Her2 antigen binding molecules and uses thereof

Abstract

The application discloses a HER2 antigen binding molecule and application thereof. The HER2 antigen binding molecule comprises at least one VHH chain, and the VHH chain comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 13, the CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 14, and the CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 15. The HER2 antigen binding molecule provided by the application shows a HER2 binding activity equivalent to that of trastuzumab and has a HER2 binding activity competitive with that of trastuzumab; and the HER2 antigen binding molecule provided by the application shows a better tumor cell killing effect through ADCC and endocytosis activity detection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a HER2 antigen-binding molecule and its applications. Background Technology

[0002] The HER2 gene is a proto-oncogene that encodes the p185 transmembrane protein with tyrosine kinase activity. It belongs to the human epidermal growth factor receptor (HER) family, which includes HER1, HER2, HER3, and HER4. The HER family regulates the growth and development of normal breast tissue, but HER2 overexpression is associated with breast cancer. HER2 overexpression leads to increased formation of HER2 homodimers and heterodimers, thereby activating the PI3K and MAPK pathways, resulting in cell proliferation, anti-apoptosis, invasion, and angiogenesis. Approximately 15%-30% of breast cancers and 10%-30% of gastric / esophageal cancers exhibit HER2 gene amplification or overexpression. HER2 overexpression is also observed in other tumors such as ovarian cancer, lung cancer, and colon cancer. Antibody drugs targeting HER2 include monoclonal antibodies, bispecific antibodies, and antibody-drug conjugates (ADCs).

[0003] Trastuzumab is the first humanized monoclonal antibody targeting HER2. It binds to the extracellular domain IV of the HER2 receptor, thereby blocking ligand-independent HER2 isodimerization in cells with high HER2 overexpression, and to some extent also blocking heterodimerization of HER2 with other family members. Most bispecific antibodies and ADCs developed targeting HER2 are based on trastuzumab or antibodies with similar epitopes. Therefore, developing effective single-domain antibodies specifically targeting HER2 with epitopes similar to trastuzumab should be of significant value. Summary of the Invention

[0004] To address the lack of effective single-domain antibodies targeting HER2 with epitopes similar to trastuzumab in existing technologies, this invention provides a HER2 antigen-binding molecule and its applications. Using HER2 as a target, this invention obtains nanobody molecules with HER2 antigen-binding activity and competition with trastuzumab through antigen immunization of alpacas, construction of nanobody phage display libraries, and library screening. Cellular-level FACS affinity, endocytosis, and ADCC assays show that these molecules exhibit good binding and tumor cell-killing effects.

[0005] To solve the above-mentioned technical problems, one of the technical solutions provided by the present invention is: a HER2 antigen-binding molecule, wherein the antigen-binding molecule comprises at least one VHH chain, wherein the VHH chain comprises CDR1, CDR2 and CDR3 selected from the following: CDR1 comprises the amino acid sequence GFTLSTYX1MT as shown in SEQ ID NO:13, wherein X1 is T or S; CDR2 comprises the amino acid sequence TIAPGX2VTG as shown in SEQ ID NO:14, wherein X2 is D or G; and CDR3 comprises the amino acid sequence PHRX3X4 as shown in SEQ ID NO:15, wherein X3 is R or V and X4 is F or Y.

[0006] In some embodiments, CDR1 comprises an amino acid sequence as shown in SEQ ID NO:6 or SEQ ID NO:7, CDR2 comprises an amino acid sequence as shown in SEQ ID NO:8 or SEQ ID NO:9, and CDR3 comprises an amino acid sequence as shown in SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0007] In some embodiments, the amino acid sequence of CDR1 is as shown in SEQ ID NO:6, the amino acid sequence of CDR2 is as shown in SEQ ID NO:8, and the amino acid sequence of CDR3 is as shown in SEQ ID NO:10; or, the amino acid sequence of CDR1 is as shown in SEQ ID NO:6, the amino acid sequence of CDR2 is as shown in SEQ ID NO:9, and the amino acid sequence of CDR3 is as shown in SEQ ID NO:11; or, the amino acid sequence of CDR1 is as shown in SEQ ID NO:7, the amino acid sequence of CDR2 is as shown in SEQ ID NO:9, and the amino acid sequence of CDR3 is as shown in SEQ ID NO:11; or, the amino acid sequence of CDR1 is as shown in SEQ ID NO:7, the amino acid sequence of CDR2 is as shown in SEQ ID NO:8, and the amino acid sequence of CDR3 is as shown in SEQ ID NO:12.

[0008] In some embodiments, the VHH chain comprises an amino acid sequence or a variant thereof as shown in any of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5;

[0009] The variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence from which it originates.

[0010] In some embodiments, the HER2 antigen-binding molecule further comprises the Fc region of an immunoglobulin;

[0011] In a preferred embodiment of the present invention, the Fc region is the Fc region of human IgG1;

[0012] In a more preferred embodiment of the present invention, the amino acid sequence of the Fc region is shown in SEQ ID NO:16.

[0013] The second technical solution provided by the present invention is: an isolated nucleic acid, wherein the nucleic acid encodes the HER2 antigen-binding molecule as described in the first technical solution.

[0014] The third technical solution provided by the present invention is: a recombinant expression vector, wherein the recombinant expression vector comprises the isolated nucleic acid as described in the second technical solution;

[0015] In a preferred embodiment of the present invention, the recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector; the backbone of the plasmid is, for example, pcDNA3.4.

[0016] The fourth technical solution provided by the present invention is: a host cell, wherein the host cell comprises the recombinant expression vector as described in the third technical solution;

[0017] In a preferred embodiment of the present invention, the host cell is a prokaryotic cell or a eukaryotic cell;

[0018] In a more preferred embodiment of the present invention, the eukaryotic cell is a yeast cell or a mammalian cell; wherein the mammalian cell is, for example, a HEK293 cell.

[0019] The fifth technical solution provided by this invention is: a method for preparing a HER2 antigen-binding molecule, the method comprising the following steps:

[0020] The host cells are cultured under suitable conditions for host cell growth and fermentation as described in technical solution four, and HER2 antigen-binding molecules are obtained from the culture.

[0021] The sixth technical solution provided by the present invention is: a multispecific antibody, which includes the HER2 antigen-binding molecule as described in one of the technical solutions, and an antigen-binding molecule having another antigen-binding property operably linked to the HER2 antigen-binding molecule.

[0022] The seventh technical solution provided by the present invention is: a pharmaceutical composition comprising a HER2 antigen-binding molecule as described in one of the technical solutions, and a pharmaceutically acceptable carrier;

[0023] In a preferred embodiment of the present invention, the pharmaceutical composition further comprises one or more of the following groups: hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0024] The eighth technical solution provided by the present invention is the application of the HER2 antigen-binding molecule as described in technical solution one, the nucleic acid as described in technical solution two, the recombinant expression vector as described in technical solution three, the host cell as described in technical solution four, the multispecific antibody as described in technical solution six, or the pharmaceutical composition as described in technical solution seven in the preparation of a drug for the prevention and / or treatment of tumors.

[0025] The ninth technical solution provided by the present invention is: a kit comprising the HER2 antigen-binding molecule as described in the first technical solution, the nucleic acid as described in the second technical solution, the recombinant expression vector as described in the third technical solution, the host cell as described in the fourth technical solution, the multispecific antibody as described in the sixth technical solution, or the pharmaceutical composition as described in the seventh technical solution;

[0026] In a preferred embodiment of the invention, the kit further includes (i) a means for administering the antibody or pharmaceutical composition; and / or (ii) instructions for use.

[0027] The tenth technical solution provided by this invention is: a set of medicine boxes, wherein the set of medicine boxes includes medicine box A and medicine box B, wherein,

[0028] The kit A contains the HER2 antigen-binding molecule as described in one of the technical solutions, the multispecific antibody as described in six of the technical solutions, or the pharmaceutical composition as described in seven of the technical solutions.

[0029] The kit B contains other anti-tumor antibodies or a pharmaceutical composition containing said other anti-tumor antibodies, and / or is one or more of the following groups: hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0030] The eleventh technical solution provided by the present invention is: a method for immunoassay or determination of HER2, wherein the method comprises mixing the sample to be tested with the HER2 antigen-binding molecule as described in one technical solution, the multispecific antibody as described in six technical solutions, or the pharmaceutical composition as described in seven technical solutions;

[0031] In a preferred embodiment of the present invention, the detection is a non-diagnostic detection.

[0032] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0033] The positive and progressive effects of this invention are as follows: the HER2 antigen-binding molecule provided by this invention exhibits HER2 binding activity comparable to trastuzumab and has competitive HER2 binding activity with trastuzumab; the HER2 antigen-binding molecule provided by this invention, as detected by ADCC and endocytosis activity assays, shows that it has a good effect on killing tumor cells. Attached Figure Description

[0034] Figure 1 The affinity activity of the candidate antibody for HER2 was determined using an ELISA method.

[0035] Figure 2 The results showed that the competitive activity of the candidate antibody against trastuzumab (Trmab) in binding to HER2 was determined using an ELISA method.

[0036] Figures 3A-3C The study demonstrated the affinity activity of candidate antibodies for binding to HER2 on the surface of tumor cells, as determined by the FACS method. Figure 3A , Figure 3B , Figure 3C The results of FACS detection on SK-BR-3 cells, BT-474 cells, and SK-OV-3 cells are shown respectively.

[0037] Figure 4A and Figure 4B The candidate antibody was shown to exhibit ADCC activity on SK-BR-3 cells.

[0038] Figure 5A and Figure 5B The candidate antibody was shown to have endocytic activity on SK-BR-3 cells. Detailed Implementation

[0039] The present invention is further illustrated below by way of examples, but these examples do not limit the invention to the scope of the embodiments described. Experimental methods not specifically described in the following examples were performed according to conventional methods and conditions, or as selected according to the product instructions. All reagents and raw materials used in this invention are commercially available.

[0040] The terms used in this invention are explained in detail below:

[0041] As used herein, the term “antibody” is used in the broadest sense, encompassing monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, diabody, triabody, and tetrabody, tandem di-scFv, tandem tri-scFv), conventional antibodies (tetrapeptide chain antibodies consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds), as well as Fab, Fab', F(ab')2, Fv, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptide antibodies, and domain antibodies (heavy chain (VH) antibodies, light chain (VL) antibodies).

[0042] The term "Fc" region refers to two heavy chain segments containing the CH2 and CH3 domains of an antibody, held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain. Various Fc constant region variants have been disclosed in the prior art, such as Fc regions of antibody heavy chain constant regions having substitutions of one or more amino acids at 238, 265, 269, 270, 297, 327, and 329 (using the EU numbering system) (US Patent No. 6,737,056), or Fc regions of antibody heavy chain constant regions having substitutions of one or more amino acids at 234, 235, 265, and 329 (using the EU numbering system). Alternatively, the Fc region of the antibody's heavy chain constant region may have one or more amino acid substitutions at positions 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (using the EU numbering system) (see U.S. Patent No. 7,371,826). These mutations have been shown to endow antibodies with new properties without altering the function of the antibody's variable region.

[0043] As used herein, the term "HER2" (also known as ErbB-2, HER-2, and CD340) refers to human epidermal growth factor receptor 2 (SwissProt P04626), including any variant, isotype, and species homolog of HER2 expressed naturally on cells (including tumor cells) or on cells transfected with the HER2 gene. Species homologs include rhesus macaque HER2 (rhesus macaque; GenBank accession number GI:109114897).

[0044] As used herein, the terms "VHH" and "nanobody" have the same meaning and are used interchangeably. A VHH is a small, stable, and highly efficient antigen-recognizing unit formed by a single heavy chain variable domain, consisting of only one chain from the C-terminus to the N-terminus: FR4-CDR3-FR3-CDR2-FR2-CDR1-FR1. VHHs specifically bind to epitopes without requiring other antigen-binding domains for recognition (unlike conventional tetrapeptide chain antibodies, where the epitope is recognized by the structure pair formed by VL and VH). Nanobodies possess excellent biological properties, with a molecular weight of 12-15 kDa, one-tenth the size of a complete antibody. They exhibit excellent tissue penetration, high specificity, and good water solubility. Due to their unique structural properties, they combine the advantages of traditional antibodies and small molecule drugs, almost overcoming the shortcomings of traditional antibodies such as long development cycles, low stability, and demanding storage conditions. They are gradually becoming an emerging force in next-generation antibody therapy, showing broad application prospects in immunodiagnosis and treatment. VHHs include, but are not limited to, natural antibodies produced by camelids, or antibodies produced by camelids that have been humanized, or those obtained through phage display technology. Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in, for example, the following literature: R. van der Linden et al., Journal of Immunological Methods, 240(2000)185-195; Li et al., J Biol Chem., 287(2012)13713-13721; Deffar et al., African Journal of Biotechnology Vol.8(12), pp.2645-2652, June 17, 2009 and WO94 / 04678.

[0045] The term "complementarity-determining region" or "CDR region" or "CDR" refers to a region within the variable domain of an antibody that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes and are sequentially numbered from the N-terminus as CDR1, CDR2, and CDR3. In a given heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of known antibody CDR assignment systems or combinations thereof, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (http: / / imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0046] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing manner.

[0047] The CDR can also be determined based on having the same AbM numbering position as a reference CDR sequence (e.g., any of the CDR sequences in the examples of this invention). In one embodiment, the CDR position of the single-domain antibody of this invention is determined according to the AbM numbering scheme.

[0048] Unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region and CDR (including heavy chain variable region residues), it means the numbering position according to the AbM numbering system.

[0049] Antibodies with different specificities (i.e., different binding sites against different antigens) have different core binding receptors (CDRs). However, although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR are directly involved in antigen binding. Minimal overlapping regions can be determined using at least two of the Kabat, Chothia, AbM, IMGT, and Contact methods, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining CDR sequence can be determined by the antibody’s structure and protein folding. Therefore, the present invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat, Chothia, AbM, IMGT, or Contact may be substituted with conserved amino acid residues.

[0050] As used herein, the term "percentage (%) sequence identity" or "sequence identity" has a generally accepted definition in the art, referring to the percentage of identical amino acid sequences between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a known algorithm). This can be determined using methods known to those skilled in the art, such as publicly available computer software like BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0051] In this article, amino acid sequences “derived from” or “consistent with” the reference amino acid sequence are partially or entirely identical or homologous to the reference amino acid sequence.

[0052] As used herein, the term "nucleic acid" refers to a nucleotide chain of any length and includes both DNA and RNA. A nucleotide can be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or its analogues, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0053] As used herein, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, permits the expression of the mRNA, protein, polypeptide, or peptide by the host cell. The vectors of the present invention are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the present invention may contain any type of nucleotide, including but not limited to DNA and RNA that may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vector may contain naturally occurring or non-naturally occurring nucleotide linkages, or both. In an exemplary aspect, modified nucleotides or non-naturally occurring nucleotide linkages do not impede transcription or replication of the vector.

[0054] The recombinant expression vector of the present invention can be any suitable recombinant expression vector capable of being used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably to express the genes or sequences in the host cell. Suitable vectors include those designed for amplification and expansion or for expression or both of the above, and examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0055] As used herein, the term "host cell" refers to any type of cell that may contain the nucleic acids or vectors described herein. Host cells may be eukaryotic cells, such as plants, animals, fungi, or algae; or host cells may be prokaryotic cells, such as bacteria or protozoa. As described herein, host cells may be cells originating from or obtained from an individual. Host cells may be derived from or obtained from mammals. As used herein, the term "mammal" means any mammal, including but not limited to rodents such as mice and hamsters; and lagomorphs such as rabbits. Preferably, the mammal is from the order Carnivora, including felines (cats) and canines (dogs). More preferably, the mammal is from the order Artiodactyla, including bovines (cattle) and suidae (pigs), or belongs to the order Perssodactyla, including equines (horses). Most preferably, the mammal belongs to the order Primate, Cebooids, or Simoids (monkeys) or the suborder Anthropoids (humans and apes). Humans are particularly preferred.

[0056] Expression vectors can be transfected or introduced into suitable host cells. Various techniques can achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene editing (CRISPR-Cas system, ZFN system, or TALEN system), transposons (Sleeping Beauty or PiggyBAC), gene guns, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, cells are cultured in a medium and screened for suitable activity. The methods and conditions used to culture the resulting transfected cells and to recover the generated antibody molecules are known to those skilled in the art and can be varied or optimized based on methods known in this specification and the prior art, depending on the specific expression vector used and the mammalian host cells. Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. Markers can, for example, provide protrophic, biocidal (e.g., antibiotic) or heavy metal (e.g., copper) resistance to auxotrophic hosts. Selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cells via co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0057] As used herein, the term "multispecific antibody" refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. A multispecific antibody is an antibody that has binding specificity to at least two different antigenic epitopes. In one embodiment, such a multispecific antibody, having binding specificity against a first antigen and a second antigen, is also provided herein, also referred to as a "bispecific antibody".

[0058] As used herein, the term "pharmaceutically acceptable carrier" refers to any of those carriers commonly used and is limited only by physicochemical considerations (such as solubility and lack of reactivity with antigen-binding molecules targeting HER2) and by route of administration. Pharmaceutically acceptable carriers described herein, such as mediators, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public. In one aspect, a pharmaceutically acceptable carrier is a carrier that is chemically inert to the active ingredient of a pharmaceutical composition and does not have adverse side effects or toxicity under the conditions of use. In some embodiments, the carrier does not produce adverse, allergic, or other inappropriate reactions when administered to animals or humans. In some aspects, the pharmaceutical composition is free of pyrogens and other impurities that could be harmful to humans or animals. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonics, and absorption delay agents, etc., the uses of which are well known in the art.

[0059] Therapeutic formulations, such as peptides, polynucleotides, or antibodies, suitable for carrying out the methods disclosed herein, can be prepared for storage by mixing a selected composition of desired purity with an optional physiologically and pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized cake or aqueous solution (Remington's Pharmaceutical Sciences, 18th ed., ARGennaro, Mack Publishing Company (1990)). Pharmaceutical compositions can be manufactured by incorporating one or more suitable carriers or adjuvants, such as water, mineral oil, polyethylene glycol, starch, talc, lactose, thickeners, stabilizers, suspending agents, etc. Such compositions can be in the form of solutions, suspensions, tablets, capsules, creams, ointments, ointments, or other conventional forms.

[0060] Compositions intended for internal administration should be sterile. This is readily achieved by filtration through a sterile filter membrane before or after lyophilization and reconstitution. Therapeutic compositions are generally housed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be punctured by a subcutaneous needle. Pharmaceutical forms suitable for injectable applications include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In some cases, the form should be sterile and fluid to facilitate injection. It should be stable under the conditions of manufacture and storage and should be protected from contamination by microorganisms such as bacteria and fungi. Compositions intended for parenteral administration are typically stored in lyophilized or solution form.

[0061] The carrier can be a solvent or dispersion medium containing, for example, water or a suitable mixture thereof, and vegetable oil. Appropriate flowability can be maintained, for example, by using a coating such as lecithin, or, in the case of a dispersion, by maintaining the desired particle size, and by using a surfactant.

[0062] The choice of carrier can be determined by the specific type of pharmaceutical composition targeting the HER2 antigen-binding molecule and the route of administration of the pharmaceutical composition. Accordingly, there are various formulations of suitable pharmaceutical compositions.

[0063] The pharmaceutical compositions of the present invention may contain any pharmaceutically acceptable ingredients, including, for example, acidifiers, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anti-caking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, matrices, binders, buffers, chelating agents, coating agents, colorants, desiccants, detergents, diluents, disinfectants, disinfectants, disintegrants, dispersants, solubilizers, dyes, emollients, emulsifiers, emulsion stabilizers, fillers, film-forming agents, flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, heat-insulating agents, lubricants, mucosal adhesives, ointment matrices, ointments, oily mediators, organic bases, lozenge matrices, pigments, plasticizers, polishing agents, preservatives, multivalent chelating agents, skin penetrants, solubilizers, solvents, stabilizers, suppository matrices, and surfactants. Agents, surfactants, suspending agents, sweeteners, therapeutic agents, thickeners, tension agents, toxic agents, viscous agents, water absorbents, water-miscible cosolvents, water softeners, or wetting agents.

[0064] The pharmaceutical compositions targeting HER2 antigen-binding molecules described herein are formulated for parenteral, subcutaneous, intravenous, intramuscular, intra-arterial, intrathecal, or intraperitoneal administration. The pharmaceutical compositions can be administered via nasal, spray, oral, aerosol, rectal, or vaginal administration. The compositions can also be administered via infusion, rapid injection, or through an implantable device.

[0065] Those skilled in the art will understand that, in addition to the pharmaceutical compositions described above, the compositions of the present invention can be formulated into inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.

[0066] As used herein, the term "monoclonal antibody" refers to an antibody derived from a single cloned cell line, which is not limited to eukaryotic, prokaryotic, or bacteriophage cloned cell lines.

[0067] As used herein, the term "epitope" refers to a region on an antigen that is capable of specifically binding to an antibody. Epitopes can be formed from a continuous string of amino acids (linear epitopes) or contain discontinuous amino acids (conformal epitopes), for example, due to the folding of the antigen (i.e., tertiary folding of the antigen as a protein). The difference between conformational and linear epitopes is that antibody binding to a conformational epitope is lost in the presence of a denaturing solvent. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463).

[0068] As used herein, the term "specific binding" refers to an antibody binding to an antigen or an epitope within that antigen with a higher affinity than it would to other antigens or epitopes. Typically, antibodies bind with an affinity of approximately 1 × 10⁻⁶. -7 M or smaller (e.g., about 1×10⁻⁶) -8 M or smaller, approximately 1×10 -9 M or smaller, approximately 1×10 -10 M or smaller, approximately 1×10 -11 M or smaller, or about 1×10 -12 The equilibrium dissociation constant (K) of M or less D The antibody binds to the antigen or an epitope within the antigen. In some embodiments, the antibody binds to the K+ of the antigen. D This antibody binds to the K+ of non-specific antigens (such as BSA, casein). D 10%, or 1%. K can be measured using standard procedures. D For example, through Surface plasmon resonance assays are used to measure this. However, antibodies that specifically bind to an antigen or an epitope within an antigen may be cross-reactive to other related antigens, for example, to the same antigens from other species (homologous) (such as humans or monkeys, such as cynomolgus (cyno) and chimpanzee (chimp)) or Callithrix jacchus (commonmarmoset (marmoset)).

[0069] As used herein, the term "HER2 antigen-binding molecule" (including antibodies) refers to a molecule that specifically binds to the antigen HER2.

[0070] Example 1: Raw material preparation

[0071] 1.1 Antigen Preparation

[0072] In this embodiment, human epidermal growth factor receptor 2 (HER2) antigen proteins with His and Fc tags were prepared, hereinafter referred to as HER2-His and HER2-Fc, respectively. The HER2 (UniProt NO: P04626) gene (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number: HG10004-ACG) was used to amplify the target fragment HER2 extracellular domain (ECD, 23-652AA, SEQ ID NO: 1) by PCR. The C-terminus of the ECD region sequence of the HER2 gene was ligated with either a His tag or a human Fc tag (Uniprot No: P0DOX5, 218-449AA) sequence. The resulting fusion gene containing the tag sequence was then constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen) by homologous recombination. The constructed recombinant protein expression vector plasmids were transformed into Escherichia coli DH5α and cultured overnight at 37°C. Then, the plasmids were extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids for eukaryotic expression.

[0073] Both HER2-His and HER2-Fc were expressed using the Expi293 transient transduction expression system (ThermoFisher, A14635). For the transient transduction method, please refer to Expi293. TMExpression System USER GUIDE. Seven days after transfection, the cell expression supernatant was centrifuged at 15000g for 10 min. The resulting Fc-tagged protein expression supernatant was affinity purified using a MabSelectSuRe LX (GE, 17547403), followed by elution with 100mM sodium acetate (pH 3.0) and neutralization with 1M Tris-HCl. The resulting His-tagged protein expression supernatant was affinity purified using a NiSmart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), followed by elution with imidazole buffers of varying concentrations. The eluted proteins were then transferred to PBS buffer via ultrafiltration concentrators (Millipore, UFC901096). After passing SDS-PAGE and activity tests, the proteins were stored at -80℃ for later use.

[0074] 1.2 Preparation of control antibody

[0075] In this embodiment, the control antibodies used are anti-HER2 trastuzumab (hereinafter referred to as Trmab) and pertuzumab (hereinafter referred to as Pemab), with sequences from patent applications WO1992022653A1 and WO2001000245A2, respectively.

[0076] The DNA sequences of the antibody heavy and light chains were synthesized by Genewiz Biotechnology Co., Ltd. Each target fragment was amplified by PCR and then constructed into the eukaryotic expression vector pcDNA3.4 (Invitrogen) via homologous recombination. The constructed recombinant protein expression vector was transformed into *E. coli* DH5α and cultured overnight at 37°C. The plasmid was then extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids for eukaryotic expression. Expression was performed using the ExpiCHO transient transduction system (Thermo Fisher, A29133) (method described in WO2020238730A1). The cell suspension was centrifuged at high speed, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter and purified by affinity chromatography using a MabSelect SuRe LX (GE, 17547403). The target protein was eluted with 100mM glycine hydrochloric acid (pH 3.0), concentrated, replaced with buffer, aliquoted, identified by SDS-PAGE and its activity was tested, and then stored frozen.

[0077] Example 2: Detection of HER2 antigen protein in alpacas and its serum titer

[0078] 2.1 Animal Immunization

[0079] Immunization was administered subcutaneously to one alpaca, NSY011 (Nanchang Dajia Technology Co., Ltd.), using the HER2-Fc immunogen. The single immunization dose was 500 μg, supplemented with CFA / IFA (French complete adjuvant and Freund's incomplete adjuvant), with immunizations every two weeks for a total of four doses.

[0080] 2.2 Serum titer detection

[0081] After the second, third, and fourth immunizations, serum titers targeting HER2 were measured, with pre-immunization serum serving as a negative control. The specific detection method was as follows: HER2-His recombinant protein was diluted with PBS to a final concentration of 2 μg / mL. 30 μL of the diluent was added to an ELISA plate and incubated overnight at 4°C. On the day of titer assay, the plate was washed three times with PBST, then blocked with PBST containing 5% skim milk at room temperature for 2 hours, followed by three more washes with PBST. On another dilution plate, unimmunized negative serum and post-immunization serum were diluted with PBS, with the first well being a 2000-fold dilution, followed by 2-fold serial dilutions in the subsequent seven wells. The diluted serum was added to the first ELISA plate and incubated at room temperature for 1 hour. After washing three times with PBST, the secondary antibody Anti IgG-HRP (Millipore, MAC129) was added at a dilution of 1:10000 and incubated at room temperature for 0.5 hours. After incubation, wash the plate six times with PBST, add TMB (SurModics, TMBS-1000-01) for color development, and add 2M HCl to terminate the reaction according to the color development results. Read the plate at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

[0082] The results are shown in Table 1. At the end of the fourth immunization, the titer of the HER2-His antibody in the alpaca serum reached 1:256K or higher (when the detected value is 1.65 times higher than the background value, it is judged as positive).

[0083] Table 1: Detection of HER2-immunized alpaca serum IgG titer (OD450)

[0084] Sample / Dilution Ratio negative serum Second immune serum 3 immune serum 4 immune serum 1:2K 0.1284 2.3438 2.8244 2.8412 1:4K 0.1262 1.8275 2.6672 2.7957 1:8K 0.0722 1.2533 2.1639 2.6516 1:16K 0.0984 0.8655 1.4027 2.1032 1:32K 0.1031 0.4809 0.8567 1.3408 1:64K 0.0702 0.2862 0.4695 0.774 1:128K 0.0693 0.1814 0.3042 0.4697 1:256K 0.0938 0.1507 0.1731 0.2701

[0085] Example 3: Phage display library construction and HER2-targeting nanobody screening

[0086] In this embodiment, the nanobody gene of alpaca peripheral blood B cells immunized with HER2-Fc was cloned, a nanobody gene phage display library was constructed, and the library was screened using HER2-Fc and HER2-His (self-made in Example 1) as screening antigens to obtain multiple nanobodies that specifically bind to HER2.

[0087] 3.1 Construction of a phage display library for camel-derived nanobodies

[0088] Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque density gradient separation buffer (GE, catalog number: 17144003S), and total RNA was extracted from the isolated PBMCs. The extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, catalog number: 6210A). Based on the VHH antibody germline, degenerate primers were designed at the front end of the V region and the middle of the second constant region (CH2) of the VHH antibody. PCR amplification yielded the VHH-CH2 and VH-CH1-CH2 fragments of the antibody. The PCR products were identified by agarose gel electrophoresis based on the length difference between the two fragments, and the VHH-CH2 fragment was recovered. The recovered VHH-CH2 fragment was amplified using a secondary PCR method with forward and reverse primers for VHH amplification and VHH-CH2 as a template to amplify the VHH antibody fragment (Sabir JS, El-Domyati FM et al. CR Biol. 20 March 2014; 337(4): 244-249). Next, the PCR product and the phage display vector were digested, recovered, and ligated. The ligation product was recovered using a recovery kit (Omega, catalog number: D6492-02). Finally, the transformed E. coli SS320 (Lucigen, MC1061 F) was transformed into competent E. coli SS320 using an electroporator (Bio-Rad, MicroPulser), and the transformed E. coli SS320 culture was plated on ampicillin-resistant 2-YT solid plates. The library size was determined to be 10^6 cells / mL by serial dilution. 8 Level. The bacterial culture of the nanobody gene library was added to fresh 2-YT liquid medium and cultured in a shaker at 37°C and 220 rpm until the logarithmic growth phase. Then, VSCM13 helper phage (purchased from Stratagene) was added at a quantity 50 times that of the bacterial count (i.e., the multiple of infection (MOI) was approximately 50) to finally obtain the phage display library of camel-derived nanobodies.

[0089] 3.2 Screening of antibody gene phage display libraries

[0090] 3.2.1 Screening of antibody gene phage display libraries using magnetic bead method

[0091] Magnetic bead screening is based on biotin labeling of antigen proteins (HER2-Fc and HER2-His) and then binding them to magnetic beads conjugated with streptavidin. The screening process involves incubating, washing, and eluting the magnetic beads bound to the antigen and the phage display library containing the antibody gene. This process can enrich a large number of specific monoclonal antibodies against the antigen.

[0092] The specific method is as follows: First, biotin-labeled HER2-Fc protein was incubated with streptavidin-conjugated magnetic beads to allow the biotin-labeled HER2-Fc protein to bind to the magnetic beads. The magnetic beads bound to HER2-Fc protein and the constructed phage library were incubated at room temperature for 2 hours. After washing 6-8 times with PBST to remove non-specifically adsorbed phages, Trypsin (Gibco, 25200072) was added, gently mixed, and reacted for 20 minutes to elute specifically bound antibodies and display the phages. Subsequently, the eluted phages were used to infect SS320 cells in the logarithmic growth phase and incubated for 30 minutes, then cultured at 220 rpm for 1 hour. VSCM13 helper phage was then added and incubated for 30 minutes, followed by another 1 hour of culture at 220 rpm. The cells were then centrifuged and transferred to C1. + / K + In 2-YT medium, the resulting bacteriophages were used in the next round of panning. The second and third rounds of panning were performed using biotin-labeled HER2-His and HER2-Fc assays, respectively, to achieve cross-selection and remove antibodies binding to the Fc fragment of the antigen as much as possible.

[0093] 3.2.2 Screening of antibody gene phage display libraries using the immunotube method

[0094] Both the immunotube method and the magnetic bead method aim to enrich specific antibodies against antigens, and are two complementary and mutually reinforcing experimental methods.

[0095] The principle of immunotube screening is to coat the HER2 protein onto the surface of an immunotube with high adsorption capacity. By adding a phage display antibody library into the immunotube and incubating, washing, and eluting the antigen protein adsorbed on the surface of the immunotube, a panning process is carried out, and finally, specific monoclonal antibodies against the antigen are enriched.

[0096] The specific method is as follows: In the first round of screening, 1 mL of 30 μg / mL HER2-Fc was added to the immunotube and coated overnight at 4°C. The coating solution was discarded the next day, and 5% milk PBS was added for blocking for 2 hours. After washing twice with PBS, a phage library containing anti-HER2-Fc nanobody was added and incubated for 2 hours. After washing to remove non-specifically bound phages, 0.8 mL of 0.05% EDTA trypsin digestion solution was added to the immunotube to wash away phages that specifically bind to the target antigen. Then, it was used to infect SS320 cells in the logarithmic growth phase, incubated at 37°C for 30 minutes, and then cultured at 220 rpm for 1 hour. VSCM13 helper phage was added, incubated for 30 minutes, and then cultured at 220 rpm for another 1 hour. After centrifugation, the culture was transferred to C64. + / K + In 2-YT medium, the phages were cultured overnight at 30°C and 220 rpm. The next day, phages were precipitated for subsequent rounds of screening. The second and third rounds of screening used biotin-labeled HER2-His and HER-Fc assays, respectively, to achieve cross-selection and remove antibodies binding to the Fc fragment of the antigen as much as possible.

[0097] 3.3 Selection of Monoclonal Cells

[0098] The enrichment effect was evaluated by ELISA detection of the phage pools eluted in each round. For the third round with better enrichment, a large number of monoclonal antibodies were selected for ELISA screening. After sequencing analysis and ELISA screening, multiple nanobodies with sequence diversity and binding to HER2 antigen were obtained.

[0099] Candidate nanobodies were named using clone numbers, and the complementarity-determining region (CDR) sequence of the heavy chain single-domain antibody was determined using the AbM definition method. The CDR and variable region (VHH) sequences of the four exemplary anti-HER2 nanobodies of this invention are shown in Tables 2 and 3.

[0100] Table 2: CDR sequences of exemplary anti-HER2 nanobodies of the present invention

[0101]

[0102] Table 3: VHH sequence of exemplary anti-HER2 nanobody of the present invention

[0103]

[0104]

[0105] Note: The bolded and italicized sequences in the table are CDR sequences; the bolded and underlined sequences are amino acids that are different from NB46-1.

[0106] Example 4: Construction, expression, and purification of candidate antibodies

[0107] The four nanobodies obtained in Example 3 were used to construct human IgG1 subtypes to form antibodies in the form of VHH-Fc, wherein the amino acid sequence of the Fc region is shown below.

[0108] From the selected candidate monoclonal strains, the antibody heavy chain variable region fragment, VHH, was obtained by PCR amplification. Using homologous recombination, this fragment was constructed into a modified eukaryotic expression vector plasmid pcDNA3.4-TOPO (Invitrogen) containing a human IgG1 Fc fragment, forming the complete VHH-Fc full-length gene. The expression and purification methods were consistent with those in Example 1.2.

[0109] Fc region sequence:

[0110]

[0111] Example 5: Identification of the physicochemical properties of candidate antibodies

[0112] 5.1 SDS-PAGE identification of candidate antibody (VHH-Fc)

[0113] Preparation of non-reducing solution: 1 μg of the candidate antibody and the quality control IPI (IPI is an abbreviation for ipilimumab, prepared according to the method in Example 4) were added to 5×SDS loading buffer and 40 mM iodoacetamide. The mixture was heated in a dry bath at 75°C for 10 min, cooled to room temperature, and centrifuged at 12000 rpm for 5 min. The supernatant was collected. Preparation of reducing solution: 2 μg of the candidate antibody and the quality control IPI were added to 5×SDS loading buffer and 5 mM DTT. The mixture was heated in a dry bath at 100°C for 10 min, cooled to room temperature, and centrifuged at 12000 rpm for 5 min. The supernatant was collected. Add the supernatant to a Bis-tris 4-15% gradient gel (purchased from GenScript) and perform electrophoresis at a constant voltage of 110V. When Coomassie Brilliant Blue migrates to the bottom of the gel, stop the electrophoresis, remove the gel slide and place it in Coomassie Brilliant Blue staining solution for 1-2 hours. Discard the staining solution, add destaining solution, and change the destaining solution 2-3 times as needed. Destain until the gel background is transparent, then store in deionized water. After destaining, scan with an EPSON V550 color scanner, and calculate the purity of reduced and non-reduced bands using ImageJ according to the peak area normalization method.

[0114] The results showed that the bands of the candidate antibody and the quality control IPI non-reducing gel were around 80kD and 150kD, respectively, while the band of the candidate antibody on the reducing gel was around 40kD. The quality control IPI bands were around 55kD and 25kD, respectively, which were in line with the expected size, and the purity of all bands was greater than 90%.

[0115] 5.2 SEC-HPLC purity identification of candidate antibody (VHH-Fc)

[0116] Materials preparation: (1) Mobile phase: 150 mmol / L phosphate buffer, pH 7.4; (2) Sample preparation: Candidate antibody and quality control IPI were diluted to 0.5 mg / mL with the mobile phase solution. The flow rate of the Agilent HPLC 1100 column (XBridge BEHSEC 3.5 μm, 7.8 mm ID×30 cm, Waters) was set to 0.8 mL / min, the injection volume was 20 μL, and the VWD detector wavelengths were 280 nm and 214 nm. Blank solution, IPI quality control solution and sample solution were injected sequentially.

[0117] The SEC-HPLC results of the candidate antibody (VHH-Fc) are shown in Table 4: the percentages of high molecular weight polymers, antibody monomers and low molecular weight substances in the sample were calculated by the area normalization method, and the purity of the candidate antibody monomers was 100%.

[0118] Table 4: Physicochemical data of candidate antibodies

[0119]

[0120] Example 6: Determination of affinity and competitive activity of candidate antibodies using ELISA method

[0121] 6.1 Determination of Affinity Activity of Candidate Antibodies Based on ELISA Method

[0122] HER2-His (2 μg / mL, 30 μL / well) was coated onto 96-well ELISA plates and incubated overnight at 4°C. The next day, the plates were washed three times with PBST, blocked with 5% skim milk for 2 hours, washed three times with PBST, and then serially diluted candidate antibodies were added and incubated for 1 hour. Afterward, the plates were washed three times with PBST, and anti-Fc secondary antibody (Jackson Immuno Research, 109-035-008) was added and incubated for 1 hour. After incubation, the plates were washed six times with PBST and then developed with TMB (SurModics, TMBS-1000-01). Based on the colorimetric results, the reaction was terminated by adding 2M HCl, and the plates were read at OD450 using a microplate reader (Molecular Devices, SpecterMax 190).

[0123] The results show Figure 1 As shown in Table 5, the results indicate that, except for NB46-73, which has slightly weaker affinity activity than trastuzumab (Trmab), the other three antibodies, NB46-P-165, NB46-88, and NB46-1, all showed slightly better ELISA-based affinity activity than Trmab.

[0124] Table 5: Affinity activity of candidate antibodies determined by ELISA method

[0125] Antibody name <![CDATA[EC 50 (μg / mL)]]> NB46-1 0.1240 NB46-73 0.2211 NB46-88 0.08929 NB46-P-165 0.1183 Trmab 0.1816

[0126] 6.2 Determination of HER2 competitive binding activity between candidate antibodies and Trmab using ELISA method

[0127] Human HER2-His (4 μg / mL, 30 μL / well) was coated onto the plates and incubated overnight at 4°C. The next day, the plates were washed three times with PBST and blocked with 5% skim milk for 2 h. Then, serially diluted candidate antibodies or Trmab were mixed with 1 μg / mL biolabeled Trmab and added to 96-well ELISA plates after blocking and washing, and incubated for 1 h. After washing three times with PBST, the secondary antibody NeutrAvidin-HRP (Therofisher, 31001) was added and incubated for 1 h. After incubation, the plates were washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added for color development. Based on the color development results, 2M HCl was added to stop the reaction, and the plates were read at OD450 using a microplate reader (Molecular Devices, SpecterMax190).

[0128] The results show Figure 2 As shown in Table 6, the results indicate that NB46-P-165, NB46-88, NB46-73, and NB46-1 competitively bind to the HER2 antigen with the control antibody Trmab, exhibiting similar binding epitopes. Among them, NB46-1 and NB46-P-165 showed better competitive HER2 binding activity against Trmab.

[0129] Table 6: ELISA method for determining the competitive binding activity of candidate antibodies to Trmab for HER2.

[0130] Antibody name <![CDATA[IC 50 (μg / mL)]]> NB46-1 1.417 NB46-73 4.494 NB46-88 2.127 NB46-P-165 1.687 Trmab 2.111

[0131] Example 7: Determination of antibody binding to HER2 antigen on tumor cell surface using FACS method

[0132] In this embodiment, the binding activity of the candidate antibody and the Trmab control to the HER2 antigen on the surface of tumor cells SK-OV-3, BT-474 and SK-BR-3 was detected using the FACS method.

[0133] Human SK-OV-3, BT-474, and SK-BR-3 tumor cells in the exponential growth phase were collected, centrifuged at 300g to remove the supernatant, resuspended in prepared FACS buffer, counted, and the cell suspension density was adjusted to 2 × 10⁻⁶. 6Cells were added at a rate of 100 μL / mL to each well of a 96-well round-bottom plate and centrifuged at 300 g to remove the supernatant. Serially diluted candidate nanobody and positive control Trmab dilutions were added to the corresponding wells, cells were resuspended, and incubated at 4°C for 30 min. The incubated cell mixture was centrifuged at 300 g to remove the supernatant, and 200 μL of FACS buffer was added to the corresponding well to resuspend the cells. This process was repeated twice, centrifuging at 300 g to remove the supernatant. FITC-labeled anti-human-IgG-Fc flow cytometry antibody (Jackson, 109-095-008) was added, cells were resuspended, and incubated at 4°C for 30 min. The cells were then centrifuged at 300 g to remove the supernatant. FACS buffer was added and the cells were resuspended. This centrifugation and resuscitation process was repeated twice, and 200 μL of FACS buffer was added to each well to resuspend the cells. Finally, the cells were analyzed using a flow cytometer (Beckman, CytoFLEX AOO-1-1102).

[0134] The results show Figures 3A-3C As shown in Table 7, under the same experimental conditions and antibody concentrations, the candidate antibodies NB46-P-165, NB46-88, NB46-73, and NB46-1 exhibited comparable HER2 binding activity to tumor cell surfaces to the control antibody Trmab.

[0135] Table 7: FACS method for determining the competitive binding activity of candidate antibodies to Trmab for HER2

[0136]

[0137] Example 8: Antibody-dependent cell-mediated cytotoxicity (ADCC)

[0138] The ADCC effect was detected using the lactate dehydrogenase (LDH) release assay. The principle is as follows: the variable region of an antibody binds to the target antigen on the target cell. When the Fc fragment of the antibody binds to FcRIIIa (also known as CD16a) on NK effector cells in PBMCs, the NK cells release perforin, granzyme, etc., to lyse the target cells. The release of lactate dehydrogenase in the cell supernatant can then be detected using an LDH lactate dehydrogenase kit (Takara, MK401), thereby determining the degree of NK cell killing of the target cells. The specific procedure is as follows:

[0139] Add 50 μL of a 1×10⁻⁶ solution to each well of a 96-well cell culture plate. 5 SK-BR-3 cells were cultured at 37°C overnight (16-20 h) at a concentration of 10 cells / mL. Serially diluted candidate antibodies NB46-1 and NB46-P-165 and control antibody Trmab were added at 50 μL / well, mixed well, and incubated at 37°C for 30 min. Then, 4 × 10⁴ cells of revived SK-BR-3 were added.5 Human PBMCs (effect cell / target cell ratio of 40:1) were incubated at 37°C for 16 hours. The supernatant was then obtained by centrifugation at 300g, followed by the addition of LDH detection reagent and incubation for 60 minutes. Finally, the OD value at 492nm was measured using a microplate reader (Molecular Devices, SpectraMax 190) for analysis. 10% Triton X-100-treated target cells served as a complete lysis control, target cells alone served as a blank negative control, and PBMCs treated with target cells served as a background negative control. Cell killing rate was calculated using the following formula: Killing rate (%) = (OD value of candidate antibody well - OD value of background well) / (OD value of completely lysed well - OD value of blank well) × 100%.

[0140] ADCC results compared with the positive control monoclonal antibody are as follows Figure 4A and Figure 4B As shown, the candidate antibodies NB46-1 and NB46-P-165 exhibit comparable cytotoxic activity to Trmab. Among them, Figure 4A The study showed that NB46-1 and Trmab induced ADCC cell killing of EC1 cells. 50 The concentrations were 0.023 μg / mL and 0.011 μg / mL, respectively. Figure 4B The study showed that NB46-P-165 and Trmab induced ADCC cell killing of EC1 cells. 50 The concentrations were 0.024 μg / mL and 0.013 μg / mL, respectively.

[0141] Example 9: Antibody endocytosis efficiency detection

[0142] This experiment assesses antibody endocytic activity by detecting the cytotoxicity of Fab-ZAP endocytosis mediated by the antibody. Fab-ZAP is a Fab fragment linked to saporin, a ribosome inhibitor that inhibits protein synthesis, leading to cell death. The Fab-ZAP used in this experiment is a Fab fragment capable of binding to the Fc of a human antibody. Incubation of the Fab-ZAP with the antibody carrying the human Fc imbues the antibody with a toxin. When the antibody is endocytosed, the toxin enters the cell along with the antibody, causing cell death. Cell viability is then detected using an MTS (Promega, G3580) assay to determine whether the human antibody has undergone endocytosis. The control antibody in this example is pertuzumab, due to its superior endocytic activity compared to Trmab.

[0143] The specific experimental method is as follows: First, Fab-ZAP was diluted to 4.5 nM with DMEM complete medium. Then, candidate antibodies NB46-1, NB46-88, and control antibody pertuzumab were serially diluted with 4.5 nM Fab-ZAP. Logarithmic growth phase SK-BR-3 cells were prepared into single-cell suspensions and the density was adjusted to 1 × 10⁻⁶ cells / cells. 5 At a concentration of 1,000 cells / mL, 50 μL was seeded into each well of a 96-well plate. Then, 50 μL of the previously prepared antibody dilution was added to each well of the cell culture plate, and the mixture was thoroughly pipetted and mixed. The cell culture plate was incubated at 37°C for 72 h. After incubation, 7.5 μL of Triton X-100 solution was added to each well, and the mixture was gently tugged and mixed. The cell culture plate was then incubated at 37°C for 0.5 h. Next, 20 μL of MTS was added to each well, and the plate was incubated at 37°C for 1–4 h. Finally, the cell culture plate was centrifuged at 1000 rpm for 5 min, and the data were read using a microplate reader at OD492.

[0144] The results are as follows Figure 5A and Figure 5B As shown, under the antibody concentration conditions defined in this embodiment, the candidate antibody exhibits superior endocytic activity in SK-BR-3 tumor cells compared to the control antibody pertuzumab. 50 The values ​​were Pemab vs. NB46-1 = 0.0145 μg / mL vs. 0.0006 μg / mL, and Pemab vs. NB46-88 = 0.0152 μg / mL vs. 0.0043 μg / mL. SEQUENCE LISTING <110> Sanyou Biopharmaceutical (Shanghai) Co., Ltd. <120> A HER2 antigen-binding molecule and its application <130> P22012419C <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 630 <212> PRT <213> Artificial Sequence <220> <223> HER2 extracellular domain (23-652AA) <400> 1 Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser 1 5 10 15 Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln 20 25 30 Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser 35 40 45 Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile 50 55 60 Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val 65 70 75 80 Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp 85 90 95 Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro 100 105 110 Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys 115 120 125 Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr 130 135 140 Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr 145 150 155 160 Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met 165 170 175 Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser 180 185 190 Leu Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro 195 200 205 Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly 210 215 220 Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly 225 230 235 240 Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr 245 250 255 Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser 260 265 270 Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser 275 280 285 Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp 290 295 300 Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys 305 310 315 320 Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser 325 330 335 Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu 340 345 350 Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala 355 360 365 Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile 370 375 380 Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu 385 390 395 400 Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn 405 410 415 Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly 420 425 430 Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His 435 440 445 Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe 450 455 460 Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp 465 470 475 480 Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg Gly 485 490 495 His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe 500 505 510 Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly Leu 515 520 525 Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro Glu 530 535 540 Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp 545 550 555 560 Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala 565 570 575 Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp 580 585 590 Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn Cys 595 600 605 Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu Gln 610 615 620 Arg Ala Ser Pro Leu Thr 625 630 <210> 2 <211> 113 <212> PRT <213> Artificial Sequence(人工序列) <220> <223> VHH sequence of antibody NB46-1 <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Thr Tyr 20 25 30 Thr Met Thr Trp Tyr Arg Gln Val Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ala Thr Ile Ala Pro Gly Asp Val Thr Gly Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Asp Pro His Arg Arg Phe Gln Gly Gln Gly Thr Gln Val Thr Val Ser 100 105 110 Ser <210> 3 <211> 113 <212> PRT <213> Artificial Sequence(人工序列) <220> <223> VHH sequence of antibody NB46-73 <400> 3 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Thr Tyr 20 25 30 Thr Met Thr Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ala Thr Ile Ala Pro Gly Gly Val Thr Gly Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Asp Pro His Arg Val Tyr Gln Gly Gln Gly Thr Gln Val Thr Val Ser 100 105 110 Ser <210> 4 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> VHH sequence of antibody NB46-88 <400> 4 Glu Val Gln Val Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Thr Tyr 20 25 30 Ser Met Thr Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ala Thr Ile Ala Pro Gly Gly Val Thr Gly Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Asp Pro His Arg Val Tyr Gln Gly Gln Gly Thr Gln Val Thr Val Ser 100 105 110 Ser <210> 5 <211> 113 <212> PRT <213> Artificial Sequence(人工序列) <220> <223> VHH sequence of antibody NB46-P-165 <400> 5 Glu Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Thr Tyr 20 25 30 Ser Met Thr Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val 35 40 45 Ala Thr Ile Ala Pro Gly Asp Val Thr Gly Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Asp Pro His Arg Arg Tyr Gln Gly Gln Gly Thr Gln Val Thr Val Ser 100 105 110 Ser <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of antibodies NB46-1 and NB46-73 <400> 6 Gly Phe Thr Leu Ser Thr Tyr Thr Met Thr 1 5 10 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of antibodies NB46-88 and NB46-P-165 <400> 7 Gly Phe Thr Leu Ser Thr Tyr Ser Met Thr 1 5 10 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of antibodies NB46-1 and NB46-P-165 <400> 8 Thr Ile Ala Pro Gly Asp Val Thr Gly 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of antibodies NB46-73 and NB46-88 <400> 9 Thr Ile Ala Pro Gly Gly Val Thr Gly 1 5 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of antibody NB46-1 <400> 10 Pro His Arg Arg Phe 1 5 <210> 11 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of antibodies NB46-73 and NB46-88 <400> 11 Pro His Arg Val Tyr 1 5 <210> 12 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of antibody NB46-P-165 <400> 12 Pro His Arg Arg Tyr 1 5 <210> 13 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR1 upper sequence <220> <221> misc_feature <222> (8)..(8) <223> Xaa is X1, where X1 is T or S. <400> 13 Gly Phe Thr Leu Ser Thr Tyr Xaa Met Thr 1 5 10 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR2 upper sequence <220> <221> misc_feature <222> (6)..(6) <223> Xaa is X2, and X2 is D or G. <400> 14 Thr Ile Ala Pro Gly Xaa Val Thr Gly 1 5 <210> 15 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR3 upper sequence <220> <221> misc_feature <222> (4)..(4) <223> Xaa is X3, and X3 is R or V. <220> <221> misc_feature <222> (5)..(5) <223> Xaa is X4, and X4 is either F or Y. <400> 15 Pro His Arg Xaa Xaa 1 5 <210> 16 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Fc region sequence <400> 16 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230

Claims

1. An antibody targeting HER2, characterized in that, The antibody is VHH or VHH-Fc; the antibody contains at least one VHH chain, the VHH chain comprising CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR1 is as shown in SEQ ID NO:6, the amino acid sequence of CDR2 is as shown in SEQ ID NO:8, and the amino acid sequence of CDR3 is as shown in SEQ ID NO:10; or, The amino acid sequence of CDR1 is shown in SEQ ID NO:6, the amino acid sequence of CDR2 is shown in SEQ ID NO:9, and the amino acid sequence of CDR3 is shown in SEQ ID NO:11; or, The amino acid sequence of CDR1 is shown in SEQ ID NO:7, the amino acid sequence of CDR2 is shown in SEQ ID NO:9, and the amino acid sequence of CDR3 is shown in SEQ ID NO:11; or, The amino acid sequence of CDR1 is shown in SEQ ID NO:7, the amino acid sequence of CDR2 is shown in SEQ ID NO:8, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

12.

2. The antibody as described in claim 1, characterized in that, The amino acid sequence of the VHH chain is as shown in any of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or a variant thereof; The variant has at least 80% sequence identity compared to the sequence from which it originates.

3. The antibody as described in claim 1, characterized in that, The Fc region of VHH-Fc is the Fc region of human IgG1.

4. The antibody as described in claim 3, characterized in that, The amino acid sequence of the Fc region is shown in SEQ ID NO:

16.

5. An isolated nucleic acid, characterized in that, The nucleic acid encodes the antibody as described in any one of claims 1 to 4.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid as described in claim 5.

7. The recombinant expression vector as described in claim 6, characterized in that, The recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector.

8. The recombinant expression vector as described in claim 7, characterized in that, The backbone of the plasmid is pcDNA3.

4.

9. A host cell, characterized in that, The host cell contains the nucleic acid as described in claim 5 or the recombinant expression vector as described in any one of claims 6 to 8.

10. The host cell as described in claim 9, characterized in that, The host cell is a prokaryotic cell or a eukaryotic cell.

11. The host cell as described in claim 10, characterized in that, The eukaryotic cells are yeast cells or mammalian cells.

12. The host cell as described in claim 11, characterized in that, The mammalian cells in question are HEK293 cells.

13. A method for preparing an antibody, characterized in that, The method includes the following steps: The host cells are cultured under conditions suitable for host cell growth and fermentation as described in any one of claims 9 to 12, and antibodies are obtained from the culture.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody as described in any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14, characterized in that, The pharmaceutical composition further comprises one or more of the following groups: hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

16. The use of an antibody targeting HER2 in the preparation of a medicament for treating breast cancer, wherein the antibody is VHH or VHH-Fc, and the amino acid sequence of the VHH chain of the antibody is shown in SEQ ID NO:2 or SEQ ID NO:

5.

17. A reagent kit, characterized in that, The kit comprises the antibody as described in any one of claims 1 to 4, the nucleic acid as described in claim 5, the recombinant expression vector as described in any one of claims 6 to 8, the host cell as described in any one of claims 9 to 12, or the pharmaceutical composition as described in claim 14 or 15.

18. The kit according to claim 17, characterized in that, The kit also includes (i) a device for administering the antibody or pharmaceutical composition; and / or (ii) instructions for use.

19. A medicine box set, characterized in that, The medicine box set includes medicine box A and medicine box B, wherein... The medicine box A contains the antibody as described in any one of claims 1 to 4 or the pharmaceutical composition as described in claim 14 or 15; The kit B contains other anti-tumor antibodies or a pharmaceutical composition containing said other anti-tumor antibodies, and / or is one or more of the following groups: hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

20. A method for immunoassay or determination of HER2, characterized in that, The method includes mixing the antibody according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 14 or 15 with the sample to be tested; wherein the test is a non-diagnostic test.

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