evaluation. These methods have been widely used for antibody production, but they also have some drawbacks, such as being time-consuming, batch-to-batch instability, requiring purification of animal-produced antibodies, poor reproducibility, and the need for humanization before clinical use. Summary of the Invention

[0004] The technical problems to be solved by the present invention are how to specifically identify the HER2 protein and / or how to prepare a kit for detecting HER2 and / or how to prepare a kit for detecting HER2 with high sensitivity and high specificity and / or how to obtain novel antibodies against the HER2 protein.

[0005] To address the aforementioned technical problems, the present invention first provides an antibody that specifically binds to the HER2 protein. The antibody may be a monoclonal antibody DY23HER202 or its antigen-binding portion. The monoclonal antibody DY23HER202 or its antigen-binding portion contains a heavy chain variable region and a light chain variable region. The amino acid sequence of the third complementarity-determining region CDRH3 in the heavy chain variable region may be SEQ ID NO.1 in the sequence listing.

[0006] In the aforementioned antibodies, the amino acid sequence of the heavy chain variable region of the monoclonal antibody DY23HER22 or its antigen-binding moiety may be SEQ ID NO.9 in the sequence listing or have at least 80% identity with SEQ ID NO.9. Inconsistencies in the amino acid sequence may occur in the frame region (FR).

[0007] In the aforementioned antibodies, the amino acid sequence of the light chain variable region of the monoclonal antibody DY23HER22 or its antigen-binding portion may be SEQ ID NO.7 in the sequence listing or have at least 80% identity with SEQ ID NO.7. Inconsistencies in the amino acid sequence may occur in the frame region (FR). The aforementioned at least 80% identity may be at least 80%, 85%, or 95%. In this document, identity refers to the identity of the amino acid sequence or nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can be obtained. In this document, the at least 80% identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0008] Variants of the antibodies described in this invention, exhibiting improved affinity and / or titer, can be obtained using methods known in the art and are included within the scope of this invention. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can also be used to improve translation efficiency in expression systems used to generate antibodies. Furthermore, polynucleotides comprising sequences whose antibody specificity or neutralizing activity is optimized by applying directed evolution to any nucleic acid sequence of this invention are also within the scope of this invention.

[0009] Of the antibodies mentioned above, the monoclonal antibody may be any of the following:

[0010] a) Single-chain antibodies;

[0011] b) A fusion antibody containing the single-chain antibody described in a);

[0012] c) Fab fragment;

[0013] d) Fv fragment.

[0014] The term "Fab fragment" refers to a heterodimer composed of a heavy chain Fd and a complete light chain linked by disulfide bonds, containing only one antigen-binding site. By linking the coding genes for the heavy chain Fd and the complete light chain and fusing them with a bacterial protein signal peptide gene, Fab antibodies (Fab fragments) can be secreted and expressed in *E. coli*, exhibiting complete stereofolding and intra- and inter-chain disulfide bonds. The heavy chain Fd refers to approximately half of the H chain portion of Fab (containing approximately 225 amino acid residues, including VH, CH1, and part of the hinge region). The term "Fv fragment" refers to vectors containing VH and VL genes, which can be constructed separately, co-transfected into cells for separate expression, and then assembled into functional Fv antibodies; alternatively, a stop codon can be placed between VH and VL in the vector to express two small protein fragments, which are then non-covalently linked to form Fv antibodies (Fv fragments). The term "Fab′ fragment" comprises a light chain and a heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of two Fab′ fragments to form the F(ab′)2 molecule. The term "F(ab′)2 fragment" comprises two light chains and two heavy chains containing the constant region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains. Interchain disulfide bonds are formed. Because... Therefore, the F(ab′)2 segment consists of two segments held together by disulfide bonds between the two heavy chains. Fab' fragment composition. The term "single-chain antibody (ScFv)" refers to an antibody that expresses a single polypeptide chain by linking the light and heavy chain variable region genes with appropriate oligonucleotide linkers. The polypeptide chain can spontaneously fold into its native conformation, maintaining the specificity and affinity of the Fv. The term "antigen-binding fragment" refers to the antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antigen-binding fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antigen-binding fragment retains at least 10% of the parent antibody binding activity. Specifically, the antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target.

[0015] The term "nanobody (single-domain antibody)" refers to an antibody containing only the VH fragment obtained by expressing the V region of the antibody heavy chain through genetic engineering methods. The binding ability and stability of single-domain antibodies to antigens are essentially the same as those of complete antibodies. The term "bispecific antibody" refers to a bispecific antibody obtained by introducing two sets of light and heavy chain genes into myeloma cells and selecting appropriate antibody constant regions and Ig types, resulting in high yield, uniformity, and purity. Alternatively, bispecific antibodies can also be obtained using chemical cross-linking techniques or hybridization-hybridoma techniques. The term "minimum recognition unit (MRU)" refers to a single CDR structure containing only the variable region, with a molecular weight of only about 1% of that of a complete antibody, capable of binding to the corresponding antigen. The antibodies of this invention can be prepared using various methods known in the art, such as through genetic engineering recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of this invention can be obtained through chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector, then transfected into host cells, cultured under specific conditions, and the antibodies of this invention are expressed. As is well known to those skilled in the art, the antigen-binding fragment can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody to produce an antigen-binding fragment of the antibody.

[0016] To address the aforementioned technical problems, the present invention also provides a nucleic acid molecule encoding the monoclonal antibody DY23HER202 described above or its antigen-binding portion; the nucleic acid molecule may be any of the following DNA molecules:

[0017] c1) The DNA molecule encoding the monoclonal antibody, wherein the coding sequence of the third complementarity-determining region CDRH3 in the heavy chain variable region of the monoclonal antibody is the DNA molecule shown in SEQ ID NO.4 in the sequence listing; and the coding sequence of the light chain variable region of the monoclonal antibody is SEQ ID NO.15 in the sequence listing;

[0018] c2) The DNA molecule encoding the monoclonal antibody, wherein the coding sequence of the heavy chain variable region of the monoclonal antibody is the DNA molecule shown in SEQ ID NO.12 of the sequence listing; and the coding sequence of the light chain variable region of the monoclonal antibody is SEQ ID NO.15 of the sequence listing;

[0019] c3) is a DNA molecule that has more than 90% identity with the DNA molecule defined by c1) or c2) and encodes the monoclonal antibody or its antigen-binding portion thereof.

[0020] In this document, the phrase "at least 90% identity" can mean at least 90%, 91%, 92%, 93%, 94%, 95%, etc. 96%, 97%, 98%, or 99% similarity.

[0021] To address the aforementioned technical problems, the present invention also provides biological materials, which may be expression cassettes containing the nucleic acid molecules described above, recombinant vectors, recombinant microorganisms, and / or recombinant animal cell lines.

[0022] The recombinant animal cell line can be eukaryotic or prokaryotic. The eukaryotic cells can be selected from any one of Expi293F, CHO, and HEK293. The prokaryotic cells can be Escherichia coli.

[0023] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), and viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, or herpesviruses (such as herpes simplex virus)). In one embodiment of the present invention, the vector may specifically be a pcDNA3.4 vector containing the constant regions of the human IgG1 heavy and light chains.

[0024] The microorganisms described herein may be yeast, bacteria, or fungi. Among them, bacteria may be derived from genera such as *Escherichia*, *Erwinia*, *Agrobacterium*, *Flavobacterium*, *Alcaligenes*, *Pseudomonas*, and *Bacillus*; yeast may be *Pichia pastoris*. The cell line (host cell) refers to cells that can be used to introduce the vector, including but not limited to: eukaryotic cells (such as yeast cells, *Aspergillus*), animal cells (such as mammalian cells, insect cells), or prokaryotic cells. In one embodiment of the present invention, the cell line may specifically be the Expi293F cell line.

[0025] The terms “cell” and “cell line” are used interchangeably, and all such names include their descendants.

[0026] To address the aforementioned technical problems, this invention also provides the application of the antibodies and / or biomaterials described above in the development or preparation of products for detecting HER2 protein.

[0027] The application of the antibodies and / or the biomaterials described above in the preparation of products for detecting or diagnosing HER2 protein-positive tumors also falls within the scope of protection of this invention.

[0028] The use of the antibodies and / or the biomaterials described above in the development or preparation of products that distinguish between HER2 protein-positive cells and HER2 protein-negative cells or products that locate HER2 protein-positive cells is also within the scope of protection of this invention.

[0029] The use of the antibodies and / or the biomaterials described above in the preparation or development of drugs for treating HER2 protein-positive tumors is also within the scope of protection of this invention.

[0030] The antibody described above can target the extracellular domain of the HER2 protein, and the amino acid sequence information of the extracellular domain of the HER2 protein can be found in SEQ ID NO.8 of the sequence listing.

[0031] The products mentioned above may be reagent kits and / or test kits.

[0032] This invention initially established a novel antibody generation platform based on artificial intelligence (AI) algorithms and biochemical screening technology, enabling a rapid, efficient, and animal-immune-free antibody discovery process. Using this platform, this invention screened suitable monoclonal antibodies targeting HER2.

[0033] The purpose of this invention is to propose several AI-predicted, designed, and in vitro detection methods for the HER2 protein. Validated monoclonal antibodies that can function in multiple in vitro assays.

[0034] To achieve the above objectives, the present invention utilizes a previously established platform to screen three monoclonal antibodies targeting the human HER2 protein. The amino acid sequences (CDRH3 region) of the monoclonal antibodies are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0035] The present invention also provides a nucleic acid sequence encoding the above-mentioned monoclonal antibody.

[0036] The present invention also provides an expression vector containing the above-mentioned nucleic acid sequence.

[0037] Preferably, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector. The eukaryotic expression vector is selected from either pcDNA3.1 or pcDNA3.4 expression vectors. The prokaryotic expression vector is selected from the pET28a vector.

[0038] The present invention also provides host cells for the above-mentioned nucleic acid or expression vector.

[0039] Preferably, the host cell is a eukaryotic cell or a prokaryotic cell, wherein the eukaryotic cell is selected from any one of CHO and HEK293, and the prokaryotic cell is Escherichia coli.

[0040] The present invention also provides a kit for detecting human HER2 protein, wherein the kit contains any one of the above-mentioned monoclonal antibodies, which are within the scope of protection of the present invention.

[0041] The applications or methods described above are not for disease diagnosis. They are not intended to directly obtain disease diagnoses or health status results from living humans or animals.

[0042] The above applications or methods are for non-disease treatment purposes. They are not intended to restore or restore health or reduce suffering in living human or animal bodies. Attached Figure Description

[0043] Figure 1 The image shows the results of SDS-PAGE gel electrophoresis followed by Coomassie brilliant blue staining to detect antibody purity for screening monoclonal antibodies.

[0044] Figure 2 This graph shows the results of ELISA screening to detect the specificity of different antibodies binding to the HER2 protein. The horizontal axis represents the names of different antibodies, and the vertical axis represents the absorbance value at 450 nm. The higher the value, the stronger the binding ability of the antibody to the antigen.

[0045] Figure 3 This graph shows the results of ELISA testing to detect the ability of different antibodies to bind to the HER2 protein. The horizontal axis represents the logarithm of different antibody incubation concentrations, and the vertical axis represents the absorbance value at 450 nm. A higher value indicates a stronger binding ability between the antibody and the antigen.

[0046] Figure 4 This graph shows the results of screening monoclonal antibody DY23HER202 for binding to HER2-His protein using a biomembrane interference assay (BLI). The horizontal axis represents the time of the interference experiment, and the vertical axis represents the interaction reaction during the interference experiment.

[0047] Figure 5 This figure shows the results of using flow cytometry to verify the ability of the screening antibody DY23HER202 to detect HER2-positive cell lines. The horizontal axis represents the logarithm of the AF488 fluorescence intensity of the detected cells, and the vertical axis represents the forward scattered light value of the detected cells.

[0048] Figure 6 To verify the ability of the screening antibody DY23HER202 to detect HER2-positive cell lines using immunofluorescence assays. The result image.

[0049] Figure 7 The figure shows the results of immunohistochemical experiments to verify the ability of the screening antibody DY23HER202 to detect HER2-positive cell lines. Detailed Implementation

[0050] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0052] Example 1: Screening, preparation, and functional testing of HER2 protein monoclonal antibodies

[0053] 1. CDRH3 diversity sequence design, monoclonal antibody screening and sequence analysis.

[0054] 1.1 Construction of phage display antibody library

[0055] The antibodies involved in this invention all target the extracellular domain of the HER2 protein, and their amino acid sequence information is as follows (SEQ ID NO.8 in the sequence listing):

[0056] .

[0057] Generative AI technology was used to analyze the extracellular domain of the HER2 protein, predicting and designing the sequence of the third complementarity-determining region (CDRH3) of the heavy chain variable region for potential specific antibodies targeting the HER2 extracellular domain. Corresponding diverse sequences were synthesized, and a phage display antibody library was constructed using the sequence of the marketed HER2 monoclonal antibody trastuzumab (Herceptin) as a backbone. The amino acid sequence of the heavy chain variable region of Herceptin is as follows; the underlined sequence is the CDRH3 region sequence of the heavy chain variable region of Herceptin:

[0058] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYP TNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMD YWG QGTLVTVSS;

[0059] The amino acid sequence of the light chain variable region of herceptin monoclonal antibody is as follows (SEQ ID NO.7):

[0060] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFL YSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK.

[0061] The nucleotide sequence encoding the light chain variable region of the herceptin monoclonal antibody is as follows (SEQ ID NO.15, 5'-3').

[0062] GACATCCAAATGACTCAGAGTCCCTCCAGCTTGTCTGCTTCAGTGGGAGACAGAGTGACTATCACCTGCCGAGCTTCACAGGACGTGAACACAGCCGTGGCCTGGTACCAGCAGAAACCCGGAAAGGCTCCAAAACTCCTGATCTACAGCGCAAGCTTTCT GTATTCCGGTGTCCCAAGCCGCTTTAGCGGTTCTAGGTCTGGTACGGATTTCACCCTGACGATCTCTTCTCTGCAGCCTGAGGACTTCGCCACTTATTATTGTCAGCAACACTATACTACACCCCCCACCTTCGGCCAGGGCACAAAGGTAGAGATCAAA.

[0063] 1.2 Screening of phage display antibody libraries

[0064] Three rounds of solid-phase screening were performed on phage display antibody libraries using HER2-His protein expressed in Expi293F cells. After monocloning the selected libraries, the specificity of different phages for HER2 protein was verified by ELISA.

[0065] To identify the antibody sequence in positive monoclonal phages, this invention extracts the plasmid from the corresponding bacterial culture and uses pCANTAB-R1 / R2 primers for sequencing to determine the specific sequence of CDRH3. Three unique sequences were ultimately obtained.

[0066] The amino acid sequences of the CDRH3 region of these three unique sequences are as follows:

[0067] The amino acid sequence (SEQ ID NO.1) of the CDRH3 region of the DY23HER202 antibody is: SYGYPPYFDS.

[0068] The amino acid sequence (SEQ ID NO.2) of the CDRH3 region of the DY23HER204 antibody is: ALGRGYNYYGFDS.

[0069] The amino acid sequence (SEQ ID NO.3) of the CDRH3 region of the DY23HER205 antibody is: GRDNERGDIGFDL.

[0070] Correspondingly, the coding nucleic acid sequences for the CDRH3 regions of these three unique sequences are as follows:

[0071] The CDRH3 region nucleic acid sequence (SEQ ID NO.4) of the DY23HER202 antibody is: 5'-TCTTACGGTTACCCGCCGTACTTCGACTCT-3'.

[0072] The CDRH3 region nucleic acid sequence (SEQ ID NO.5) of the DY23HER204 antibody is: 5'-GCGCTGGGTCGTGGTTACAACTACTACGGTTTCGACTCT-3'.

[0073] The CDRH3 region nucleic acid sequence (SEQ ID NO.6) of the DY23HER205 antibody is: 5'-GGTCGTGACAACGAACGTGGTGACATCGGTTTCGACCTG-3'.

[0074] The amino acid sequence (SEQ ID NO.9) of the heavy chain variable region of the DY23HER202 antibody is:

[0075] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYP TNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SYGYPPYFDS QGT LVTVSS;

[0076] The amino acid sequence (SEQ ID NO.10) of the heavy chain variable region of the DY23HER204 antibody is:

[0077] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYP TNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC ALGRGYNYYGFD S QGTLVTVSS;

[0078] The amino acid sequence (SEQ ID NO. 11) of the heavy chain variable region of the DY23HER205 antibody is: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNG YTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC GRDNERGDIGFDL QGT LVTVSS;

[0079] Correspondingly, the coding nucleic acid sequence (SEQ ID NO.12, 5'-3') for the heavy chain variable region of the DY23HER202 antibody is:

[0080] GAAGTACAATTGGTCGAGAGTGGAGGAGGCCTCGTTCAACCTGGAGGCTCTCTGAGGCTTTCATGCGCTGCCAGTGGGTTCAACATCAAGGACACCTACATACATTGGGTTAGGCAGGCACCGGGGAAAGGACTGGAATGGGTA GCTAGGATTTAACCCACCAACGGCTACACAAGGTACGCTGACTCAGTGAAGGGCCGGTTCACCATTTCAGCAGATACGTCAAAGAATACCGCTTATCTGCAGATGAATAGTCTTCGGGCGGAGGATACAGCCGTGTATTACTGT TCTTACGG TTACCCGCCGTACTTCGACTCT CAAGGAACCCTGGTGACCGTGTCTTCC;

[0081] The coding nucleic acid sequence (SEQ ID NO.13, 5'-3') for the heavy chain variable region of the DY23HER204 antibody is:

[0082] GAAGTACAATTGGTCGAGAGTGGAGGAGGCCTCGTTCAACCTGGAGGCTCTCTGAGGCTTTCATGCGCTGCCAGTGGGTTCAACATCAAGGACACCTACATACATTGGGTTAGGCAGGCACCGGGGAAAGGACTGGAATGGGTA GCTAGGATTTAACCCACCAACGGCTACACAAGGTACGCTGACTCAGTGAAGGGCCGGTTCACCATTTCAGCAGATACGTCAAAGAATACCGCTTATCTGCAGATGAATAGTCTTCGGGCGGAGGATACAGCCGTGTATTACTGT GCGCTGGG TCGTGGTTACAACTACTACGGTTTCGACTCT CAAGGAACCCTGGTGACCGTGTCTTCC;

[0083] The coding nucleic acid sequence (SEQ ID NO.14, 5'-3') for the heavy chain variable region of the DY23HER205 antibody is:

[0084] GAAGTACAATTGGTCGAGAGTGGAGGAGGCCTCGTTCAACCTGGAGGCTCTCTGAGGCTTTCATGCGCTGCCAGTGGGTTCAACATCAAGGACACCTACATACATT GGGTTAGGCAGGCACCGGGGAAAGGACTGGAATGGGTA GCTAGGATTTACCCCA CCAACGGCTACACAAGGTACGCTGACTCAGTGAAGGGCCGGTTCACCATTTCAGCAGATACGTCAAAGAATACCGCTTATCTGCAGATGAATAGTCTTCGGGCGGAGGATACAGCCGTGTATTACTGT GGTCGTGA CAACGAACGTGGTGACATCGGTTTCGACCTG CAAGGAACCCTGGTGACCGTGTCTTCC.

[0085] 2. Monoclonal antibody preparation.

[0086] Based on the Sanger sequencing results, the coding nucleotide sequences (SEQ ID NO.12-SEQ ID NO.14, SEQ ID NO.15) of the variable regions of the heavy and light chains of the above three monoclonal antibodies were synthesized and ligated into the pcDNA3.4 vector containing the constant regions of the human IgG1 heavy and light chains to construct three fully human IgG antibody expression plasmids. The corresponding three monoclonal antibodies were then expressed using the Expi293F (Invitrogen, USA) cell line. Specifically, the three fully human IgG antibody expression plasmids were transformed into the Expi293F cell line to obtain three recombinant cell lines.

[0087] Three recombinant cell lines were cultured separately to obtain three cell culture supernatants. These three cell culture supernatants were then processed using HiTrap. TM The monoclonal antibodies were purified using an rProtein A FF affinity chromatography column (GE Healthcare, USA) to obtain three different monoclonal antibodies against the HER2 protein with a Herceptin backbone: DY23HER202, DY23HER204, and DY23HER205.

[0088] The amino acid sequences of the light chain variable region of monoclonal antibodies DY23HER202, DY23HER204 and DY23HER205 are all SEQ ID NO.7.

[0089] SDS-PAGE gels were prepared, and the purity of the three purified monoclonal antibodies was detected by electrophoresis. Figure 1 The results of SDS-PAGE electrophoresis show that the purity of the three purified monoclonal antibodies is greater than 95%.

[0090] 3. Enzyme-linked immunosorbent assay (ELISA).

[0091] To determine the specificity of the three monoclonal antibodies obtained in step 2 in binding to HER2 protein, 200 ng / well of HER2-His protein, control CD123-His (Sinochem, China), and control bovine serum albumin (BSA) were coated in micro-ELISA plates (Nunc, USA) and incubated overnight at 4°C. The plates were then blocked with 5% skim milk powder dissolved in PBS-Tween 20 (PBST). After washing with PBST, 100 μL / well of 1 μg / mL monoclonal antibody was added, and the plates were incubated at 37°C for 2 hours. After washing, horseradish peroxidase (HRP)-conjugated goat anti-human IgG-Fc (1:10,000 dilution; Abcam, USA) was added and the plates were incubated at 37°C for 1 hour. After color development, OD was monitored using a microplate reader. 450nm The absorbance value at that location.

[0092] The results of ELISA assays to detect the specificity of the binding of the three monoclonal antibodies to the HER2 protein are as follows: Figure 2 As shown, the ELISA results indicate that the DY23HER202 and DY23HER204 monoclonal antibodies exhibit OD values ​​in their reaction with HER2-His protein. 450nm The two monoclonal antibodies showed stronger absorption peaks than the control group (CD123-His and BSA), indicating that they can specifically bind to the HER2-His protein and do not bind to the control CD123-His and BSA. This fully demonstrates that these two monoclonal antibodies can specifically recognize the HER2 protein and have no cross-reactivity with the His tag. However, the binding reaction of the DY23HER205 monoclonal antibody to the CD123-His protein was stronger than that to the HER2-His protein, indicating that the binding of this monoclonal antibody to the HER2-His protein is not specific to HER2 but to the His tag. Furthermore, based on... Figure 2 The results also show that DY23HER202 binds to the HER2 protein better than other antibodies, OD 450nm The reading was significantly higher than that of other antibodies. This result is consistent with the subsequent EC50 analysis.

[0093] To compare the binding affinity of different monoclonal antibodies to the HER2 protein, an EC50 assay was performed. 100 ng / well of HER2-His protein was coated onto micro-ELISA plates (Nunc, USA) and incubated overnight at 4°C. The plates were then blocked with 5% skim milk dissolved in PBS-Tween 20 (PBST). After washing with PBST, add 100 μL / well of serially diluted monoclonal antibody starting at 1 μg / mL, in 12 dilution gradients (1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, 0.03125 μg / mL, 0.015625 μg / mL, 0.0078125 μg / mL, 0.00390625 μg / mL, 0.001953125 μg / mL, 0.000976563 μg / mL, and 0.000488281 μg / mL). Incubate at 37°C for 2 hours. After washing, add horseradish peroxidase (HRP)-conjugated goat anti-human IgG-Fc (1:10,000 dilution; Abcam, USA) and incubate at 37°C for 1 hour. After color development, the absorbance at 450 nm was monitored using an ELISA reader.

[0094] like Figure 3 As shown, the EC50 results indicate that DY23HER202 has a stronger binding affinity to HER2 than other antibodies, with an EC50 value of 12.90 ng / mL, which is significantly higher than that of DY23HER204 and DY23HER205.

[0095] 4. Biomembrane Interference Measurement (BLI)

[0096] To determine the binding affinity of antibodies DY23HER202 and DY23HER208 to the HER2 protein, a BLI assay was performed on an Octet RED96 machine (PallFortéBio, USA) according to the manufacturer's instructions. In short, the HER2-His protein was immobilized on a Ni-coated biosensor (PallFortéBio) until saturation. The antigen-bound biosensor was placed in wells containing a series of diluted monoclonal antibody samples to allow antigen-antibody binding, and then immersed in dissociation buffer (0.01M PBS supplemented with 0.1% bovine serum albumin and 0.02% Tween 20) for dissociation. The equilibrium dissociation constant (KD) was calculated using Octet data analysis software (PallFortéBio).

[0097] Experimental results are as follows Figure 4 As shown, the equilibrium dissociation constant K of antibody DY23HER202 binding to HER2 protein is... D The value reached 1.182 nM, indicating that DY23HER202 binds to HER2 protein with high affinity and has high detection sensitivity.

[0098] 5. Flow cytometry

[0099] Human breast cancer cells (SK-BR-3) and human embryonic kidney cells (HEK293) were cultured to an appropriate density in T25 culture flasks. The cells were washed once with PBS, digested with 0.25% trypsin until the cells were rounded, and the process was terminated with DMEM medium. After washing twice with PBS, equal amounts of the two cell lines were mixed to form the experimental group samples. Untreated mixed cells were used as a blank control, and HEK293 cells alone were used as a negative control. 2 x 10-1 cells were collected from each tube. 5 Each cell was aliquoted into a 1.5 ml EP tube. Centrifuged at 300 g for 5 min. During centrifugation, the antibody to be tested was diluted to a final concentration of 1 μg / ml with flow cytometry buffer. According to the experimental design, the cells in the corresponding EP tube were resuspended in 100 μl of diluted antibody or 100 μl of flow cytometry buffer. Incubated at room temperature in the dark for 30 min. Washed three times with flow cytometry buffer. Then, according to the experimental design, the cells in the corresponding EP tube were resuspended in 100 μl of AF488 fluorescently labeled anti-human IgG Fc secondary antibody (#410706, BioLegend) or 100 μl of flow cytometry buffer. Incubated at room temperature in the dark for 30 min. Washed three times with flow cytometry buffer. Repeat three times. Add 500 μL of flow cytometry buffer to each group, mix thoroughly, and use a flow cytometer. (BDCanto2) test Measure the AF488 index in cells.

[0100] The results are as follows Figure 5As shown, the monoclonal antibody DY23HER202 can specifically recognize the HER2 protein on the surface of SK-BR-3 cells. Figure 5 (middle right image), but does not recognize HEK293 cells ( Figure 5 (The negative control is represented in the middle of the graph). Therefore, DY23HER202 can clearly distinguish between HER2-positive SK-BR-3 cell populations and HER2-negative HEK293 cell populations, with no overlap in their signal peaks, resulting in high detection accuracy.

[0101] 6. Immunofluorescence assay (IFA)

[0102] SK-BR-3 cells were cultured to a suitable density on treated coverslips, and the tissue was evenly covered with 3% BSA and blocked at room temperature for 30 min. The antibody to be tested was diluted with PBS to a final concentration of 20 μg / ml and added to the cell sample for incubation at 4°C overnight. The cells were washed three times with PBS, 5 min each time. The cells were covered with diluted Goat anti-Human IgG Fc-HRP (#ab97225, Abcam) and incubated at room temperature for 50 min. The cells were washed three times with PBS, 5 min each time. The cells were covered with TSA reagent (Aifang) and incubated, then washed three times with PBS, 5 min each time. DAPI staining solution was added to the cell sample and incubated at room temperature in the dark for 10 min. The cells were washed three times with PBS, 5 min each time. After slightly drying the sections, they were mounted with anti-fluorescence quenching mounting medium. The samples were observed and images were acquired under a Nikon inverted fluorescence microscope. The cell nuclei stained with DAPI were blue under UV excitation, and the emission wavelength of CY3 red light was 510-560 nm.

[0103] Test results as follows Figure 6 As shown, the monoclonal antibody DY23HER202 can clearly stain HER2-positive SK-BR-2 cells. Figure 6 (Middle right image) The red signal on the membrane represents the localization of the HER2 protein on the cell membrane. Cells not incubated with the selection antibody show no red fluorescent signal. Figure 6 (Left image). Blue fluorescence represents the cell nucleus.

[0104] 7. Immunohistochemical assay (IHC)

[0105] Culture SK-BR-3 cells to a suitable density on treated coverslips. Incubate the coverslips in 3% hydrogen peroxide solution at room temperature in the dark for 25 min. Wash the coverslips three times with PBS (pH 7.4) on a decolorizing shaker for 5 min each time. Cover the cells evenly with 3% BSA and block at room temperature for 30 min. Dilute the antibody to be tested with PBS to a final concentration of 1 μg / ml and add to the cell sample. Incubate overnight at 4°C. Wash three times with PBS for 5 min each time. Cover the cells with diluted Goat anti-Human IgG Fc-HRP and incubate at room temperature for 50 min. Wash three times with PBS for 5 min each time. Add DAB chromogenic solution and incubate until a brownish-yellow color is visible under a microscope. Stop the reaction with water. Counterstain the sample with Harris hematoxylin for 3 min and wash with water to stop the reaction. Reverse the blue color with ammonia and rinse with running water. The sections were sequentially immersed in 75% ethanol for 6 min, 85% ethanol for 6 min, anhydrous ethanol I for 6 min, anhydrous ethanol II for 6 min, and xylene I for 5 min to dehydrate and clear. The sections were then removed from the xylene and allowed to air dry slightly before mounting with neutral resin. Microscopic examination and image acquisition and analysis were then performed.

[0106] The results are as follows Figure 7 As shown, the DY23HER202 antibody can clearly stain HER2-positive SK-BR-3 cells. Figure 7 (Middle right image) The brown signal on the membrane represents the localization of the HER2 protein on the cell membrane. Cells not incubated with the selection antibody show no brown signal. Figure 7 (Left image). Blue represents the cell nucleus.

[0107] As the results above indicate, the monoclonal antibody DY23HER202 can function effectively in various in vitro detection experiments, exhibiting high specificity and accuracy, and can clearly distinguish HER2 positivity in all in vitro detection experiments. and negative fine This allows for the convenient and accurate counting of HER2-positive cells, or the localization of HER2-positive cells. This is very important for the auxiliary diagnosis of HER2-positive tumor cells in clinical practice.

[0108] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. An AI-designed antibody that specifically binds to the HER2 protein, characterized in that: The antibody is a monoclonal antibody DY23HER202 or its antigen-binding fragment; the monoclonal antibody DY23HER202 or its antigen-binding fragment contains a heavy chain variable region and a light chain variable region, the amino acid sequence of the third complementarity-determining region CDRH3 in the heavy chain variable region is SEQ ID NO.1 in the sequence listing; the amino acid sequence of the heavy chain variable region is SEQ ID NO.9 in the sequence listing; the amino acid sequence of the light chain variable region is SEQ ID NO.7 in the sequence listing.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that: The monoclonal antibody or its antigen-binding fragment is any one of the following: a) Single-chain antibodies; b) Fab fragment; c) Fv segment.

3. A nucleic acid molecule encoding the monoclonal antibody DY23HER202 or its antigen-binding fragment as described in claim 1 or 2; The nucleic acid molecule is a DNA molecule as described below: c1) A DNA molecule encoding a monoclonal antibody, wherein the coding sequence of the heavy chain variable region of the monoclonal antibody is the DNA molecule shown in SEQ ID NO.12 of the sequence listing; and the coding sequence of the light chain variable region of the monoclonal antibody is SEQ ID NO.15 of the sequence listing; c2) is a DNA molecule that has more than 90% identity with the DNA molecule defined in c1) and encodes the monoclonal antibody or its antigen-binding fragment.

4. A biomaterial, characterized in that: The biological material is an expression cassette, recombinant vector, recombinant microorganism, and / or recombinant animal cell line containing the nucleic acid molecule described in claim 3.

5. The use of the monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2 in the development or preparation of products for detecting HER2 protein.

6. The use of the monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2 and / or the biomaterial as described in claim 4 in the preparation of products for detecting or diagnosing HER2 protein-positive tumors.

7. The use of the monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2 and / or the biomaterial as described in claim 4 in the development or preparation of products that distinguish between HER2 protein-positive cells and HER2 protein-negative cells or products that target HER2 protein-positive cells.

Images