Anti-HER2 antibodies and uses thereof

Abstract

The invention relates to an anti-HER2 antibody and application thereof, and particularly provides a series of anti-HER2 antibodies which are high in binding affinity with HER2, have cross reaction activity with human and cynomolgus monkey HER2, are good in thermal stability, and have an excellent anti-tumor effect in various tumors (such as neurocytoma, breast cancer, ovarian cancer, gastric cancer, lung cancer, kidney cancer, intestinal cancer, pancreatic cancer, bladder cancer and the like).

Description

Anti-HER2 antibodies and their uses Technical Field

[0001] The present invention relates to the field of antibody drugs, and in particular to anti-HER2 antibodies and uses thereof. Background Art

[0002] HER2, also known as ErbB2, belongs to the HER subfamily of the type I receptor tyrosine kinase family, which also includes three members: HER1 (ErbB1 or EGFR), HER3 (ErbB3), and HER4 (ErbB4). HER subfamily members can form homodimers and heterodimers, and HER2 is the strongest dimerization partner of other ErbB receptors. HER2 activation leads to receptor phosphorylation, which can trigger downstream signaling cascades through multiple signaling pathways such as MAPK, PI3K / AKT, JAK / STAT, and PKC, playing an important regulatory role in cell proliferation, differentiation, development, adhesion, and migration. Researchers have found that high expression of HER2 is associated with many tumors, such as neuroblastoma, breast cancer, ovarian cancer, gastric cancer, lung cancer, kidney cancer, intestinal cancer, pancreatic cancer, bladder cancer, and so on.

[0003] Currently, trastuzumab and pertuzumab are the main HER2-targeted therapeutic antibodies available on the market. Trastuzumab recognizes the HER2 extracellular domain IV, while pertuzumab recognizes the HER2 extracellular domain II heterodimerization site, significantly improving patient survival. However, researchers have also found that trastuzumab has no therapeutic effect on many patients whose tumors also overexpress HER2.

[0004] Therefore, there is still a need in the art to develop new anti-HER2 antibodies.

[0005] Summary of the Invention

[0006] After extensive research, the inventors of this application have screened and obtained a series of anti-HER2 antibodies with high binding affinity to HER2, cross-reactivity with human and cynomolgus monkey HER2, good thermal stability, and excellent anti-tumor effects in various tumors (such as neuroblastoma, breast cancer, ovarian cancer, gastric cancer, lung cancer, kidney cancer, intestinal cancer, pancreatic cancer, bladder cancer, etc.). Therefore, the present invention provides the following aspects.

[0007] Antibodies or antigen-binding fragments thereof

[0008] In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to HER2, wherein the antibody or antigen-binding fragment thereof comprises:

[0009] (1) The following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0010] (a) VH CDR1 having the structure shown as GFNIKDTY (SEQ ID NO: 10);

[0011] (b) VH CDR2 having the structure shown as IYPTQGYT (SEQ ID NO: 11);

[0012] (c) VH CDR3 having the structure shown as SRWGGEGFYAMDY (SEQ ID NO: 12);

[0013] and / or,

[0014] (2) The following three light chain variable region (VL) complementarity determining regions (CDRs):

[0015] (d) VL CDR1 having the structure shown as X1X2VQX3A (SEQ ID NO: 33);

[0016] (e) VL CDR2 having the structure shown as SAS (SEQ ID NO: 19, SEQ ID NO: 23 or SEQ ID NO: 27);

[0017] (f) VL CDR3 having the structure shown as QQHX4X5TPPT (SEQ ID NO: 34);

[0018] in:

[0019] X1 is selected from amino acid residues Q and N;

[0020] X2 is selected from amino acid residues N, Y, and S;

[0021] X3 is selected from amino acid residues G and T;

[0022] X4 is selected from amino acid residues Y, F, and S;

[0023] X5 is selected from the group consisting of amino acid residues S, M, and T.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0025] VH CDR1 as shown in SEQ ID NO: 10, VH CDR2 as shown in SEQ ID NO: 11, and VH CDR3 as shown in SEQ ID NO: 12; and

[0026] The VL CDR1 is set forth in any one of SEQ ID NOs: 18, 22 or 26, the VL CDR2 is set forth in any one of SEQ ID NOs: 19, 23 or 27, and the VL CDR3 is set forth in any one of SEQ ID NOs: 20, 24 or 28.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 10, a VH CDR2 as set forth in SEQ ID NO: 11, and a VH CDR3 as set forth in SEQ ID NO: 12; and a VL CDR1 as set forth in SEQ ID NO: 18, a VL CDR2 as set forth in SEQ ID NO: 19, and a VL CDR3 as set forth in SEQ ID NO: 20.

[0028] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 10, a VH CDR2 as set forth in SEQ ID NO: 11, and a VH CDR3 as set forth in SEQ ID NO: 12; and a VL CDR1 as set forth in SEQ ID NO: 22, a VL CDR2 as set forth in SEQ ID NO: 23, and a VL CDR3 as set forth in SEQ ID NO: 24.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a VH CDR1 as set forth in SEQ ID NO: 10, a VH CDR2 as set forth in SEQ ID NO: 11, and a VH CDR3 as set forth in SEQ ID NO: 12; and a VL CDR1 as set forth in SEQ ID NO: 26, a VL CDR2 as set forth in SEQ ID NO: 27, and a VL CDR3 as set forth in SEQ ID NO: 28.

[0030] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a VH having a sequence as shown in SEQ ID NO: 9 or a variant thereof, and a VL having a sequence as shown in any one of SEQ ID NOs: 17, 21 or 25 or a variant thereof;

[0031] Wherein, the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having 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%, at least 99%, or 100% sequence identity; preferably, the substitutions are conservative substitutions.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a VH having a sequence as shown in SEQ ID NO:9, and a VL having a sequence as shown in SEQ ID NO:17.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a VH having a sequence as shown in SEQ ID NO:9, and a VL having a sequence as shown in SEQ ID NO:21.

[0034] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a VH having a sequence as shown in SEQ ID NO:9, and a VL having a sequence as shown in SEQ ID NO:25.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a constant region derived from a human immunoglobulin.

[0036] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3, or IgG4).

[0037] In some embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).

[0038] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO:29 and / or a light chain constant region as shown in SEQ ID NO:31.

[0039] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody, and single domain antibody (sdAb).

[0040] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0041] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab, or F(ab')2 fragments can also be directly isolated from recombinant host cell culture fluid. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0042] The CDR of the present invention is obtained by classification according to the IMGT classification website http: / / aligncdr.labshare.cn / aligncdr / abrsa.php.

[0043] One-armed antibody

[0044] In a second aspect, the present invention provides a one-armed antibody comprising:

[0045] (1) A first peptide chain comprising the following three heavy chain variable region (VH) complementarity determining regions (CDRs):

[0046] (a) VH CDR1 having the structure shown as GFNIKDTY (SEQ ID NO: 10);

[0047] (b) VH CDR2 having the structure shown as IYPTQGYT (SEQ ID NO: 11);

[0048] (c) VH CDR3 having the structure shown as SRWGGEGFYAMDY (SEQ ID NO: 12);

[0049] (2) a second peptide chain, wherein the second peptide chain comprises the following three light chain variable region (VL) complementarity determining regions (CDRs):

[0050] (d) VL CDR1 having the structure shown as X1X2VQX3A (SEQ ID NO: 33);

[0051] (e) VL CDR2 having the structure shown as SAS (SEQ ID NO: 19, SEQ ID NO: 23 or SEQ ID NO: 27);

[0052] (f) VL CDR3 having the structure shown as QQHX4X5TPPT (SEQ ID NO: 34);

[0053] and,

[0054] (3) a third peptide chain, wherein the third peptide chain is capable of forming a dimer with the first peptide chain;

[0055] in:

[0056] X1 is selected from amino acid residues Q and N;

[0057] X2 is selected from amino acid residues N, Y, and S;

[0058] X3 is selected from amino acid residues G and T;

[0059] X4 is selected from amino acid residues Y, F, and S;

[0060] X5 is selected from the group consisting of amino acid residues S, M, and T.

[0061] In some embodiments, the first peptide chain comprises a VH CDR1 as set forth in SEQ ID NO:10, a VH CDR2 as set forth in SEQ ID NO:11, and a VH CDR3 as set forth in SEQ ID NO:12.

[0062] In some embodiments, the second peptide chain comprises a VL CDR1 as shown in any one of SEQ ID NOs: 18, 22 or 26, a VL CDR2 as shown in any one of SEQ ID NOs: 19, 23 or 27, and a VL CDR3 as shown in SEQ ID NOs: 20, 24 or 28.

[0063] In some embodiments, the second peptide chain comprises a VL CDR1 as set forth in SEQ ID NO:18, a VL CDR2 as set forth in SEQ ID NO:19, and a VL CDR3 as set forth in SEQ ID NO:20.

[0064] In some embodiments, the second peptide chain comprises a VL CDR1 as set forth in SEQ ID NO:22, a VL CDR2 as set forth in SEQ ID NO:23, and a VL CDR3 as set forth in SEQ ID NO:24.

[0065] In some embodiments, the second peptide chain comprises a VL CDR1 as set forth in SEQ ID NO:26, a VL CDR2 as set forth in SEQ ID NO:27, and a VL CDR3 as set forth in SEQ ID NO:28.

[0066] In some embodiments, the first peptide chain comprises a heavy chain variable region (VH) of a sequence as shown in SEQ ID NO: 9 or a variant thereof, wherein the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence with 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%, at least 99%, or 100% sequence identity; preferably, the substitutions are conservative substitutions.

[0067] In some embodiments, the first peptide chain comprises a heavy chain variable region (VH) having a sequence as shown in SEQ ID NO:9.

[0068] In some embodiments, the second peptide chain comprises a light chain variable region (VL) of a sequence as shown in any one of SEQ ID NOs: 17, 21 or 25, or a variant thereof, wherein the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence with 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%, at least 99%, or 100% sequence identity; preferably, the substitutions are conservative substitutions.

[0069] In some embodiments, the second peptide chain comprises a light chain variable region (VL) having a sequence as shown in any one of SEQ ID NOs: 17, 21 or 25.

[0070] In some embodiments, the second peptide chain further comprises a constant region derived from a human immunoglobulin.

[0071] In some embodiments, the second peptide chain comprises a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).

[0072] In some embodiments, the second peptide chain comprises a light chain constant region as shown in SEQ ID NO:31.

[0073] In some embodiments, the first peptide chain further comprises a constant region derived from a human immunoglobulin. In some embodiments, the constant region derived from a human immunoglobulin is a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the heavy chain constant region has a first modification to promote dimerization of the first peptide chain and the third peptide chain.

[0074] In some embodiments, the third peptide chain comprises an Fc domain monomer. In some embodiments, the Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain monomer has a second modification to promote dimerization of the third peptide chain and the first peptide chain.

[0075] In some embodiments, any one of the first modification and the second modification is a "knob" modification and the other is a "hole" modification to form a "knob-into-hole" modification to promote dimerization of the first peptide chain and the third peptide chain.

[0076] In some embodiments, the first modification is a "knob" modification, and the second modification is a "hole" modification, to form a "knob-into-hole" modification, which promotes dimerization of the first peptide chain and the third peptide chain.

[0077] In some embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:30, and the Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO:32.

[0078] The knob-into-hole technique is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance ("knot") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide so that the protuberance can be placed in the cavity to promote heterodimer formation and hinder homodimer formation. The protuberance is constructed by replacing the small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protuberance is created in the interface of the second polypeptide by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0079] Isolated nucleic acid molecules

[0080] In a third aspect, the present invention provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the first aspect, or its heavy chain variable region and / or light chain variable region; or, it encodes the one-armed antibody of the second aspect, or its heavy chain variable region and / or light chain variable region.

[0081] In some embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding a light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecule of the present invention comprises a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

[0082] In some embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the first peptide chain of the one-armed antibody of the present invention or its heavy chain variable region, a second nucleotide sequence encoding the second peptide chain of the one-armed antibody or its light chain variable region, and a third nucleic acid sequence encoding the third peptide chain of the one-armed antibody, wherein the first nucleotide sequence, the second nucleotide sequence, and the third nucleic acid sequence are present on the same or different isolated nucleic acid molecules. When the first nucleotide sequence, the second nucleotide sequence, and the third nucleic acid sequence are present on different isolated nucleic acid molecules, the isolated nucleic acid molecule of the present invention comprises a first nucleic acid molecule containing the first nucleotide sequence, a second nucleic acid molecule containing the second nucleotide sequence, and a third nucleic acid molecule containing the third nucleic acid sequence.

[0083] carrier

[0084] In a fourth aspect, the present invention provides a vector comprising the nucleic acid molecule of the third aspect. In some embodiments, the vector is a cloning vector or an expression vector.

[0085] In some embodiments, the vector comprises a first nucleotide sequence encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the present invention and a second nucleotide sequence encoding the light chain or light chain variable region of the antibody or antigen-binding fragment thereof, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present on different vectors, the vector of the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0086] In some embodiments, the vector comprises a first nucleotide sequence encoding the first peptide chain of the one-armed antibody of the present invention or its heavy chain variable region, a second nucleotide sequence encoding the second peptide chain of the one-armed antibody or its light chain variable region, and a third nucleic acid sequence encoding the third peptide chain of the one-armed antibody, wherein the first nucleotide sequence, the second nucleotide sequence, and the third nucleic acid sequence are present on the same or different vectors. When the first nucleotide sequence, the second nucleotide sequence, and the third nucleic acid sequence are present on different vectors, the vector of the present invention comprises a first vector containing the first nucleotide sequence, a second vector containing the second nucleotide sequence, and a third vector containing the third nucleic acid sequence.

[0087] host cells

[0088] In a fifth aspect, the present invention provides a host cell comprising the nucleic acid molecule of the third aspect or the vector of the fourth aspect. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., Escherichia coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.).

[0089] Preparation method

[0090] In the sixth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the first aspect or the one-armed antibody of the second aspect, comprising culturing the host cell of the fifth aspect under conditions allowing expression of the antibody or antigen-binding fragment thereof or the one-armed antibody, and recovering the antibody or antigen-binding fragment thereof or the one-armed antibody from the cultured host cell culture.

[0091] Therapeutic uses

[0092] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the first aspect or the one-armed antibody of the second aspect, and optionally a pharmaceutically acceptable carrier and / or excipient.

[0093] In certain exemplary embodiments, the pharmaceutically acceptable carrier and / or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0094] In an eighth aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof of the first aspect, the one-armed antibody of the second aspect, the isolated nucleic acid molecule of the third aspect, the vector of the fourth aspect, or the host cell of the fifth aspect for preparing a drug, wherein the drug is used to activate HER2 in a subject, increase immune cell activity, enhance immune response, and / or prevent and / or treat tumors or infections.

[0095] In some embodiments, the immune cells are T cells, B cells, DC cells, macrophages, and / or NK cells.

[0096] In some embodiments, the immune response is a HER2-mediated immune response.

[0097] In some embodiments, the tumor is selected from a solid tumor or a hematological tumor (eg, leukemia, lymphoma, myeloma). In certain embodiments, the tumor is selected from a solid tumor or a lymphoma.

[0098] In some embodiments, the tumor is selected from neuroblastoma, breast cancer, ovarian cancer, gastric cancer, lung cancer, kidney cancer, intestinal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, uterine / cervical cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, head and neck cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, tumors of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, melanoma.

[0099] In some embodiments, the infection is selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, and a parasitic infection.

[0100] In some embodiments, the subject is a mammal, such as a human.

[0101] In some embodiments, the antibody or antigen-binding fragment thereof or the one-armed antibody is used alone or in combination with another pharmaceutically active agent.

[0102] In the ninth aspect, the present invention provides a method for enhancing an immune response in a subject, and / or preventing and / or treating a tumor or infection, the method comprising: administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of the first aspect or the one-armed antibody of the second aspect or the pharmaceutical composition of the seventh aspect.

[0103] In some embodiments, the immune response is a HER2-mediated immune response.

[0104] In some embodiments, the tumor is selected from a solid tumor or a hematological tumor (eg, leukemia, lymphoma, myeloma). In certain embodiments, the tumor is selected from a solid tumor or a lymphoma.

[0105] In some embodiments, the tumor is selected from neuroblastoma, breast cancer, ovarian cancer, gastric cancer, lung cancer, kidney cancer, intestinal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, uterine / cervical cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, head and neck cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, tumors of the central nervous system, lymphoma, leukemia, myeloma, sarcoma, melanoma.

[0106] In some embodiments, the infection is selected from the group consisting of a viral infection, a bacterial infection, a fungal infection, and a parasitic infection.

[0107] In some embodiments, the subject is a mammal, such as a human.

[0108] The antibodies or antigen-binding fragments thereof or one-armed antibodies or pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended route of administration and therapeutic use. The antibodies or antigen-binding fragments thereof or one-armed antibodies or pharmaceutical compositions of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: incorporating the necessary dose of the antibody or antigen-binding fragment thereof or one-armed antibody of the present invention into an appropriate solvent, and optionally, incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0109] Antibody of the present invention or its Fab or one-armed antibody of the present invention or pharmaceutical composition of the present invention can be used by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, inguinal, intravesical, topical (such as, powder, ointment or drops), or nasal route. However, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). It will be understood by those skilled in the art that route of administration and / or mode will change according to intended purpose. In certain embodiments, antibody of the present invention or its Fab or one-armed antibody or pharmaceutical composition is given by intravenous injection or push injection.

[0110] Detection purpose

[0111] In a tenth aspect, the present invention provides a conjugate comprising the antibody or antigen-binding fragment thereof of the first aspect or the one-armed antibody of the second aspect, and optionally a detectable label linked to the antibody or antigen-binding fragment thereof or the one-armed antibody.

[0112] In some embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0113] In an eleventh aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof of the first aspect, the one-armed antibody of the second aspect, or the conjugate of the tenth aspect.

[0114] In some embodiments, the kit comprises the conjugate of the tenth aspect.

[0115] In some embodiments, the kit comprises the antibody or antigen-binding fragment thereof of the first aspect, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof. In certain embodiments, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0116] In some embodiments, the kit comprises the one-armed antibody of the second aspect, and a second antibody that specifically recognizes the one-armed antibody. In certain embodiments, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0117] In a twelfth aspect, the present invention provides a method for detecting the presence or level of HER2 in a sample, comprising using the antibody or antigen-binding fragment thereof of the first aspect, the one-armed antibody of the second aspect, or the conjugate of the tenth aspect. In certain embodiments, the method is used for therapeutic purposes or diagnostic purposes. In other embodiments, the method is used for non-therapeutic and non-diagnostic purposes.

[0118] In some embodiments, the method is an immunological assay, such as immunoblotting, an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0119] In some embodiments, the method comprises using the conjugate of the tenth aspect.

[0120] In some embodiments, the method comprises using the antibody or antigen-binding fragment thereof of the first aspect, and the method further comprises detecting the antibody or antigen-binding fragment thereof using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin).

[0121] In some embodiments, the method comprises using the one-armed antibody of the second aspect, and the method further comprises detecting the one-armed antibody using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin).

[0122] In certain embodiments, the method comprises: (1) contacting the sample with an antibody or antigen-binding fragment thereof or a one-armed antibody of the present invention; and (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. The formation of the immune complex indicates the presence of HER2 or cells expressing HER2.

[0123] In the thirteenth aspect, the present invention provides use of the antibody or antigen-binding fragment thereof of the first aspect, the one-armed antibody of the second aspect, or the conjugate of the third aspect in the preparation of a detection reagent for detecting the presence or level of HER2 in a sample.

[0124] In some embodiments, the detection reagent detects the presence or level of HER2 in a sample by the method for detecting the presence or level of HER2 in a sample of the twelfth aspect.

[0125] In some embodiments, the sample is a cell sample (eg, immune cell) from a subject (eg, a mammal, preferably a human or a cynomolgus monkey).

[0126] Definition of terms

[0127] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0128] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0129] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0130] The term "antibody," as used herein, refers to an immunoglobulin-derived molecule that is capable of specifically binding to a target antigen through at least one antigen-binding site located in its variable region. When referring to the term "antibody," unless the context clearly indicates otherwise, it includes not only intact antibodies but also antigen-binding fragments that are capable of specifically binding to a target antigen. "Intact antibodies" are typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ). Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain CL. The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each V H and V LIt consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site. The allocation of amino acids within each region or domain can be based on the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0131] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable region of each of the heavy and light chains contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0132] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0133] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0134] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), probodies, and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen binding ability on the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0135] As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Wherein, "full-length heavy chain" refers to a polypeptide chain that, in the direction from N-terminus to C-terminus, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; and, when the full-length antibody is an IgE isotype, optionally further comprises a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the direction from N-terminus to C-terminus. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N-terminus to C-terminus. The two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibodies of the present invention can be derived from a single species, such as humans; they can also be chimeric antibodies or humanized antibodies. The full-length antibodies of the present invention comprise two antigen-binding sites formed by a VH and a VL pair, respectively, and the two antigen-binding sites specifically recognize / bind to the same antigen.

[0136] As used herein, the term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means the fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of one complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0137] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., an Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of the complete binding site.

[0138] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments of the invention, scFv can form a di-scFv, which refers to two or more single scFvs in series to form an antibody. In certain embodiments of the invention, scFv can form a (scFv)2, which refers to two or more single scFvs in parallel to form an antibody.

[0139] As used herein, the term "diabodies" means antibodies whose VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0140] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody. Single-domain antibodies are also called nanobodies.

[0141] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0142] Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

[0143] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light chain or / and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain or / and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851 6855 (1984)). In certain embodiments, the term "chimeric antibody" may include antibodies in which the heavy and light chain variable regions of the antibody are derived from a first antibody, and the heavy and light chain constant regions of the antibody are derived from a second antibody.

[0144] As used herein, the term "Fc domain" or "Fc region" refers to a portion of the heavy chain constant region comprising CH2 and CH3. The Fc fragment of an antibody has a variety of different functions but is not involved in antigen binding. The "effector functions" mediated by the Fc region include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. The Fc region can be of any antibody heavy chain constant region isotype, such as IgG1, IgG2, IgG3, or IgG4.

[0145] The Fc domain may include both a native Fc region and a variant Fc region. A native Fc region comprises an amino acid sequence that is consistent with the amino acid sequence of an Fc region found in nature, for example, a native sequence human Fc region includes a native sequence human IgG1 Fc region (non-A and A allotypes); a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. A variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification. In some embodiments, a variant Fc region may have effector functions (e.g., Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function) that are altered compared to a native Fc region. In some embodiments, a variant Fc region may have modifications that promote dimerization.

[0146] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions × 100%). In certain embodiments, the two sequences are the same length.

[0147] The determination of percent identity between two sequences can also be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403.

[0148] As used herein, the term "variant" also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing amino acid residue substitutions, deletions, or additions in the context of a polypeptide (including polypeptides). In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived.

[0149] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D ) indicates. In the present invention, the term "K D ” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0150] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant K D (See Davies et al., Annual Rev Biochem, 1990; 59: 439-473). K can be measured by any effective method. D , kon and kdis values. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0151] As used herein, the detectable label of the present invention can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium ester compounds, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels.

[0152] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0153] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0154] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0155] The twenty conventional amino acids referred to herein are written in accordance with conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0156] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0157] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0158] As used herein, the term "subject" refers to a mammal, such as a human. In certain embodiments, the subject (eg, a human) suffers from a tumor, infection, or autoimmune disease, or is at risk of suffering from the above diseases.

[0159] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor, an infection, or an autoimmune disease) refers to an amount sufficient to prevent, stop, or delay the occurrence of the disease; an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0160] Advantageous Effects of the Invention

[0161] The present invention provides an anti-HER2 antibody that has high binding affinity to HER2, cross-reactivity with human and cynomolgus macaque HER2, good thermal stability, and excellent anti-tumor effects in various tumors (such as neuroblastoma, breast cancer, ovarian cancer, gastric cancer, lung cancer, kidney cancer, intestinal cancer, pancreatic cancer, bladder cancer, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0162] FIG1 shows a schematic diagram of the structure of the monoclonal antibody and the one-armed antibody of the present invention.

[0163] FIG2A and FIG2B show the binding affinity of the monoclonal antibody of the present invention to the HER2 protein.

[0164] FIG3 shows the binding of the monoclonal antibody of the present invention to human HER2 detected on N87 cells overexpressing HER2.

[0165] FIG4 shows the detection of the monoclonal antibody of the present invention blocking HER2 signaling-dependent cell proliferation in N87 cells overexpressing HER2.

[0166] FIG5 shows the ADCC effect of the monoclonal antibody of the present invention mediated by HER2.

[0167] Figure 6 shows the charge heterogeneity analysis of the monoclonal antibodies of the present invention after high temperature treatment, wherein 0H represents the sample before high temperature treatment; HT2W represents the sample treated with high temperature for 2 weeks; and HT4W represents the sample treated with high temperature for 4 weeks.

[0168] Figure 7 shows the activity verification of the monoclonal antibody of the present invention after high temperature treatment, wherein 0H represents the sample before high temperature treatment; HT2W represents the sample treated with high temperature for 2 weeks; HT4W represents the sample treated with high temperature for 4 weeks.

[0169] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments.

[0170] Sequence information

[0171] Post-translational modification sites (PTMs) can negatively impact drug stability and safety, reducing their effectiveness. The present invention optimizes the trastuzumab sequence, removes its PTMs, and constructs an antibody library by randomly mutagenizing the heavy and light chain CDR regions of the antibody. High-affinity antibodies are then screened from the library using yeast display technology. The present invention specifically discloses the amino acid sequence encoding the antibody and further presents data related to the candidate antibodies.

[0172] Descriptions of the sequences involved in the present invention are provided in Tables 1-1 and 1-2 below.

[0173] Table 1-1. Summary of sequence variable regions and CDRs of the present invention

[0174] Table 1-2. Summary of the constant regions and other sequences of the present invention DETAILED DESCRIPTION

[0175] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0176] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically based on the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Molecular Biology: A Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995. Restriction endonucleases were used according to the conditions recommended by the product manufacturers. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0177] Example 1: Construction and screening of a monoclonal antibody affinity maturation library

[0178] 1.1. Affinity maturation library construction

[0179] Trastuzumab heavy chain variable region gene fragment (amino acid sequence shown in SEQ ID NO: 1), light chain variable region gene fragment (amino acid sequence shown in SEQ ID NO: 5), heavy chain variable region gene fragment with N62Q and D110E mutations (amino acid sequence shown in SEQ ID NO: 9), and light chain variable region gene fragment with N36Q mutations (amino acid sequence shown in SEQ ID NO: 13) were synthesized. Using PTM-removed VH and VK as templates, degenerate primers were designed for the CDR2 and CDR3 regions of VH and the CDR3 region of VK, respectively. To ensure that each amino acid could be mutated into any of the 20 amino acids, PCR amplification was performed using NNK as the mutated base form. The target fragments were recovered using a PCR purification kit (purchased from QIAGEN). The linearized yeast display vector and the PCR products of VH and VK were mixed and then electroporated into Saccharomyces cerevisiae to construct affinity maturation libraries for heavy chain mutations and light chain mutations, respectively, and the library capacity was measured.

[0180] 1.2. Screening of anti-HER2 antibodies

[0181] 1.2.1 Biotinylation of HER2 Protein

[0182] Human HER2 protein (purchased from AcroBiosystems) was dissolved in an appropriate volume of double-distilled water. Following the instructions for the biotin labeling kit (purchased from Thermo), the biotin was dissolved and mixed with the protein solution, followed by incubation at 4°C for 2 hours. Excess biotin was removed using a desalting column (purchased from Thermo). Desalting column pretreatment and sample collection procedures were performed according to the product instructions.

[0183] 1.2.2 MACS enrichment of yeast that specifically bind to human HER2

[0184] The antibody library constructed in Example 1.1 was inoculated into SD-CAA amplification medium (1 L SD-CAA amplification medium containing 6.7 g YNB, 5 g tyrosine, 13.62 g Na2HPO4·12H2O, 7.44 g NaH2PO4, and 2% glucose) and cultured overnight at 30°C and 225 rpm. An appropriate amount of yeast cells was taken and centrifuged at 3000 rpm for 5 min (the same centrifugation operation applies to all subsequent centrifugation operations). The culture medium was removed and the yeast cells were resuspended in SD-CAA induction medium and induced overnight. The library concentration after induction was determined, and an appropriate amount of yeast cells were taken and the culture medium was removed by centrifugation. The yeast cells were resuspended in 50 ml of PBS and the supernatant was removed by centrifugation. The yeast cells were resuspended in 10 ml of PBS.

[0185] Add biotin-labeled human HER2 protein (final concentration 100nM), incubate at room temperature for 30 minutes, collect yeast cells by centrifugation, and wash the yeast three times with 50ml PBS. Resuspend the yeast cells in 5ml of washing solution, add 200μl SA magnetic beads (purchased from Miltenyi Biotec), and incubate upside down for 10 minutes. Wash the yeast and magnetic bead mixture three times with PBS, and add the mixture to the LS purification column (purchased from Miltenyi Biotec). Place the LS purification column on a magnetic stand and wash with PBS to remove non-specifically bound yeast cells. Remove the purification column from the magnetic stand and add PBS to elute the yeast. After centrifugation, transfer the eluted yeast into SD-CAA amplification medium for amplification.

[0186] 1.2.3 Flow cytometry sorting to obtain high-affinity yeast cells

[0187] Yeast cells enriched by MACS were inoculated into SD-CAA expansion medium. Cultured overnight at 30°C and 225 rpm. Yeast cells were resuspended in SD-CAA induction medium and induced overnight. Anti-c-Myc mouse antibody (purchased from Thermo) and 100 nM biotin-labeled HER2 antigen were added and incubated for 10 minutes. The yeast were washed three times with PBS, and goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 fluorescent antibody (purchased from Invitrogen) and streptavidin-APC conjugate fluorescent antibody (purchased from Invitrogen) were added and incubated for 15 minutes. Cells were resuspended in PBS and sorted using a BD FACS AriaIII instrument to obtain yeast with high binding ability to the HER2 antigen.

[0188] 1.2.4 Retrieval of HER2 Antibody Candidate Molecular Antibody Genes

[0189] The yeast liquid with high binding ability to human HER2 antigen obtained by MACS and FACS enrichment was spread on SD-CAA solid culture plates, and then single clones were picked and cultured overnight in SD-CAA amplification medium at 30°C and 225 rpm. The amplified single clones were treated with 0.1% SDS, centrifuged, and PCR amplification was performed using the supernatant as a template. The PCR products were sent for sequencing to obtain the gene sequence.

[0190] Example 2: Construction, expression and purification of monoclonal antibodies

[0191] 2.1. Construction of monoclonal antibody gene into pCDNA3.1 expression vector

[0192] The heavy chain variable region gene sequence was linked to the human IgG1 constant region (amino acid sequence shown in SEQ ID NO:29) and constructed using homologous recombination enzymes (purchased from Vazyme) into the pCDNA3.1 vector linearized with EcoR I / Not I. The light chain variable region gene sequence was linked to the human kappa constant region (amino acid sequence shown in SEQ ID NO:31) and constructed into the pCDNA3.1 vector linearized with EcoR I / Xhol I, following the manufacturer's instructions. The homologous recombination products were transformed into Top10 competent cells, plated on ampicillin-resistant plates, and cultured overnight at 37°C. Single colonies were picked for sequencing, and plasmids were extracted.

[0193] 2.2. Cell transfection and protein purification

[0194] HEK293 cells were used for transfection and expression. The cell density was adjusted to 2.5×10 6 cells / ml, diluted to 3.0×10 cells / ml for transfection the next day 6 cells / ml, using MEM medium as the transfection buffer. PEI was added at a PEI:plasmid ratio of 3:1, and the extracted heavy chain and light chain plasmids were co-transfected into HEK293 cells. After 5 days of cell culture, the supernatant was collected and the target protein was purified using a Protein A column. 10 column volumes of PBS were added to the column to equilibrate the column. The cell supernatant was added to the gravity column and allowed to flow through by gravity. After loading, unbound contaminants were washed away with 20 column volumes of PBS. After no liquid flowed out, the column was placed in a collection tube pre-added with neutralization buffer (1M Tris, pH 8.54) and the target protein was eluted with 3-5 column volumes of elution buffer (0.1M sodium citrate, pH 3.2).

[0195] Example 3: Construction, expression and purification of anti-HER2 single-arm antibody

[0196] To further confirm the affinity of the monovalent antibody, the heavy chain variable region gene sequence was linked to a human IgG1 constant region containing a Knob mutation (amino acid sequence shown in SEQ ID NO:30) and constructed using homologous recombination enzymes (purchased from Vazyme) into the EcoRI / NotI double-digested, linearized pCDNA3.1 vector. The light chain variable region gene sequence was linked to a human kappa light chain constant region (amino acid sequence shown in SEQ ID NO:31) and constructed into the EcoRI / XholI double-digested, linearized pCDNA3.1 vector. The gene encoding the human Fc region containing a Hole mutation (amino acid sequence shown in SEQ ID NO:32) was constructed into the EcoRI / XholI double-digested, linearized pCDNA3.1 vector. The procedure was as per the manufacturer's instructions. The homologous recombination product was transformed into Top10 competent cells, plated on ampicillin-resistant plates, and cultured overnight at 37°C. Single clones were picked for sequencing, and plasmids were extracted. Cell transfection and protein purification were performed as described in Example 2.2.

[0197] Example 4: Purity determination of anti-HER2 antibodies

[0198] In this study, protein purity was determined using HPLC. The HPLC method was as follows: mobile phase: 150 mM Na₂HPO₄·12H₂O, pH 7.0. Chromatographic conditions: detection wavelength: 280 nm, column temperature: 25°C, flow rate: 0.35 ml / min, detection time: 20 min, Zenix-C SEC-300 column (SEPAX 4.6 × 300 mm, 3 μm).

[0199] The results are shown in Table 2. The antibody molecules of the present invention after affinity purification have good purity and meet the requirements of downstream process development.

[0200] Table 2. Purity test results of Anti-HER2 antibodies

[0201] Example 5: Thermal stability of anti-HER2 monoclonal antibodies

[0202] The thermal stability of different antibodies was tested using DSC (Differential Scanning Calorimetry). The sample was concentrated and diluted to 1 mg / ml with PBS. 5000× fluorescent colorimetric agent Cypro Orange (purchased from Bio-Rad) was diluted 50-fold with ultrapure water to obtain 100× fluorescent colorimetric agent Sypro Orange. 50 μl of the 1 mg / ml sample was added to 10 μl of 100× fluorescent colorimetric agent Sypro Orange and 40 μl of ultrapure water. After mixing, 30 μl was added to a 96-well PCR plate. Each sample was replicated in triplicate. The plate was placed in a PCR instrument and the temperature program was set as follows: 25°C for 5 minutes, then heated to 99°C at a rate of 0.5°C / min. After the program was completed, the temperature at the lowest point of the curve was read from the "Melt Curve" graph, which was the Tm value of the sample. The specific results are shown in Table 3 below. Point mutations and affinity optimization did not reduce the thermal stability of the antibody.

[0203] Table 3. Tm values ​​of anti-HER2 monoclonal antibodies

[0204] Example 6: Affinity determination of anti-HER2 antibodies

[0205] ForteBio affinity assays were performed according to existing methods (Estep, P et al., Antibody-antigen affinity and epitope binding determination based on high-throughput methods. MAbs, 2013. 5(2): p. 270-8). Briefly, the sensor was equilibrated offline in assay buffer for 30 minutes, followed by online detection for 60 seconds to establish a baseline. The purified antibody obtained as described above was then loaded online onto the AHQ sensor. The sensor was then exposed to 100 nM human or cynomolgus macaque HER2 antigen (purchased from AcroBiosystems) for 5 minutes, after which the sensor was transferred to assay buffer for dissociation for 5 minutes. Finally, kinetic analysis was performed using a 1:1 binding model.

[0206] The experimental results, as shown in Figures 2A and 2B, show that the affinity of the optimized anti-HER2 antibody of the present invention is comparable to that of pre-optimized trastuzumab. The affinity of the trastuzumab PTM removal single-arm antibody is significantly weaker than that of the trastuzumab single-arm antibody, while the anti-HER2 single-arm antibody of the present invention, after PTM modification and affinity optimization, binds to the human HER2 antigen with an affinity comparable to that of the trastuzumab single-arm antibody.

[0207] Example 7: Monoclonal Antibody Binding to Human HER2

[0208] In this experiment, N87 cells (which express HER2) were cultured and digested with 0.25% EDTA trypsin, washed once with culture medium, and then the cell density was adjusted to 2×10 6 Cells were diluted 100 μl / well in a 96-well flow cytometry plate and centrifuged for later use. Serially diluted antibodies were added to the 96-well flow cytometry plate at 100 μl / well and incubated at 4°C for 60 min. After washing twice with PBS, 100 μl / well of Goat anti-human IgG-Fc (PE) (Abcam, ab98596) diluted 1000-fold in 2% BSA solution was added and incubated at 4°C for 60 min. After washing twice with PBS, cells were resuspended in 100 μl / well of PBS and analyzed on a CytoFlex (Beckman) flow cytometer, and the corresponding mean fluorescence intensity (MFI) was calculated.

[0209] As shown in Figure 3, the optimized anti-HER2 antibody of the present invention exhibited comparable binding activity to HER2 expressed on human gastric cancer cell line N87 compared to trastuzumab. The cell-binding activity of the trastuzumab PTM removal single-arm antibody was significantly weaker than that of the trastuzumab single-arm antibody, while the affinity-optimized anti-HER2 single-arm antibody bound to the HER2 antigen on N87 cells with an affinity comparable to that of the trastuzumab single-arm antibody and comparable to that of the monoclonal antibody.

[0210] Example 8. Anti-HER2 antibodies block HER2 signaling-dependent cell proliferation

[0211] In this experiment, the expanded cultured N87 cells (which express HER2) were digested with 0.25% EDTA trypsin, washed once with culture medium, and then the cell density was adjusted to 5×10 4 cells / ml, 80μl / well was added to the 96-well plate and set aside. The gradient diluted antibody was added to the 96-well plate with cells at 80μl / well and placed in a cell culture incubator for 3-5 days. The chemiluminescent signal was collected using a microplate reader after color development using the Luminescent Cell Viability Assay (Promega, G7572) kit.

[0212] The results are shown in FIG4 . The optimized anti-HER2 monoclonal antibodies of the present invention can significantly inhibit the proliferation of N87 cells, and the cell proliferation inhibition effect is comparable to that of the trastuzumab monoclonal antibody.

[0213] Example 9. Anti-HER2 Antibody Induces ADCC Effect (Reporter Gene)

[0214] In this experiment, N87 cells (which express HER2) were cultured and cultured at a rate of 3×10 4 / hole and 1.2×10 5 A mixture of NFAT Luciferase / Jurkat CD16a (overexpressing CD16a and NFAT-Luc) effector cells was seeded into a 96-well white-bottom cell culture plate. A serially diluted HER2 monoclonal antibody was then added to the 96-well plate and mixed thoroughly. The plate was then incubated in a cell culture incubator for 6 hours. The color was developed using the Bio-Glo luciferase assay system (Promega, G7940), and the chemiluminescent signal was collected using a microplate reader.

[0215] The experimental results are shown in Figure 5. The optimized anti-HER2 monoclonal antibodies of the present invention can mediate ADCC through HER2 expressed on N87 cells, thereby activating the CD16a-NFAT signaling pathway on Jurkat cells. In addition, the ADCC effect of the anti-HER2 (NO3-46) antibody is comparable to that of the trastuzumab antibody.

[0216] Example 10. Stability study of anti-HER2 monoclonal antibodies

[0217] 10.1. Sample Preparation for Anti-HER2 Monoclonal Antibody Stability Studies

[0218] After purification, the protein samples were diluted to 1 mg / ml in diluent, aliquoted into vials, and incubated in a 4°C refrigerator and a 40°C incubator for 2 and 4 weeks, respectively. After 2 and 4 weeks of high-temperature treatment, the samples were subjected to antibody characterization and activity validation, including charge heterogeneity analysis, PTM analysis, and activity validation.

[0219] 10.2 Charge Heterogeneity Analysis of Anti-HER2 Antibodies After High-Temperature Treatment

[0220] The charge heterogeneity of the anti-HER2 antibody samples after high-temperature treatment was determined using cation exchange chromatography (CEX-HPLC). The CEX-HPLC method was as follows: mobile phase A: 20 mM MES / MES-Na, pH 6.7; mobile phase B: 20 ​​mM MES / MES-Na + 200 mM NaCl, pH 6.7; chromatographic conditions: detection wavelength: 280 nm; column temperature: 40°C; flow rate: 1 ml / min; detection time: 20 min; gradient: 0% B to 100% B, 3-35 min; column: ProPac WCX-10 (4×250 mm, 10 μm).

[0221] The experimental results are shown in Figure 6. After high-temperature accelerated treatment of trastuzumab, CEX-HPLC observed a significant increase in basic and acidic charge variants. Compared to trastuzumab, the basic and acidic charge variants of the anti-HER2 antibody of the present invention after PTM modification and affinity optimization remained unchanged, demonstrating significantly improved stability.

[0222] 10.3 PTM Analysis of Anti-HER2 Antibody after High-Temperature Treatment

[0223] 200 μg of each high-temperature accelerated sample of trastuzumab and the modified molecule was placed in a 1.5 ml centrifuge tube. 100 μl of protein denaturation solution (8 M guanidine hydrochloride, pH 6.0) was added, along with 2 μl of 1 M DTT. The cells were then incubated at 37°C for 30 minutes for protein denaturation and reduction. After the incubation period, the cells were centrifuged for 30 seconds. 4.4 μL of 1 mol / L IAM solution was added, and the mixture was vortexed for 30 seconds to mix. The cells were centrifuged for 30 seconds and then stored in the dark for 30 minutes. After incubation in the dark, the samples were transferred to a 10 kDa ultrafiltration tube, 300 μl of 20 mmol / L His-HCl (pH 6.0) digestion buffer was added, and the cells were centrifuged at 13,000 rpm for 15 minutes. After desalting, the samples were centrifuged at 3,000 g for 3 minutes. The samples were recovered and the protein concentration was determined. Take 40 μg of desalted protein, add 2 μl of 0.5 mg / ml Trypsin / Lys-C mix (enzyme:protein ratio 1:40), and add cleavage buffer to 40 μl. Gently vortex to mix, centrifuge for a few seconds, and avoid creating bubbles. Incubate at 37°C in a water bath for 4 hours. After digestion, add 2 μl of 20% formic acid to terminate the reaction. Mix thoroughly, centrifuge at 13,000 rpm / min for 5 minutes, and collect the supernatant for RP-UHPLC-MS analysis.

[0224] The experimental results are shown in Table 4. Trastuzumab has deamidation and isomerization at the LC-Asn-30 / HC-Asn-55 / HC-Asp-102 sites, and the deamidation and isomerization of the sample show a certain degree of improvement after high-temperature accelerated treatment. RP-UHPLC-MS analysis results show that the degree of deamidation of Trastuzumab at the LC-Asn-30 site is significantly increased, while the changes in deamidation or isomerization at the HC-Asn-55 / HC-Asp-102 sites are relatively small. Compared to Trastuzumab, the anti-HER2 antibody of the present invention, after PTM modification and affinity optimization, has no detectable deamidation or isomerization at these sites, and has significantly better stability.

[0225] Table 4. PTM analysis results of Anti-HER2 antibodies after high temperature treatment

[0226] N / A means not detected

[0227] 10.4 Verification of Anti-HER2 Antibody Activity After High-Temperature Treatment

[0228] ForteBio affinity assays were performed according to existing methods (Estep, P et al., Antibody-antigen affinity and epitope binding determination based on high-throughput methods. MAbs, 2013. 5(2): p. 270-8). Briefly, the sensor was equilibrated offline in assay buffer for 30 minutes, followed by online detection for 60 seconds to establish a baseline. The purified antibody obtained as described above was then loaded online onto the AHQ sensor. The sensor was then exposed to 100 nM human HER2 antigen (purchased from AcroBiosystems) for 5 minutes, after which the sensor was transferred to assay buffer for dissociation for 5 minutes. Finally, kinetic analysis was performed using a 1:1 binding model.

[0229] The experimental results, as shown in Figure 7, show that after 2 and 4 weeks of high-temperature treatment, trastuzumab's binding affinity for the human HER2 antigen showed a significant decrease, and the longer the high-temperature treatment, the weaker the binding activity. Compared to trastuzumab, the anti-HER2 antibody of the present invention, which has undergone PTM modification and affinity optimization, showed no significant change in its binding activity to the HER2 antigen before and after high-temperature treatment, and exhibited significantly better stability.

[0230] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to HER2, wherein the antibody or antigen-binding fragment thereof comprises: (1) The following three heavy chain variable region (VH) complementarity determining regions (CDRs): (a) VH CDR1 having the structure shown as GFNIKDTY (SEQ ID NO: 10); (b) VH CDR2 having the structure shown as IYPTQGYT (SEQ ID NO: 11); (c) VH CDR3 having the structure shown as SRWGGEGFYAMDY (SEQ ID NO: 12); and / or, (2) The following three light chain variable region (VL) complementarity determining regions (CDRs): (d) VL CDR1 having the structure shown as X1X2VQX3A (SEQ ID NO: 33); (e) VL CDR2 having the structure shown as SAS (SEQ ID NO: 19, SEQ ID NO: 23 or SEQ ID NO: 27); (f) VL CDR3 having the structure shown as QQHX4X5TPPT (SEQ ID NO: 34); in: X1 is selected from amino acid residues Q and N; X2 is selected from amino acid residues N, Y, and S; X3 is selected from amino acid residues G and T; X4 is selected from amino acid residues Y, F, and S; X5 is selected from the group consisting of amino acid residues S, M, and T.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: VH CDR1 as shown in SEQ ID NO: 10, VH CDR2 as shown in SEQ ID NO: 11, and VH CDR3 as shown in SEQ ID NO: 12; and The VL CDR1 is set forth in any one of SEQ ID NOs: 18, 22 or 26, the VL CDR2 is set forth in any one of SEQ ID NOs: 19, 23 or 27, and the VL CDR3 is set forth in any one of SEQ ID NOs: 20, 24 or 28.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, comprising: (1) VH CDR1 as set forth in SEQ ID NO: 10, VH CDR2 as set forth in SEQ ID NO: 11, and VH CDR3 as set forth in SEQ ID NO: 12; and VL CDR1 as set forth in SEQ ID NO: 18, VL CDR2 as set forth in SEQ ID NO: 19, and VL CDR3 as set forth in SEQ ID NO: 20; or (2) VH CDR1 as set forth in SEQ ID NO: 10, VH CDR2 as set forth in SEQ ID NO: 11, and VH CDR3 as set forth in SEQ ID NO: 12; and VL CDR1 as set forth in SEQ ID NO: 22, VL CDR2 as set forth in SEQ ID NO: 23, and VL CDR3 as set forth in SEQ ID NO: 24; or (3) VH CDR1 as set forth in SEQ ID NO: 10, VH CDR2 as set forth in SEQ ID NO: 11, and VH CDR3 as set forth in SEQ ID NO: 12; and VL CDR1 as set forth in SEQ ID NO: 26, VL CDR2 as set forth in SEQ ID NO: 27, and VL CDR3 as set forth in SEQ ID NO:

28.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising: A VH of the sequence shown in SEQ ID NO: 9 or a variant thereof, and a VL of the sequence shown in any one of SEQ ID NOs: 17, 21 or 25 or a variant thereof; in, The variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, or a sequence having 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%, at least 99%, or 100% sequence identity; preferably, the substitutions are conservative substitutions.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising: (1) a VH sequence as shown in SEQ ID NO: 9, and a VL sequence as shown in SEQ ID NO: 17; or (1) a VH sequence as shown in SEQ ID NO: 9, and a VL sequence as shown in SEQ ID NO: 21; or (1) VH of the sequence shown in SEQ ID NO: 9, and, VH of the sequence shown in SEQ ID NO: 25 The sequence of VL.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, further comprising a constant region derived from a human immunoglobulin; Preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (eg, IgG1, IgG2, IgG3 or IgG4); Preferably, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, further comprising a heavy chain constant region as shown in SEQ ID NO: 29 and / or a light chain constant region as shown in SEQ ID NO:

31.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein The antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody and single domain antibody (sdAb).

9. A one-armed antibody comprising: (1) a first peptide chain, wherein the first peptide chain comprises three heavy chain variable region (VH) complementarity determining regions (CDRs), wherein the three heavy chain variable region (VH) complementarity determining regions (CDRs) are as defined in any one of claims 1 to 3, (2) a second peptide chain, wherein the second peptide chain comprises three light chain variable region (VL) complementarity determining regions (CDRs), wherein the three light chain variable region (VL) complementarity determining regions (CDRs) are as defined in any one of claims 1 to 3, and (3) a third peptide chain, wherein the third peptide chain is capable of forming a dimer with the first peptide chain; Preferably, the first peptide chain comprises a heavy chain variable region (VH), and the heavy chain variable region (VH) is as defined in any one of claims 4-5; Preferably, the second peptide chain comprises a light chain variable region (VL), and the light chain variable region (VL) is as defined in any one of claims 4-5.

10. The one-armed antibody according to claim 9, wherein The second peptide chain further comprises a constant region derived from a human immunoglobulin; Preferably, the second peptide chain comprises a light chain constant region derived from a human immunoglobulin (eg, κ or λ); Preferably, the second peptide chain comprises a light chain constant region as shown in SEQ ID NO:

31.

11. The one-armed antibody according to any one of claims 9 to 10, wherein The one-armed antibody further has at least one technical feature selected from the following (1)-(2): (1) The first peptide chain further comprises a constant region derived from a human immunoglobulin; preferably, the constant region derived from a human immunoglobulin is a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4); preferably, the heavy chain constant region has a first modification to promote dimerization of the first peptide chain and the third peptide chain; (2) the third peptide chain comprises an Fc domain monomer; preferably, the Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1, IgG2, IgG3 or IgG4; preferably, the Fc domain monomer has a second modification to promote dimerization of the third peptide chain and the first peptide chain; Preferably, either one of the first modification and the second modification is a "knob" modification and the other is a "hole" modification, so as to form a "knob-into-hole" modification to promote dimerization of the first peptide chain and the third peptide chain; Preferably, the first modification is a "knob" modification, and the second modification is a "hole" modification, so as to form a "knob-into-hole" modification to promote dimerization of the first peptide chain and the third peptide chain; Preferably, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 30, and the Fc domain monomer comprises the amino acid sequence shown in SEQ ID NO:

32.

12. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the heavy chain variable region and / or light chain variable region thereof; or, encoding the one-armed antibody according to any one of claims 9 to 11, or the heavy chain variable region and / or light chain variable region thereof.

13. A vector comprising the nucleic acid molecule according to claim 12; preferably, the vector is a cloning vector or an expression vector.

14. A host cell comprising the nucleic acid molecule of claim 12 or the vector of claim 13.

15. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 or the one-armed antibody according to any one of claims 9 to 11, comprising culturing the host cell according to claim 14 under conditions that allow expression of the antibody or antigen-binding fragment thereof or the one-armed antibody, and recovering the antibody or antigen-binding fragment thereof or the one-armed antibody from the cultured host cell culture.

16. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 or the one-armed antibody according to any one of claims 9 to 11, and optionally a pharmaceutically acceptable carrier and / or excipient.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the one-armed antibody according to any one of claims 9 to 11, the isolated nucleic acid molecule according to claim 12, the vector according to claim 13, or the host cell according to claim 14 for preparing a medicament for activating HER2, increasing immune cell activity, enhancing immune response, and / or preventing and / or treating tumors or infections in a subject; Preferably, the immune cells are T cells, B cells, DC cells, macrophages, and / or NK cells; Preferably, the immune response is a HER2-mediated immune response; Preferably, the subject is a mammal, such as a human; Preferably, the antibody or antigen-binding fragment thereof or the one-armed antibody is used alone or in combination with another pharmaceutically active agent.

18. A method for enhancing an immune response in a subject, and / or preventing and / or treating a tumor or infection, the method comprising: administering an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the one-armed antibody according to any one of claims 9 to 11, or the pharmaceutical composition according to claim 16 to a subject in need thereof; Preferably, the immune response is a HER2-mediated immune response; Preferably, the subject is a mammal, such as a human.

19. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 or the one-armed antibody according to any one of claims 9 to 11, and optionally a detectable label attached to the antibody or antigen-binding fragment thereof or the one-armed antibody; Preferably, the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

20. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the one-armed antibody according to any one of claims 9 to 11, or the conjugate according to claim 19; Preferably, the kit comprises the conjugate of claim 19; Preferably, the kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide, or biotin; Preferably, the kit comprises the one-armed antibody according to any one of claims 9 to 11, and a second antibody that specifically recognizes the one-armed antibody; optionally, the second antibody further comprises a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (such as fluorescein or fluorescent protein), a radionuclide or biotin.

21. A method for detecting the presence or level of HER2 in a sample, comprising using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the one-armed antibody according to any one of claims 9 to 11, or the conjugate according to claim 19; Preferably, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (e.g. ELISA), chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay; Preferably, the method comprises using the conjugate of claim 19; Preferably, the method comprises using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and the method further comprises detecting the antibody or antigen-binding fragment thereof using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin); Preferably, the method comprises using the one-armed antibody according to any one of claims 9 to 11, and the method further comprises using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide or biotin) to detect the one-armed antibody.

22. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the one-armed antibody according to any one of claims 9 to 11, or the conjugate according to claim 19 in the preparation of a detection reagent for detecting the presence or level of HER2 in a sample; Preferably, the detection reagent detects the presence or level of HER2 in a sample by the method of claim 21; Preferably, the sample is a cell sample (eg, immune cell) from a subject (eg, a mammal, preferably a human or a cynomolgus monkey).

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