Her2 protein-specific binding antibody obtained by ai-based de novo design, screening and synthesis and use thereof

Abstract

Disclosed in the present invention are a HER2 protein-specific binding antibody obtained by AI-based de novo design, screening and synthesis and a use thereof. According to the present invention, an AI prediction and phage screening technology is used to obtain monoclonal antibody DY23HER202 capable of specifically binding to HER2, and the amino acid sequence of CDRH3 in a heavy chain variable region of the monoclonal antibody is SEQ ID NO. 1. Upon verification by means of an ELISA experiment, a flow experiment, an immunofluorescence experiment and an immunohistochemical experiment, the detection results show that compared with other screened monoclonal antibodies, the binding of the monoclonal antibody DY23HER202 to HER2 has high sensitivity and high specificity. Upon BLI detection, the results show that DY23HER202 has high affinity to HER2. The monoclonal antibody DY23HER202 provided in the present invention can be used for preparing a reagent or kit for detecting HER2 or HER2 protein-positive tumors or developing a drug for treating HER2 protein-positive tumors.

Description

AI-based de novo design and screening of synthesized HER2 protein-specific binding antibodies and applications thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and specifically relates to AI-based de novo design and screening of synthesized HER2 protein-specific binding antibodies and applications thereof. BACKGROUND

[0002] Human epidermal growth factor receptor 2 (HER2) is almost not expressed in normal cells, but is highly expressed in a variety of tumor cells, such as breast cancer, gastric cancer, prostate cancer, and ovarian cancer, etc. HER2 has been considered as a potential therapeutic target for these tumors. Trastuzumab is the first monoclonal antibody targeting high-invasive HER2-positive breast cancer, which opens up the road for antibody therapy of HER2-positive tumors. However, despite the great success in treating HER2-positive tumors, there are still some serious challenges, including the generation of drug-resistant antibodies, the limited recognition of antibody HER2 epitopes, and the heterogeneous expression of HER2 tumors, etc. This indicates the need to continue to develop more new antibodies against HER2. In addition, the new antibodies can also be used for effective identification of HER2-positive tumors and biochemical research of HER2, which is helpful for the diagnosis and research of HER2-positive tumors. Therefore, the development of new HER2-targeting antibodies has become one of the important works.

[0003] There are many existing screening techniques for monoclonal antibodies, and currently the three main ones that are widely accepted and used are: hybridoma technology, phage display technology, and single B cell-based PCR technology. However, the classical antibody discovery process is mainly based on animal immune response, involving animal immunization, molecular cloning, immunological detection, antibody function evaluation, and other processes. These methods have been widely used to produce antibodies, but also have some disadvantages, such as time-consuming, batch instability, the need to purify animal-produced antibodies, poor reproducibility, the need for humanization before clinical use, etc. SUMMARY

[0004] The main technical problem to be solved by the present application is how to prepare a kit for detecting HER2 with high sensitivity and high specificity. In order to solve the technical problem, the present application provides a monoclonal antibody DY23HER202 specifically binding to HER2 protein and application thereof in preparing a kit for detecting HER2 protein. The monoclonal antibody contains a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the third complementarity determining region CDRH3 in the heavy chain variable region is shown in SEQ ID NO. 1 in the sequence listing. Experimental results show that the monoclonal antibody DY23HER202 of the present application can play a role in different in vitro detection experiments, has high specificity and accuracy, and can clearly distinguish HER2 positive and negative cells or locate HER2 positive cells in all in vitro detection experiments. Technical problem

[0005] The technical problem to be solved by the present application is how to specifically recognize 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 a novel antibody against HER2 protein. Technical solution

[0006] The technical problem to be solved by the present application is how to specifically recognize 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 a novel antibody against HER2 protein.

[0007] In order to solve the above technical problem, the present application first provides an antibody specifically binding to HER2 protein, which can be a monoclonal antibody DY23HER202 or an antigen binding portion thereof; the monoclonal antibody DY23HER202 or the antigen binding portion thereof contains a heavy chain variable region and a light chain variable region, and the amino acid sequence of the third complementarity determining region CDRH3 in the heavy chain variable region can be SEQ ID NO. 1 in the sequence listing.

[0008] In the above antibody, the amino acid sequence of the third complementarity determining region CDRH3 in the heavy chain variable region can include SEQ ID NO. 1 in the sequence listing (i.e. the 97-106th amino acid residues of SEQ ID NO. 9), the amino acid sequence of the first complementarity determining region CDRH1 can include the 26-33th amino acid residues of SEQ ID NO. 9 in the sequence listing, and the amino acid sequence of the second complementarity determining region CDRH2 can include the 51-58th amino acid residues of SEQ ID NO. 9 in the sequence listing.

[0009] In the above antibody, the amino acid sequence of the heavy chain variable region of the monoclonal antibody DY23HER202 or the antigen-binding portion thereof can be SEQ ID NO. 9 in the sequence listing or have at least 80% identity to SEQ ID NO. 9. Among them, the inconsistent place of the amino acid sequence can be in the framework region (FR).

[0010] In the above antibody, the amino acid sequence of the heavy chain variable region of the monoclonal antibody DY23HER202 or the antigen-binding portion thereof can be SEQ ID NO. 9 in the sequence listing or have at least 80% identity to SEQ ID NO. 9. Among them, the inconsistent place of the amino acid sequence can be in the framework region (FR).

[0011] In the above antibody, the amino acid sequence of the heavy chain variable region of the monoclonal antibody DY23HER202 or the antigen-binding portion thereof can be SEQ ID NO. 9 in the sequence listing or have at least 80% identity to SEQ ID NO. 9. Among them, the inconsistent place of the amino acid sequence can be in the framework region (FR).

[0012] In the above antibody, the amino acid sequence of the heavy chain variable region of the monoclonal antibody DY23HER202 or the antigen-binding portion thereof can be SEQ ID NO. 9 in the sequence listing or have at least 80% identity to SEQ ID NO. 9. Among them, the inconsistent place of the amino acid sequence can be in the framework region (FR).

[0013] Variants of the antibodies of the present application having improved affinity and / or valence can be obtained by employing methods known in the art and are included within the scope of the present application. For example, amino acid substitutions can be used to obtain antibodies having further improved affinity. Alternatively, codon optimization of the nucleotide sequence can also be used to improve the efficiency of translation in expression systems used to produce the antibodies. Furthermore, polynucleotides comprising sequences optimized for antibody specificity or neutralizing activity by applying directed evolution methods to any of the nucleic acid sequences of the present application are also within the scope of the present application.

[0014] Among the above-mentioned antibodies, the monoclonal antibody can be a full-length antibody.

[0015] The "full-length antibody" consists of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain is composed of a heavy chain variable domain (VH) and a heavy chain constant domain, which is composed of subdomains CH1, hinge, CH2 and CH3. Each light chain is composed of a light chain variable domain (VL) and a light chain constant domain (CL). The VH and VL can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FW). Each VH and VL is composed of three CDRs and four FW segments, arranged from amino- to carboxy-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3 and FW4.

[0016] "Complementarity determining region (CDR)" is the antigen binding site in an antibody. CDRs can be defined using various terms: (i) Complementarity determining regions (CDRs) (three in VH (CDRH1, CDRH2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3)) based on sequence variability (Wu and Kabat, J. Exp. Med. 132:211-50, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) "Hypervariable region," "HVR," or "HV" (three in VH (H1, H2, H3) and three in VL (L1, L2, L3)) refer to the regions of an antibody variable domain that are highly variable in sequence and form structurally defined loops, as defined by Chothia and Lesk (Chothia and Lesk, Mol. Biol. 196:901-17, 1987). The International ImMunoGeneTics (IMGT) database (http: / / www_imgt_org) provides standardized numbering and definition of antigen binding sites. The correspondence between CDR, HV, and IMGT descriptions is described in Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003. As used herein, the terms "CDR," "HCDR1," "HCDR2," "HCDR3," "LCDR1," "LCDR2," and "LCDR3" include CDRs defined by any of the methods, Kabat, Chothia, or IMGT described above, unless otherwise explicitly stated in the specification.

[0017] The monoclonal antibody can also be any one of:

[0018] a) a single chain antibody;

[0019] b) a fusion antibody containing the single chain antibody of a);

[0020] c) a Fab fragment;

[0021] d) a Fv fragment.

[0022] The term "Fab fragment" is a heterodimer with one antigen binding site, comprising the heavy chain Fd and the entire light chain connected by disulfide bonds. The coding genes of the heavy chain Fd and the entire light chain are connected and fused with the bacterial protein signal peptide gene, and the Fab antibody (Fab fragment) can be expressed and secreted in E. coli, with complete stereo-folding and intra-chain and inter-chain disulfide bonds. The heavy chain Fd refers to about 1 / 2 of the H chain part in Fab (about 225 amino acid residues, including VH, CH1 and part of the hinge region). The term "Fv fragment" refers to a vector containing VH and VL genes, respectively, co-transfected into cells to express separately, and then assembled into a functional Fv antibody; or a stop codon is set between VH and VL in the vector, and two small protein fragments are expressed separately, and then combined by non-covalent bonds to form Fv antibody (Fv fragment). The term "Fab' fragment" contains one light chain and a part of one heavy chain containing VH domain and CH1 domain and the region between CH1 and CH2 domain, so that inter-chain disulfide bonds can be formed between the two heavy chains of two Fab' fragments to form F(ab')2 molecules. The term "F(ab')2 fragment" contains two light chains and two heavy chains containing a part of the constant region between CH1 and CH2 domains, so that inter-chain disulfide bonds are formed between the two heavy chains. Therefore, the F(ab')2 fragment is composed of two Fab' fragments held together by disulfide bonds between the two heavy chains. The term "single-chain antibody (ScFv)" refers to the connection of light chain and heavy chain variable region genes with appropriate oligonucleotide linkers, so that a single polypeptide chain called single-chain antibody (ScFv) is expressed. The polypeptide chain can spontaneously fold into a natural conformation, maintaining the specificity and affinity of Fv. The term "antigen binding fragment" refers to an antigen binding fragment of an antibody and an antibody analog, which generally includes at least part of the antigen binding region or variable region (e.g., one or more CDRs) of the parental antibody. The antigen binding fragment retains at least some of the binding specificity of the parental antibody. Generally, the antigen binding fragment retains at least 10% of the binding activity of the parent. Specifically, the antigen binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody to the target.

[0023] The term "nanobody (single domain antibody)" refers to an antibody whose heavy chain V region is expressed by a genetic engineering method to obtain an antibody containing only a VH fragment. The ability of a single domain antibody to bind to an antigen and its stability are substantially the same as those of a complete antibody. The term "bispecific antibody" refers to a bispecific antibody that can be obtained in large quantities, with high uniformity and purity by introducing two sets of light chain and heavy chain genes into myeloma cells, selecting a suitable antibody constant region and Ig type. In addition, a bispecific antibody can be obtained by chemical cross-linking technology or hybrid-hybridoma technology. The term "minimal recognition unit (MRU)" refers to a structure containing only a single CDR in a variable region, with a molecular weight of only about 1% of that of a complete antibody, and can bind to the corresponding antigen. The antibody of the present application can be prepared by various methods known in the art, for example, by a genetic engineering recombination technique. For example, a DNA molecule encoding the heavy chain and light chain genes of the antibody of the present application is obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector, and then the host cell is transfected, the transfected host cell is cultured under specific conditions, and the antibody of the present application is expressed. Those skilled in the art are well aware that the antigen-binding fragment can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of the complete antibody to produce an antigen-binding fragment of the antibody.

[0024] To solve the above technical problem, the present application also provides a nucleic acid molecule encoding the monoclonal antibody DY23HER202 or an antigen-binding portion thereof described above; the nucleic acid molecule can be any one of the following DNA molecules:

[0025] c1) a DNA molecule encoding a monoclonal antibody, wherein the coding sequence of the third complementarity determining region CDRH3 in the variable region of the heavy chain of the monoclonal antibody is a DNA molecule represented by 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;

[0026] c2) a DNA molecule encoding a monoclonal antibody, wherein the coding sequence of the heavy chain variable region of the monoclonal antibody is a DNA molecule represented by SEQ ID NO. 12 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;

[0027] c3) a DNA molecule having more than 90% identity with the DNA molecule defined in c1) or c2) and encoding the monoclonal antibody or an antigen-binding portion thereof.

[0028] Herein, the at least 90% identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0029] To solve the above technical problems, the present application also provides a biological material, which can be an expression cassette, a recombinant vector, a recombinant microorganism and / or a recombinant animal cell line containing the nucleic acid molecule described above.

[0030] The recombinant animal cell line can be a eukaryotic cell or a prokaryotic cell. The eukaryotic cell can be selected from any one of Expi293F, CHO and HEK293. The prokaryotic cell can be Escherichia coli.

[0031] The vector described herein is well known to those skilled in the art, including but not limited to: plasmid, bacteriophage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e. cos plasmid), viral vector (such as baculovirus vector, retrovirus (including lentivirus), adenovirus, adeno-associated virus or herpes virus (such as herpes simplex virus), etc.). In an embodiment of the present application, the vector can be specifically a pcDNA3.4 vector containing human IgG1 heavy and light chain constant region.

[0032] The microorganism described herein can be yeast, bacteria or fungi. Among them, the bacteria can be from Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus, etc.; the yeast can be P. pastoris. The cell line (host cell) refers to a cell that can be used for introducing a vector, which includes but is not limited to: eukaryotic cells (such as yeast cells, Aspergillus), animal cells (such as mammalian cells, insect cells) or prokaryotic cells. In an embodiment of the present application, the cell line can be specifically Expi293F cell line.

[0033] The terms "cell" and "cell line" can be used interchangeably, and all such designations include their progeny.

[0034] To solve the above technical problems, the present application also provides the use of the antibody described above and / or the biological material described above in the development or preparation of a product for detecting HER2 protein.

[0035] The use of the antibody described above and / or the biological material described above in the preparation of a product for detecting or diagnosing HER2 protein positive tumor also falls within the scope of the present application.

[0036] The use of the antibody described above and / or the biomaterial described above in the development or preparation of a product for distinguishing HER2 protein positive cells and HER2 protein negative cells or a product for locating HER2 protein positive cells also falls within the protection scope of the present application.

[0037] The use of the antibody described above and / or the biomaterial described above in the preparation or development of a drug for treating HER2 protein positive tumors also falls within the protection scope of the present application.

[0038] The antibody described above can target the extracellular domain of HER2 protein, and the amino acid sequence information of the extracellular domain of HER2 protein can be SEQ ID NO. 8 in the sequence listing.

[0039] The product described above can be a reagent and / or a kit.

[0040] The present application previously established a new antibody generation platform based on artificial intelligence (AI) algorithm and biochemical screening technology, which can realize a rapid, efficient and animal-free immunization antibody discovery process. Using the platform, the present application screens suitable monoclonal antibodies targeting HER2.

[0041] The purpose of the present application is to propose some monoclonal antibodies predicted by AI, designed and verified by in vitro detection experiments for HER2 protein, which can play a role in multiple in vitro detection experiments.

[0042] In order to achieve the above purpose, the present application screens three monoclonal antibodies targeting human HER2 protein using the previously established platform, and the amino acid sequences (CDRH3 regions) of the monoclonal antibodies are shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0043] The present application also provides a nucleic acid sequence encoding the above-mentioned monoclonal antibody.

[0044] The present application also provides an expression vector containing the above-mentioned nucleic acid sequence.

[0045] Preferably, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector. The eukaryotic expression vector is selected from any one of pcDNA3.1 and pcDNA3.4 expression vectors. The prokaryotic expression vector is selected from a pET28a vector.

[0046] The present application also provides a host cell of the above-mentioned nucleic acid or the above-mentioned expression vector.

[0047] Preferably, the host cell is a eukaryotic cell or a prokaryotic cell, the eukaryotic cell is selected from any one of CHO and HEK293, and the prokaryotic cell is Escherichia coli.

[0048] The present application also provides a kit for detecting human HER2 protein, which comprises any one of the above monoclonal antibodies, i.e. falls within the scope of the present application.

[0049] The present application also provides a method of treating a subject.

[0050] The method of treating a subject provided by the present application comprises administering to the subject a composition comprising a therapeutically effective amount of an antibody that specifically binds to HER2 protein as described above, wherein the subject has a HER2 protein positive tumor.

[0051] In the present application, the HER2 protein positive tumor can be specifically breast cancer, gastric cancer, prostate cancer, and / or ovarian cancer, etc.

[0052] In the present application, the terms "subject" and "patient" are used interchangeably herein when referring to a subject. A "subject" includes a human who is being treated as a patient for a disease or to prevent a disease. The methods described herein can be used to treat animal subjects belonging to any classification. Examples of such animals include mammals. Mammals include, but are not limited to, mammals of order Rodentia, such as mice and rats, and of order Logomorpha, such as rabbits. Mammals can be of order Carnivora, including cats (Felidae) and dogs (Canidae). Mammals can be of order Artiodactyla, including bovines (Bovidae) and porcines (Suidae), or of order Perssodactyla, including equines (Equidae). Mammals can be of order Primate, Ceboid, or Simoid (monkeys) or Anthropoid (humans and apes). In some embodiments, the mammal is a human.

[0053] The term "effective" applied to dose or amount is meant to refer to that quantity of a compound or pharmaceutical composition that is sufficient to yield a desired activity after administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination can, and typically does, include the amount of each ingredient that would be effective if administered alone. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, mode of administration, and the like. Beneficial effects

[0054] In view of the problems of the existing HER2 antibody drugs, the monoclonal antibody DY23HER202 obtained by screening has a new recognition epitope of HER2 protein, the antibody can specifically and accurately detect and locate HER2 positive cells in different in vitro detection experiments, can be applied to the detection and diagnosis of HER2 positive cells or tissue samples, and can be applied to the research and development or preparation of a new drug for treating HER2 protein positive tumors. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 is a result map of detecting the purity of the screened monoclonal antibody by Coomassie brilliant blue staining after SDS-PAGE gel electrophoresis.

[0056] Figure 2 is a result map of detecting the specificity of different antibodies binding to HER2 protein by ELISA. The abscissa is the name of different antibodies, and the ordinate is the absorbance value at 450nm, and the larger the value means the stronger the binding ability of the antibody to the antigen.

[0057] Figure 3 is a result map of detecting the ability of different antibodies to bind to HER2 protein by ELISA. The abscissa is the logarithmic value of different incubation concentrations of the antibody, and the ordinate is the absorbance value at 450nm, and the larger the value means the stronger the binding ability of the antibody to the antigen.

[0058] Figure 4 is a result map of detecting the binding of monoclonal antibody DY23HER202 to HER2-His protein by biofilm interference experiment (BLI). The abscissa is the time of the interference experiment, and the ordinate is the interaction reaction in the interference experiment.

[0059] Figure 5 is a result map of verifying the ability of the screened antibody DY23HER202 to detect HER2 positive cell lines by flow cytometry. The abscissa is the logarithmic value of the AF488 fluorescence intensity of the detected cells, and the ordinate is the forward scattering light value of the detected cells.

[0060] Figure 6 is a result map of verifying the ability of the screened antibody DY23HER202 to detect HER2 positive cell lines by immunofluorescence experiment.

[0061] Figure 7 is a result map of verifying the ability of the screened antibody DY23HER202 to detect HER2 positive cell lines by immunohistochemical experiment. Embodiments of the present application

[0062] The present application will be further described in detail below in conjunction with specific embodiments, and the examples given are only for the purpose of illustrating the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application.

[0063] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available, unless otherwise specified.

[0064] The sequences of the complementarity determining regions (CDRs) of the antibodies in the present application are defined according to the Kabat numbering system.

[0065] Example 1. Screening, preparation and functional detection of HER2 protein monoclonal antibodies

[0066] 1. CDRH3 diversity sequence design, monoclonal antibody screening and sequence analysis

[0067] 1.1 Construction of phage display antibody library

[0068] The antibodies involved in the present application all target the extracellular domain of HER2 protein, and the amino acid sequence information is as follows (SEQ ID NO. 8 in the sequence listing):

[0069] TQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLT.

[0070] The extracellular domain of HER2 protein is analyzed by generative AI technology to predict and design the sequence of potential specific antibody heavy chain variable region third complementarity determining region (CDRH3) against the extracellular domain of HER2. The corresponding diversity sequence is synthesized, and the sequence of the marketed HER2 monoclonal antibody trastuzumab (Herceptin) is used as a framework to construct a phage display antibody library. The amino acid sequence of the heavy chain variable region of Herceptin monoclonal antibody is as follows SEQ ID NO. 16, and the sequence in the underlined part is the CDRH3 region sequence of the heavy chain variable region of Herceptin monoclonal antibody:

[0071] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS;

[0072] The amino acid sequence of the light chain variable region of Herceptin monoclonal antibody is as follows (SEQ ID NO. 7, the underlined sequence is the sequence of the complementarity determining regions LCDR1-LCDR3 regions):

[0073] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK.

[0074] The amino acid sequence of the first complementarity determining region LCDR1 in the light chain variable region of Herceptin monoclonal antibody is the 27th-32nd amino acid residues of SEQ ID NO. 7, the amino acid sequence of the second complementarity determining region LCDR2 is the 50th-52nd amino acid residues of SEQ ID NO. 7, and the amino acid sequence of the third complementarity determining region LCDR3 is the 89th-97th amino acid residues of SEQ ID NO. 7.

[0075] The coding nucleotide sequence of the light chain variable region of Herceptin monoclonal antibody is as follows (SEQ ID NO. 15, 5'-3', the underlined sequence is the coding sequence of the complementarity determining regions LCDR1-LCDR3 regions):

[0076] GACATCCAAATGACTCAGAGTCCCTCCAGCTTGTCTGCTTCAGTGGGAGACAGAGTGACTATCACCTGCCGAGCTTCACAGGACGTGAACACAGCCGTGGCCTGGTACCAGCAGAAACCCGGAAAGGCTCCAAAACTCCTGATCTACAGCGCAAGCTTTCTGTATTCCGGTGTCCCAAGCCGCTTTAGCGGTTCTAGGTCTGGTACGGATTTCACCCTGACGATCTCTTCTCTGCAGCCTGAGGACTTCGCCACTTATTATTGTCAGCAACACTATACTACACCCCCCACCTTCGGCCAGGGCACAAAGGTAGAGATCAAA.

[0077] 1.2 Screening of phage display antibody library

[0078] Three rounds of solid-phase screening were performed on the phage display antibody library using HER2-His protein expressed by Expi293F cells, and the specificities of different phages to HER2 protein were verified by ELISA experiments after monoclonalization of the obtained screening library.

[0079] In order to identify the antibody sequences in the positive monoclonal phages, the plasmids in the corresponding bacterial liquid were extracted, sequence determination was performed using pCANTAB-R1 / R2 primers, and the specific sequences of CDRH3 were determined. Finally, three unique sequences were obtained.

[0080] The amino acid sequences of the CDRH3 regions of the three unique sequences are respectively:

[0081] The CDRH3 region amino acid sequence (SEQ ID NO. 1) of the DY23HER202 antibody is: SYGYPPYFDS.

[0082] The CDRH3 region amino acid sequence (SEQ ID NO. 2) of the DY23HER204 antibody is: ALGRGYNYYGFDS.

[0083] The CDRH3 region amino acid sequence (SEQ ID NO. 3) of the DY23HER205 antibody is: GRDNERGDIGFDL.

[0084] Correspondingly, the encoding nucleic acid sequences of the CDRH3 regions of the three unique sequences are respectively:

[0085] The CDRH3 region nucleic acid sequence (SEQ ID NO. 4) of the DY23HER202 antibody is: 5'-TCTTACGGTTACCCGCCGTACTTCGACTCT-3'.

[0086] The CDRH3 region nucleic acid sequence (SEQ ID NO. 5) of the DY23HER204 antibody is: 5'-GCGCTGGGTCGTGGTTACAACTACTACGGTTTCGACTCT-3'.

[0087] The CDRH3 region nucleic acid sequence (SEQ ID NO. 6) of the DY23HER205 antibody is: 5'-GGTCGTGACAACGAACGTGGTGACATCGGTTTCGACCTG-3'.

[0088] The amino acid sequence of the heavy chain variable region of the DY23HER202 antibody (SEQ ID NO. 9, the sequence in underlined part is the sequence of the complementarity determining regions CDRH1-CDRH3) is:

[0089] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSYGYPPYFDSQGTLVTVSS;

[0090] The amino acid sequence of the heavy chain variable region of the DY23HER202 antibody (SEQ ID NO. 9, the sequence in underlined part is the sequence of the complementarity determining regions CDRH1-CDRH3) is:

[0091] The amino acid sequence of the heavy chain variable region of the DY23HER202 antibody (SEQ ID NO. 9, the sequence in underlined part is the sequence of the complementarity determining regions CDRH1-CDRH3) is:

[0092] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCALGRGYNYYGFDSQGTLVTVSS;

[0093] The amino acid sequence of the heavy chain variable region of the DY23HER202 antibody (SEQ ID NO. 9, the sequence in underlined part is the sequence of the complementarity determining regions CDRH1-CDRH3) is:

[0094] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCGRDNERGDIGFDLQGTLVTVSS;

[0095] The amino acid sequence of the heavy chain variable region of the DY23HER202 antibody (SEQ ID NO. 9, the sequence in underlined part is the sequence of the complementarity determining regions CDRH1-CDRH3) is:

[0096] GAAGTACAATTGGTCGAGAGTGGAGGAGGCCTCGTTCAACCTGGAGGCTCTCTGAGGCTTTCATGCGCTGCCAGTGGGTTCAACATCAAGGACACCTACATACATTGGGTTAGGCAGGCACCGGGGAAAGGACTGGAATGGGTAGCTAGGATTTACCCCACCAACGGCTACACAAGGTACGCTGACTCAGTGAAGGGCCGGTTCACCATTTCAGCAGATACGTCAAAGAATACCGCTTATCTGCAGATGAATAGTCTTCGGGCGGAGGATACAGCCGTGTATTACTGTTCTTACGGTTACCCGCCGTACTTCGACTCTCAAGGAACCCTGGTGACCGTGTCTTCC;

[0097] DY23HER204抗体的重链可变区的编码核酸序列(SEQ ID NO.13,5’-3’)是:

[0098] GAAGTACAATTGGTCGAGAGTGGAGGAGGCCTCGTTCAACCTGGAGGCTCTCTGAGGCTTTCATGCGCTGCCAGTGGGTTCAACATCAAGGACACCTACATACATTGGGTTAGGCAGGCACCGGGGAAAGGACTGGAATGGGTAGCTAGGATTTACCCCACCAACGGCTACACAAGGTACGCTGACTCAGTGAAGGGCCGGTTCACCATTTCAGCAGATACGTCAAAGAATACCGCTTATCTGCAGATGAATAGTCTTCGGGCGGAGGATACAGCCGTGTATTACTGTGCGCTGGGTCGTGGTTACAACTACTACGGTTTCGACTCTCAAGGAACCCTGGTGACCGTGTCTTCC;

[0099] DY23HER205抗体的重链可变区的编码核酸序列(SEQ ID NO.14,5’-3’)是:

[0100] GAAGTACAATTGGTCGAGAGTGGAGGAGGCCTCGTTCAACCTGGAGGCTCTCTGAGGCTTTCATGCGCTGCCAGTGGGTTCAACATCAAGGACACCTACATACATTGGGTTAGGCAGGCACCGGGGAAAGGACTGGAATGGGTAGCTAGGATTTACCCCACCAACGGCTACACAAGGTACGCTGACTCAGTGAAGGGCCGGTTCACCATTTCAGCAGATACGTCAAAGAATACCGCTTATCTGCAGATGAATAGTCTTCGGGCGGAGGATACAGCCGTGTATTACTGTGGTCGTGACAACGAACGTGGTGACATCGGTTTCGACCTGCAAGGAACCCTGGTGACCGTGTCTTCC.

[0101] 2. Monoclonal antibody preparation.

[0102] According to the results of Sanger sequencing, the coding nucleotide sequences of the variable regions of the heavy chains and light chains of the above-mentioned three monoclonal antibodies (SEQ ID NO. 12-SEQ ID NO. 14, SEQ ID NO. 15) were synthesized and connected to the pcDNA3.4 vector containing human IgG1 heavy and light chain constant regions to construct three human IgG antibody expression plasmids, respectively; and Expi293F (Invitrogen, USA) cell lines were used to express the corresponding three monoclonal antibodies, specifically, three recombinant cell lines were obtained by transforming the three human IgG antibody expression plasmids into Expi293F cell lines.

[0103] Three cell culture supernatants were obtained by culturing the three recombinant cell lines obtained, respectively. Then the three cell culture supernatants obtained were purified by HiTrap TM rProtein A FF affinity chromatography column (GE Healthcare, USA) to obtain three Herceptin skeleton monoclonal antibodies DY23HER202, DY23HER204 and DY23HER205 against HER2 protein.

[0104] The amino acid sequences of the light chain variable regions of the monoclonal antibodies DY23HER202, DY23HER204 and DY23HER205 are all SEQ ID NO. 7.

[0105] SDS-PAGE gels were prepared and electrophoresis was performed to detect the purity of the three purified mAbs. The results of SDS-PAGE electrophoresis are shown in Figure 1, which shows that the purity of the three purified mAbs is greater than 95%.

[0106] 3. Enzyme-linked immunosorbent assay (ELISA).

[0107] To determine the specificity of the three mAbs obtained in step 2 for binding to HER2 protein, 200 ng / well of HER2-His protein, control CD123-His (Yiqiao, China) and control bovine serum albumin BSA were coated in a micro-ELISA plate (Nunc, USA) at 4°C overnight. Then 5% skimmed milk powder dissolved in PBS-Tween20 (PBST) was used for blocking. After washing with PBST, 100 μL / well of mAb at a concentration of 1 μg / mL was added, followed by incubation at 37°C for 2 hours. After washing, horseradish peroxidase (HRP)-coupled goat anti-human IgG-Fc (diluted 1:10,000; Abeam, USA) was added and incubated at 37°C for 1 hour. After color development, the absorbance value at OD 450nm was monitored using an enzyme marker.

[0108] The results of the ELISA test for detecting the specificity of the three mAbs for binding to HER2 protein are shown in Figure 2. The results of the ELISA show that the OD 450nm values of the binding reactions of DY23HER202 and DY23HER204 mAbs with HER2-His protein are stronger than those of the control groups (CD123-His and BSA), indicating that these two mAbs can specifically bind to HER2-His protein and do not bind to the control CD123-His and BSA. This fully demonstrates that these two mAbs can specifically recognize HER2 protein and have no cross-reaction with the His tag. The binding reaction of DY23HER205 mAb with CD123-His protein is stronger than that with HER2-His protein, indicating that this mAb does not bind to HER2-His protein specifically but to the His tag. At the same time, according to the results in Figure 2, it can be seen that the binding effect of DY23HER202 with HER2 protein is better than that of the other antibodies, and the OD 450nm value is significantly higher than that of the other antibodies. This result is consistent with the results of the subsequent EC50 analysis experiment.

[0109] To compare the binding ability of different mAbs to HER2 protein, EC50detection experiments were performed. 100 ng / well of HER2-His protein was coated in micro-ELISA plates (Nunc, USA) at 4°C overnight. Then blocked with 5% skim milk powder in PBS-Tween20 (PBST). After washing with PBST, 100 µL / well of mAbs diluted by 12 groups of dilution gradient starting from 1 µg / mL concentration (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, respectively) were added. Then incubated at 37°C for 2 hours. After washing, horseradish peroxidase (HRP)-conjugated goat anti-human IgG-Fc (1:10,000 dilution; Abeam, USA) was added and incubated at 37°C for 1 hour. After color development, the absorbance value at 450 nm was monitored by a microplate reader.

[0110] As shown in Figure 3, the results of EC50experiments showed that the binding ability of DY23HER202 to HER2 was stronger than that of other antibodies, with an EC50value of 12.90 ng / mL, which was significantly higher than that of DY23HER204 and DY23HER205.

[0111] 4. Bio-Layer Interferometry (BLI) assay.

[0112] To determine the binding affinity of antibodies DY23HER202 and DY23HER208 to HER2 protein, BLI assays were performed on an Octet RED96 machine (Pall ForteBio, USA) according to the manufacturer's instructions. Briefly, HER2-His protein was immobilized on Ni-coated biosensors (Pall ForteBio) until saturation. The antigen-bound biosensors were placed in wells containing a series of diluted mAb 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 (Pall ForteBio).

[0113] The experimental results are shown in Figure 4, and the equilibrium dissociation constant K D value of DY23HER202 binding to HER2 protein reached 1.182 nM, indicating that DY23HER202 has high affinity for binding to HER2 protein and high sensitivity for detection.

[0114] 5. Flow cytometry.

[0115] Human breast cancer cell SK-BR-3 cells and human embryonic kidney cell HEK293 cells were cultured in T25 culture flask to appropriate density, washed once with PBS, digested with 0.25% trypsin until the cells became round, and terminated with DMEM medium. Washed twice with PBS, and mixed equal amounts of the two cell lines to form experimental group samples. Untreated mixed cells were taken as blank control, and HEK293 cells alone were taken as negative control group. 2x10 5 centrifugation. During centrifugation, the antibody to be tested was diluted with flow buffer to a final concentration of 1 μg / ml. According to the experimental grouping design, 100 μl of diluted antibody or 100 μl of flow buffer was used to resuspend the cells in the corresponding ep tube. Incubate for 30 min at room temperature in the dark. Wash three times with flow buffer. Then according to the experimental design, 100 μl of diluted AF488 fluorescent dye labeled anti-human IgG Fc secondary antibody (#410706, BioLegend) or 100 μl of flow buffer was used to resuspend the cells in the corresponding ep tube. Incubate for 30 min at room temperature in the dark. Wash three times with flow buffer. Add 500 μL of flow buffer to each group, mix well, and detect the AF488 indicator of the cells with a flow cytometer (BD Canto2).

[0116] The results are shown in Figure 5. Monoclonal antibody DY23HER202 can specifically recognize HER2 protein on the surface of SK-BR-3 cells (right panel in Figure 5), but not HEK293 cells (negative control represented in the middle panel of Figure 5). Therefore, DY23HER202 can clearly distinguish between HER2 positive SK-BR-3 cell population and negative HEK293 cell population, with non-overlapping signal peaks and high detection accuracy.

[0117] 6. Immunofluorescence test (IFA).

[0118] SK-BR-3 cells were cultured on treated cover slips to appropriate density, cover the tissue with 3% BSA, block at room temperature for 30 min. Dilute the antibody to be tested with PBS to a final concentration of 20 μg / ml, add to the cell sample, incubate at 4°C overnight. Wash 3 times with PBS, each for 5 min. Cover the cells with diluted Goat anti-Human IgG Fc-HRP (#ab97225, Abeam), incubate at room temperature for 50 min. Wash 3 times with PBS, each for 5 min. Cover the cells with TSA reagent (Aidgen), after incubation, wash 3 times with PBS, each for 5 min. Add DAPI staining solution to the cell sample, incubate at room temperature for 10 min in the dark. Wash 3 times with PBS, each for 5 min. After the section is slightly dried, it is sealed with an anti-fluorescence quenching mounting medium. The sample is observed under a Nikon inverted fluorescence microscope and images are collected. The nuclei stained by DAPI are blue under ultraviolet excitation, CY3 red light excitation wavelength 510-560, emission wavelength 590 nm.

[0119] [Corrected according to Rule 91 on 06.01.2025] The detection results are shown in Figure 6. The monoclonal antibody DY23HER202 can clearly stain HER2-positive SK-BR-2 cells (right panel in Figure 6), and the fluorescent signal on the membrane represents the localization of HER2 protein on the cell membrane. Cells incubated without the screening antibody have no fluorescent signal (left panel in Figure 6). The fluorescent signal in the cell membrane represents the nucleus.

[0120] 7. Immunohistochemical test (IHC).

[0121] SK-BR-3 cells were cultured on treated cover slips to appropriate density, cover the tissue with 3% BSA, block at room temperature for 30 min. Dilute the antibody to be tested with PBS to a final concentration of 20 μg / ml, add to the cell sample, incubate at 4°C overnight. Wash 3 times with PBS, each for 5 min. Cover the cells with diluted Goat anti-Human IgG Fc-HRP (#ab97225, Abeam), incubate at room temperature for 50 min. Wash 3 times with PBS, each for 5 min. Cover the cells with TSA reagent (Aidgen), after incubation, wash 3 times with PBS, each for 5 min. Add DAPI staining solution to the cell sample, incubate at room temperature for 10 min in the dark. Wash 3 times with PBS, each for 5 min. After the section is slightly dried, it is sealed with an anti-fluorescence quenching mounting medium. The sample is observed under a Nikon inverted fluorescence microscope and images are collected. The nuclei stained by DAPI are blue under ultraviolet excitation, CY3 red light excitation wavelength 510-560, emission wavelength 590 nm.

[0122] [Corrected according to Rule 91 on 06.01.2025] The results are shown in Figure 7. The DY23HER202 antibody can clearly stain the HER2 positive SK-BR-3 cells (right panel in Figure 7), and the dark gray signal on the outer edge of the membrane represents the localization of HER2 protein on the cell membrane. There is no dark gray signal on the outer edge of the membrane in cells incubated with the unselected antibody (left panel in Figure 7). The dark gray signal inside the cell represents the nucleus.

[0123] As described above, the monoclonal antibody DY23HER202 can be used in different in vitro detection experiments, has high specificity and accuracy, and can clearly distinguish HER2 positive and negative cells or locate HER2 positive cells in all in vitro detection experiments. Therefore, it is convenient and accurate to count HER2 positive cells, which is very important for the auxiliary diagnosis of HER2 positive tumor cells in clinical practice.

[0124] The above describes the present application in detail. For those skilled in the art, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present application, and without unnecessary experiments. Although the present application gives a specific example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, the present application intends to include any changes, uses or improvements of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application.

[0125] Cross-reference of related applications

[0126] This application claims priority to Chinese Patent Application No. 202410440858.3, filed on April 12, 2024, the entire contents of which are hereby incorporated by reference. Industrial applicability

[0127] The monoclonal antibody DY23HER202 against HER2 protein prepared by screening in the present application can specifically and accurately detect and locate HER2 positive cells in different in vitro detection experiments, and can be applied to diagnose HER2 positive tumor cells, distinguish HER2 positive and negative cells, and conveniently and accurately count HER2 positive cells in clinical practice. It can be applied to effectively identify HER2 positive tumors and biochemical research of HER2, and can be applied to prepare products for distinguishing HER2 protein positive cells and HER2 protein negative cells and locating HER2 protein positive cells. It can also be applied to the research and development of new HER2 targeted antibodies or the preparation of new drugs for treating HER2 protein positive tumors.

Claims

1. An antibody specifically binding to HER2 protein designed based on AI, characterized by: The antibody is the 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, and 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.

2. The antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is SEQ ID NO.9 in the sequence listing.

3. The antibody according to claim 1, wherein: The amino acid sequence of the light chain variable region is SEQ ID NO.7 in the sequence listing.

4. The antibody according to claim 1, wherein: 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.

5. The antibody according to claim 1, wherein: The monoclonal antibody is any of the following: a) Single-chain antibodies; b) a fusion antibody comprising the single-chain antibody described in a); c) Fab fragment; d) Fv fragment.

6. The antibody according to claim 1, wherein: 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; the monoclonal antibody is any one of the following: a) Single-chain antibodies; b) a fusion antibody comprising the single-chain antibody described in a); c) Fab fragment; d) Fv fragment.

7. A nucleic acid molecule encoding the monoclonal antibody DY23HER202 or an antigen-binding portion thereof according to any one of claims 1 to 6; The nucleic acid molecule is a DNA molecule as described in any of the following: c1) a DNA molecule encoding a 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 shown in SEQ ID NO. 15 in the sequence listing; c2) 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 in the sequence listing; the coding sequence of the light chain variable region of the monoclonal antibody is the DNA molecule shown in SEQ ID NO. 15 in the sequence listing; c3) A DNA molecule that is more than 90% identical to the DNA molecule defined in c1) or c2) and encodes the monoclonal antibody or the antigen-binding portion thereof.

8. Biomaterial, characterized in that: The biological material is an expression cassette, a recombinant vector, a recombinant microorganism and / or a recombinant animal cell line containing the nucleic acid molecule according to claim 7.

9. Use of the antibody according to any one of claims 1 to 6 in developing or preparing a product for detecting HER2 protein.

10. Use of the antibody according to any one of claims 1 to 6 in the preparation of a product for detecting or diagnosing HER2 protein-positive tumors.

11. Use of the biomaterial according to claim 8 in preparing a product for detecting or diagnosing HER2 protein-positive tumors.

12. Use of the antibody according to any one of claims 1 to 6 in developing or preparing a product for distinguishing HER2 protein-positive cells from HER2 protein-negative cells or a product for localizing HER2 protein-positive cells.

13. Use of the biomaterial according to claim 8 in developing or preparing a product for distinguishing HER2 protein-positive cells from HER2 protein-negative cells.

14. Use of the biomaterial according to claim 8 in developing or preparing a product for localizing HER2 protein-positive cells.

15. Use of the antibody according to any one of claims 1 to 6 in the preparation or development of a drug for treating HER2 protein-positive tumors.

16. Use of the biomaterial according to claim 8 in the preparation or development of a drug for treating HER2 protein-positive tumors.

Images