Anti-her2 nanobody and preparation method and application thereof

Abstract

The application provides an anti-HER2 nanobody and a preparation method and application thereof. Specifically, the application also provides a corresponding anti-HER2 antibody, a chimeric antigen receptor, a fusion protein, a recombinant protein and nucleic acid coding same, an expression vector, a host cell and the like. The anti-HER2 nanobody of the application has high affinity, simple structure, is easy to prepare, and is different from the antigen epitope combined by trastuzumab and pertuzumab, and has a wide application prospect in the fields of tumor treatment, immune detection and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedicine or biopharmaceutical technology, and more specifically to an anti-HER2 nanobody, its preparation method, and its application. Background Technology

[0002] Human epidermal growth factor receptor 2 (HER2, ERBB2) is a tyrosine kinase receptor membrane glycoprotein encoded by the ErbB gene and belongs to the epidermal growth factor receptor family. Abnormal HER2 expression makes normal cells and tissues more susceptible to carcinogenesis, leading to tumor formation. HER2 proto-oncogene amplification or protein overexpression has been found in various human cancers, including breast cancer, gastric cancer, colon cancer, ovarian cancer, lung cancer, and prostate cancer.

[0003] HER2 comprises three domains: an extracellular ligand-binding domain (ECD), a short transmembrane region, and an intracellular tyrosine kinase domain. Its ECD consists of 630 amino acids and contains four domains (Domain I–IV). Antibodies exert their effects and biological functions differently after binding to different HER2 domains, providing new avenues for HER2 target development. For example, the classic HER2 targets trastuzumab and pertuzumab bind to different domains. Trastuzumab inhibits HER2 signaling by binding to ECD4, the extracellular juxtamembranous domain of HER2; pertuzumab binds to ECD2 of HER2, preventing homodimerization and heterodimerization with HER2 and HER3, becoming a successful approach to cancer treatment and significantly improving patient survival rates and duration. However, antibody therapy still faces limitations in use and side effects, making HER2 a crucial target for cancer treatment.

[0004] Compared to traditional monoclonal antibodies, nanobodies have smaller molecular weight, stronger penetrability, higher sensitivity, stronger specificity, and better stability. Once inside the body, they can efficiently penetrate cells to rapidly capture antigens for therapeutic purposes. In disease diagnosis, while monoclonal antibodies are used in tumor imaging, their weak tumor penetration and long serum half-life make it difficult to generate high-contrast images, thus limiting their application to some extent. Nanobodies, with their advantages of easy penetration and rapid renal clearance, can be labeled with fluorescent probes, enzyme tracers, biotin, and other molecules as tracers. Combined with molecular imaging techniques, they optimize imaging systems, making them an ideal choice for both in vitro and in vivo imaging.

[0005] Therefore, there is an urgent need in this field to develop various types of nanobody-based drugs for the specific targeting of HER2. Summary of the Invention

[0006] The purpose of this invention is to provide an anti-HER2 nanobody, its preparation method, and its application.

[0007] Another object of the present invention is to provide antibodies, chimeric antigen receptors, fusion proteins, recombinant proteins and their encoded nucleic acids, expression vectors, host cells, immunoconjugates, etc., based on anti-HER2 nanobodies, and to provide pharmaceutical compositions comprising the above active ingredients.

[0008] Another object of the present invention is to provide methods for preventing and / or treating HER2 hyperexpression diseases, as well as diagnostic methods for anti-HER2 hyperexpression diseases.

[0009] In a first aspect of the invention, an anti-HER2 nanobody is provided, the anti-HER2 nanobody having one or more complementarity-determining regions (CDRs) selected from the group consisting of:

[0010] (1) CDR1 shown in SEQ ID NO:27, CDR2 shown in SEQ ID NO:28 and CDR3 shown in SEQ ID NO:29;

[0011] (2) CDR1 shown in SEQ ID NO:34, CDR2 shown in SEQ ID NO:35 and CDR3 shown in SEQ ID NO:36;

[0012] (3) CDR1 shown in SEQ ID NO:40, CDR2 shown in SEQ ID NO:41 and CDR3 shown in SEQ ID NO:42;

[0013] (4) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47 and CDR3 shown in SEQ ID NO:48;

[0014] (5) CDR1 shown in SEQ ID NO:52, CDR2 shown in SEQ ID NO:53 and CDR3 shown in SEQ ID NO:54;

[0015] (6) CDR1 shown in SEQ ID NO:57, CDR2 shown in SEQ ID NO:58 and CDR3 shown in SEQ ID NO:59;

[0016] (7) CDR1 shown in SEQ ID NO:63, CDR2 shown in SEQ ID NO:64 and CDR3 shown in SEQ ID NO:65;

[0017] (8) CDR1 shown in SEQ ID NO:69, CDR2 shown in SEQ ID NO:70 and CDR3 shown in SEQ ID NO:71;

[0018] (9) CDR1 shown in SEQ ID NO:74, CDR2 shown in SEQ ID NO:75 and CDR3 shown in SEQ ID NO:76;

[0019] (10) CDR1 shown in SEQ ID NO:27, CDR2 shown in SEQ ID NO:28 and CDR3 shown in SEQ ID NO:80;

[0020] (11) CDR1 shown in SEQ ID NO:83, CDR2 shown in SEQ ID NO:84 and CDR3 shown in SEQ ID NO:85;

[0021] (12) CDR1 shown in SEQ ID NO:57, CDR2 shown in SEQ ID NO:58 and CDR3 shown in SEQ ID NO:89;

[0022] (13) CDR1 shown in SEQ ID NO:91, CDR2 shown in SEQ ID NO:92 and CDR3 shown in SEQ ID NO:93;

[0023] (14) CDR1 shown in SEQ ID NO:97, CDR2 shown in SEQ ID NO:98 and CDR3 shown in SEQ ID NO:99.

[0024] In another preferred embodiment, any of the amino acid sequences described above further includes a derived sequence which has optionally been added, deleted, modified and / or substituted at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and retains the ability to specifically bind to HER2.

[0025] In another preferred embodiment, the derived sequence, which has been added, deleted, modified, and / or substituted with at least one amino acid and is capable of retaining the ability to specifically bind to HER2, is an amino acid sequence with homology or sequence identity of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0026] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the backbone regions FR1, FR2, FR3 and FR4 of the VHH chain, respectively.

[0027] In another preferred embodiment, the anti-HER2 nanobody has the structure shown in formula (I):

[0028] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 formula (I)

[0029] Each of the "-" symbols independently represents a linking peptide or a peptide bond.

[0030] In another preferred embodiment, intercyclic disulfide bonds exist between the CDR1 and CDR3 regions of the anti-HER2 nanobody.

[0031] In another preferred embodiment, the anti-HER2 nanobody further includes a backbone region FR.

[0032] In another preferred embodiment, the skeleton region FR is selected from one or more of the following groups:

[0033] (1) FR1 shown in SEQ ID NO:30, FR2 shown in SEQ ID NO:31, FR3 shown in SEQ ID NO:32 and FR4 shown in SEQ ID NO:33;

[0034] (2) FR1 shown in SEQ ID NO:37, FR2 shown in SEQ ID NO:38, FR3 shown in SEQ ID NO:39 and FR4 shown in SEQ ID NO:33;

[0035] (3) FR1 shown in SEQ ID NO:43, FR2 shown in SEQ ID NO:44, FR3 shown in SEQ ID NO:45 and FR4 shown in SEQ ID NO:33;

[0036] (4) FR1 shown in SEQ ID NO:37, FR2 shown in SEQ ID NO:49, FR3 shown in SEQ ID NO:50 and FR4 shown in SEQ ID NO:33;

[0037] (5) FR1 shown in SEQ ID NO:51, FR2 shown in SEQ ID NO:31, FR3 shown in SEQ ID NO:32 and FR4 shown in SEQ ID NO:33;

[0038] (6) FR1 shown in SEQ ID NO:37, FR2 shown in SEQ ID NO:55, FR3 shown in SEQ ID NO:56 and FR4 shown in SEQ ID NO:33;

[0039] (7) FR1 shown in SEQ ID NO:60, FR2 shown in SEQ ID NO:61, FR3 shown in SEQ ID NO:62 and FR4 shown in SEQ ID NO:33;

[0040] (8) FR1 shown in SEQ ID NO:66, FR2 shown in SEQ ID NO:67, FR3 shown in SEQ ID NO:68 and FR4 shown in SEQ ID NO:33;

[0041] (9) FR1 shown in SEQ ID NO:37, FR2 shown in SEQ ID NO:72, FR3 shown in SEQ ID NO:73 and FR4 shown in SEQ ID NO:33;

[0042] (10) FR1 shown in SEQ ID NO:77, FR2 shown in SEQ ID NO:78, FR3 shown in SEQ ID NO:79 and FR4 shown in SEQ ID NO:33;

[0043] (11) FR1 shown in SEQ ID NO:30, FR2 shown in SEQ ID NO:81, FR3 shown in SEQ ID NO:82 and FR4 shown in SEQ ID NO:33;

[0044] (12) FR1 shown in SEQ ID NO:86, FR2 shown in SEQ ID NO:87, FR3 shown in SEQ ID NO:88 and FR4 shown in SEQ ID NO:33;

[0045] (13) FR1 shown in SEQ ID NO:30, FR2 shown in SEQ ID NO:61, FR3 shown in SEQ ID NO:90 and FR4 shown in SEQ ID NO:33;

[0046] (14) FR1 shown in SEQ ID NO:94, FR2 shown in SEQ ID NO:95, FR3 shown in SEQ ID NO:96 and FR4 shown in SEQ ID NO:33;

[0047] (15) FR1 shown in SEQ ID NO:100, FR2 shown in SEQ ID NO:101, FR3 shown in SEQ ID NO:102 and FR4 shown in SEQ ID NO:33.

[0048] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-HER2 nanobody is selected from one or more of SEQ ID NO:6-20.

[0049] In another preferred embodiment, the anti-HER2 nanobody includes humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0050] In a second aspect of the invention, an anti-HER2 antibody is provided, the antibody comprising one or more VHH chains of an anti-HER2 nanobody as described in the first aspect of the invention.

[0051] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-HER2 nanobody is selected from one or more of SEQ ID NO:6-20.

[0052] In another preferred embodiment, the anti-HER2 antibody may be a monomer, a bivalent antibody, and / or a multivalent antibody.

[0053] In another preferred embodiment, the anti-HER2 antibody is a bivalent antibody.

[0054] In a third aspect of the invention, a chimeric antigen receptor (CAR) is provided, the CAR containing an extracellular domain comprising an anti-HER2 nanobody as described in the first aspect of the invention, or an anti-HER2 antibody as described in the second aspect of the invention.

[0055] In another preferred embodiment, the extracellular domain further includes a signal peptide.

[0056] In another preferred embodiment, the extracellular domain also includes other exogenous proteins.

[0057] In another preferred embodiment, the CAR has the structure shown in Formula Ia:

[0058] L-Nb-H-TM-C-CD3ζ(Ia)

[0059] In the formula,

[0060] L represents the absence of a signal peptide sequence;

[0061] Nb is a specific binding domain;

[0062] H represents the area with no hinge or no connection.

[0063] TM represents a transmembrane domain;

[0064] C is the co-stimulation signal structure domain;

[0065] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type or its mutants / modifiers);

[0066] The "-" indicates a linking peptide or peptide bond.

[0067] In another preferred embodiment, the L is selected from the signal peptides of the following histones: CD8, GM-CSF, CD4, CD28, CD137, or mutants / modified forms thereof, or combinations thereof.

[0068] In another preferred embodiment, the Nb targets HER2.

[0069] In another preferred embodiment, the Nb is an anti-HER2 nanobody.

[0070] In another preferred embodiment, the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG, or a combination thereof.

[0071] In another preferred embodiment, H is the human IgG1 Fc hinge region.

[0072] In another preferred embodiment, the TM is selected from the transmembrane regions of the following histones: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or mutants / modified forms thereof, or combinations thereof.

[0073] In another preferred embodiment, C is selected from the co-stimulatory domains of the following histones: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutants / modified forms thereof, or combinations thereof.

[0074] In another preferred embodiment, the CAR includes the intracellular domain of a cytokine.

[0075] In another preferred embodiment, the cytokines include: interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), colony-stimulating factor (CSF), growth factors, chemokines, or combinations thereof; preferably, the cytokines are interleukins.

[0076] In another preferred embodiment, the CAR includes the intracellular domain of interleukin.

[0077] In another preferred embodiment, the interleukin is selected from the group consisting of IL-12, IL-2, IL-15, IL-21, or combinations thereof; preferably IL-12.

[0078] In a fourth aspect of the invention, a fusion protein is provided, the fusion protein having:

[0079] (Z1) First protein, comprising: anti-HER2 nanobody as described in the first aspect of the present invention, anti-HER2 antibody as described in the second aspect of the present invention, or an active fragment thereof;

[0080] (Z2) Second protein, which includes cytokines; and

[0081] (Z3) An optional linker located between the first protein and the second protein.

[0082] In another preferred embodiment, the cytokines include: interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), colony-stimulating factor (CSF), growth factors, chemokines, or combinations thereof.

[0083] In another preferred embodiment, the second protein is interleukin.

[0084] In another preferred embodiment, the first protein is an anti-HER2 nanobody as described in the first aspect of the present invention, and the second protein is an interleukin.

[0085] In another preferred embodiment, the interleukin is selected from the group consisting of IL-12, IL-2, IL-15, IL-21, or combinations thereof.

[0086] In another preferred embodiment, the interleukin is IL-12.

[0087] In a fifth aspect of the invention, a recombinant protein is provided, the recombinant protein having:

[0088] (i) the anti-HER2 nanobody as described in the first aspect of the present invention, the anti-HER2 antibody as described in the second aspect of the present invention, the fusion protein as described in the fourth aspect of the present invention, or an active fragment thereof; and

[0089] (ii) Optional tag sequences to assist in expression and / or purification.

[0090] In another preferred embodiment, the tag includes an Fc tag, an HA tag, a GGGS sequence, a FLAG tag, a Myc tag, a 6His tag, or a combination thereof.

[0091] In another preferred embodiment, the recombinant protein specifically binds to HER2.

[0092] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0093] In another preferred embodiment, the label is an Fc label.

[0094] In a sixth aspect of the invention, a polynucleotide is provided, the polynucleotide encoding a protein selected from the group consisting of: anti-HER2 nanobody as described in the first aspect of the invention, anti-HER2 antibody as described in the second aspect of the invention, chimeric antigen receptor as described in the third aspect of the invention, fusion protein as described in the fourth aspect of the invention, recombinant protein as described in the fifth aspect of the invention, or combinations thereof.

[0095] In another preferred embodiment, the polynucleotide is RNA, DNA, or cDNA.

[0096] In a seventh aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide as described in the sixth aspect of the invention.

[0097] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the expression vector includes viral vectors, such as lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.

[0098] In another preferred embodiment, the expression vector is selected from the group consisting of: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, etc.

[0099] In another preferred embodiment, the expression vector is pcDNA3.1 vector, pMES4 vector, or pABG1 vector (including pABG1-Fc vector).

[0100] In another preferred embodiment, the expression vector further includes a selection from the group consisting of: promoters, transcriptional enhancement elements (WPREs), long terminal repeat sequences (LTRs), etc.

[0101] In an eighth aspect of the invention, a host cell is provided, the host cell containing an expression vector as described in the seventh aspect of the invention, or having a genome containing polynucleotides as described in the sixth aspect of the invention.

[0102] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0103] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0104] In a ninth aspect of the invention, an engineered immune cell is provided, the engineered immune cell containing an expression vector as described in the seventh aspect of the invention or having an exogenous polynucleotide as described in the sixth aspect of the invention integrated into its genome, or expressing a chimeric antigen receptor as described in the third aspect of the invention.

[0105] In another preferred embodiment, the engineered immune cells are selected from the group consisting of:

[0106] (i) Chimeric antigen receptor αβ T cells (CAR-T cells);

[0107] (ii) Chimeric antigen receptor γδ T cells (CAR-T cells);

[0108] (iii) Chimeric antigen receptor NKT cells (CAR-NKT cells);

[0109] (iv) Chimeric antigen receptor NK cells (CAR-NK cells).

[0110] In another preferred embodiment, the engineered immune cells include autologous or allogeneic αβT cells, γδT cells, NKT cells, NK cells, or combinations thereof.

[0111] In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0112] In a tenth aspect of the present invention, a method for generating anti-HER2 nanobodies is provided, the method comprising the steps of:

[0113] (a) Under conditions suitable for the production of nanobodies, host cells as described in the eighth aspect of the present invention are cultured to obtain a culture containing anti-HER2 nanobodies.

[0114] (b) Isolating and / or recovering the anti-HER2 nanobody from the culture; and

[0115] (c) Optionally, the anti-HER2 nanobody obtained in step (b) is purified and / or modified.

[0116] In an eleventh aspect of the present invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0117] (a) an anti-HER2 nanobody as described in the first aspect of the present invention, an anti-HER2 antibody as described in the second aspect of the present invention, a fusion protein as described in the fourth aspect of the present invention, or a recombinant protein as described in the fifth aspect of the present invention; and

[0118] (b) The conjugate selected from the group consisting of: detectable markers, drugs, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral capsid proteins or VLPs, or combinations thereof.

[0119] In another preferred embodiment, part (a) is an anti-HER2 nanobody as described in the first aspect of the present invention, or an anti-HER2 antibody as described in the second aspect of the present invention.

[0120] In another preferred embodiment, the (a) portion is coupled to the coupling portion by a chemical bond or a connector.

[0121] In another preferred embodiment, the radionuclide includes:

[0122] (i) a diagnostic isotope selected from the group consisting of: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and / or

[0123] (ii) Therapeutic isotopes selected from the group consisting of: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or combinations thereof.

[0124] In another preferred embodiment, the coupling portion is a drug or toxin.

[0125] In another preferred embodiment, the drug is a drug for targeted treatment of HER2-overexpressing diseases.

[0126] In another preferred embodiment, the HER2-overexpressing diseases are selected from: breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small bowel cancer, large bowel cancer, bile duct cancer, cervical cancer, lymphoma, esophageal cancer, or combinations thereof.

[0127] In another preferred embodiment, the drug is a cytotoxic drug.

[0128] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0129] Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, docarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0130] In another preferred embodiment, the toxin is selected from the group consisting of: ostatins (e.g., ostatin E, ostatin F, MMAE, and MMAF), chlortetracycline, methemosiderin, pyrethroids, pyrethroid A-chain, cobustatin, docalimicin, dolalastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, and dihydroxychloroquine. Anthraxone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE)A, PE40, abrin, abrin A chain, saccharin A chain, α-Dacococcus, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxacin, jatropha toxin, croton toxin, chachomycin, Sapaonaria officinalis inhibitor, glucocorticoids, or combinations thereof.

[0131] In another preferred embodiment, the coupling portion is a detectable marker.

[0132] In another preferred embodiment, the coupling portion is selected from the group consisting of: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), or any form of nanoparticles.

[0133] In another preferred embodiment, the immunoconjugate comprises: a multivalent (e.g., bivalent) VHH chain of an anti-HER2 nanobody as described in the first aspect of the invention.

[0134] In another preferred embodiment, the polyvalent means that the amino acid sequence of the immunoconjugate contains a plurality of repeating identical or different VHH chains of the anti-HER2 nanobody as described in the first aspect of the invention.

[0135] In a twelfth aspect of the present invention, there is provided a use of an active ingredient selected from the group consisting of: anti-HER2 nanobodies as described in the first aspect of the present invention, anti-HER2 antibodies as described in the second aspect of the present invention, chimeric antigen receptors as described in the third aspect of the present invention, fusion proteins as described in the fourth aspect of the present invention, recombinant proteins as described in the fifth aspect of the present invention, polynucleotides as described in the sixth aspect of the present invention, expression vectors as described in the seventh aspect of the present invention, host cells as described in the eighth aspect of the present invention, engineered immune cells as described in the ninth aspect of the present invention, immunoconjugates as described in the eleventh aspect of the present invention, or combinations thereof, wherein the active ingredient is used to prepare:

[0136] (a) Medications for the prevention and / or treatment of diseases with high HER2 expression;

[0137] (b) Reagents for detecting diseases with high HER2 expression.

[0138] In another preferred embodiment, the reagent is a diagnostic reagent, preferably a test strip or test plate.

[0139] In another preferred embodiment, the diagnostic reagent is used to detect HER2 protein or fragments thereof in a sample.

[0140] In another preferred embodiment, the HER2-overexpressing diseases are selected from: breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small bowel cancer, large bowel cancer, bile duct cancer, cervical cancer, lymphoma, esophageal cancer, or combinations thereof.

[0141] In a thirteenth aspect of the present invention, a method for in vitro detection of HER2 protein or a fragment thereof in a sample is provided, the method comprising the steps of:

[0142] (1) In vitro, the sample is contacted with anti-HER2 nanobody as described in the first aspect of the present invention, anti-HER2 antibody as described in the second aspect of the present invention, chimeric antigen receptor as described in the third aspect of the present invention, fusion protein as described in the fourth aspect of the present invention, recombinant protein as described in the fifth aspect of the present invention, host cell as described in the eighth aspect of the present invention, engineered immune cell as described in the ninth aspect of the present invention, immunoconjugate as described in the eleventh aspect of the present invention, or a combination thereof.

[0143] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of HER2 protein or its fragments in the sample.

[0144] In another preferred embodiment, the detection includes diagnostic or non-diagnostic methods.

[0145] In a fourteenth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0146] (i) an anti-HER2 nanobody as described in the first aspect of the present invention, an anti-HER2 antibody as described in the second aspect of the present invention, a chimeric antigen receptor as described in the third aspect of the present invention, a fusion protein as described in the fourth aspect of the present invention, a recombinant protein as described in the fifth aspect of the present invention, a polynucleotide as described in the sixth aspect of the present invention, an expression vector as described in the seventh aspect of the present invention, a host cell as described in the eighth aspect of the present invention, an engineered immune cell as described in the ninth aspect of the present invention, an immunoconjugate as described in the eleventh aspect of the present invention, or a combination thereof as an active ingredient; and

[0147] (ii) Pharmaceutically acceptable carriers, diluents or excipients.

[0148] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injections and lyophilized preparations.

[0149] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of an anti-HER2 nanobody as described in the first aspect of the present invention, an anti-HER2 antibody as described in the second aspect of the present invention, a chimeric antigen receptor as described in the third aspect of the present invention, a fusion protein as described in the fourth aspect of the present invention, a recombinant protein as described in the fifth aspect of the present invention, a host cell as described in the eighth aspect of the present invention, an engineered immune cell as described in the ninth aspect of the present invention, an immunoconjugate as described in the eleventh aspect of the present invention, or a combination thereof, and 0.01 to 99.99% of a pharmaceutically acceptable carrier, wherein the percentage is a percentage by mass of the pharmaceutical composition.

[0150] In another preferred embodiment, the concentration of the engineered immune cells in the active ingredient is 1 × 10⁻⁶. 3 -1×10 8 cells / mL, preferably 1×10⁻⁶. 4 -1×10 7 Cells / mL.

[0151] According to a fifteenth aspect of the present invention, a kit is provided, the kit comprising:

[0152] (1) A first container containing an anti-HER2 nanobody as described in the first aspect of the present invention, an anti-HER2 antibody as described in the second aspect of the present invention, a chimeric antigen receptor as described in the third aspect of the present invention, a fusion protein as described in the fourth aspect of the present invention, a recombinant protein as described in the fifth aspect of the present invention, a host cell as described in the eighth aspect of the present invention, an engineered immune cell as described in the ninth aspect of the present invention, an immunoconjugate as described in the eleventh aspect of the present invention, or a combination thereof; and / or

[0153] (2) A second container, wherein the second container contains a secondary antibody against the contents of the first container;

[0154] or,

[0155] The kit contains a detection plate, which includes a substrate (support plate) and a test strip. The test strip contains an anti-HER2 nanobody as described in the first aspect of the present invention, an anti-HER2 antibody as described in the second aspect of the present invention, a fusion protein as described in the fourth aspect of the present invention, a recombinant protein as described in the fifth aspect of the present invention, an immunoconjugate as described in the eleventh aspect of the present invention, or a combination thereof.

[0156] In another preferred embodiment, the kit also includes an instruction manual, according to which the kit is used for non-invasive detection of HER2 expression in a test subject.

[0157] In another preferred embodiment, the kit is used for the detection of HER2-overexpressing diseases.

[0158] In another preferred embodiment, the HER2-overexpressing diseases are selected from: breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small bowel cancer, large bowel cancer, bile duct cancer, cervical cancer, lymphoma, esophageal cancer, or combinations thereof.

[0159] In a sixteenth aspect of the invention, a method for preventing and / or treating HER2 overexpression diseases is provided, the method comprising: administering to a desired subject an anti-HER2 nanobody as described in a first aspect of the invention, an anti-HER2 antibody as described in a second aspect of the invention, a chimeric antigen receptor as described in a third aspect of the invention, a fusion protein as described in a fourth aspect of the invention, a recombinant protein as described in a fifth aspect of the invention, a polynucleotide as described in a sixth aspect of the invention, an expression vector as described in a seventh aspect of the invention, a host cell as described in an eighth aspect of the invention, engineered immune cells as described in a ninth aspect of the invention, an immunoconjugate as described in an eleventh aspect of the invention, a pharmaceutical composition as described in a fourteenth aspect of the invention, or a combination thereof.

[0160] In another preferred embodiment, the object includes mammals, such as humans.

[0161] In another preferred embodiment, the HER2-overexpressing diseases are selected from: breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small bowel cancer, large bowel cancer, bile duct cancer, cervical cancer, lymphoma, esophageal cancer, or combinations thereof.

[0162] In another preferred embodiment, the CAR immune cells contained in the engineered immune cells or pharmaceutical composition are cells derived from the subject (autologous cells).

[0163] In another preferred embodiment, the CAR immune cells contained in the engineered immune cells or pharmaceutical composition are cells derived from a healthy individual (allogeneic cells).

[0164] In another preferred embodiment, the method may be used in combination with other treatment methods.

[0165] In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy, etc.

[0166] In a seventeenth aspect of the present invention, a diagnostic method for anti-HER2 hyperexpression diseases is provided, comprising the steps of:

[0167] (i) Obtaining a sample from a diagnostic subject and contacting the sample with an anti-HER2 nanobody as described in the first aspect of the present invention, an anti-HER2 antibody as described in the second aspect of the present invention, a chimeric antigen receptor as described in the third aspect of the present invention, a fusion protein as described in the fourth aspect of the present invention, a recombinant protein as described in the fifth aspect of the present invention, a host cell as described in the eighth aspect of the present invention, engineered immune cells as described in the ninth aspect of the present invention, an immunoconjugate as described in the eleventh aspect of the present invention, or a combination thereof; and

[0168] (ii) Detect whether an antigen-antibody complex is formed, wherein the formation of a complex indicates that the subject is a confirmed patient with HER2 hyperexpression disease.

[0169] In another preferred embodiment, the sample is a blood sample or a throat swab sample, or a sample from other tissues or organs.

[0170] In another preferred embodiment, the HER2-overexpressing diseases are selected from: breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small bowel cancer, large bowel cancer, bile duct cancer, cervical cancer, lymphoma, esophageal cancer, or combinations thereof.

[0171] In an eighteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, wherein the recombinant polypeptide is an anti-HER2 nanobody as described in the first aspect of the present invention, an anti-HER2 antibody as described in the second aspect of the present invention, a chimeric antigen receptor as described in the third aspect of the present invention, a fusion protein as described in the fourth aspect of the present invention, a recombinant protein as described in the fifth aspect of the present invention, or a combination thereof, and the method comprises:

[0172] (a) Culture host cells as described in aspect eight of the present invention under suitable expression conditions; and

[0173] (b) Isolate the recombinant polypeptide from the culture.

[0174] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0175] Figure 1 The images show the identification of purified antigen proteins by SDS-PAGE gel electrophoresis. 1: Unreduced SDS-PAGE of HER2 protein, 2: Reduced SDS-PAGE of HER2 protein, M: Protein molecular weight standard.

[0176] Figure 2 The images show the identification of purified nanobodies by non-reduced SDS-PAGE gel electrophoresis. 1-11 (left): 11 nanobodies (HER2-1, HER2-2, HER2-3, HER2-4, HER2-5, HER2-8, HER2-10, HER2-11, HER2-16, HER2-32, and HER2-34), 12 (left): protein molecular weight standard; 1-4 (right): 4 nanobodies (HER2-37, HER2-38, HER2-40, and HER2-41), 5 (right): protein molecular weight standard.

[0177] Figure 3 The image shows the identification of the purified HER2-Fc recombinant antigen protein by SDS-PAGE gel electrophoresis. 7: Non-reducing SDS-PAGE of HER2-Fc protein; 6: Protein molecular weight standards.

[0178] Figures 4-5 The identification of nanobody affinity was shown, in which Figure 4 This involves HER2-1, HER2-2, HER2-3, HER2-4, HER2-5, HER2-8, HER2-10, and HER2-11. Figure 5 This involves HER2-16, HER2-32, HER2-34, HER2-37, HER2-38, HER2-40, and HER2-41.

[0179] Figure 6 The fitted curves corresponding to the single-concentration affinity screening test of nanobodies are shown.

[0180] Figure 7 The results show the comparative results of single-concentration affinity screening tests for nanobodies using surface plasmon resonance (SPR) technology.

[0181] Figures 8-9 The results show the FACS-detected HER2 binding activity of 15 HER2 nanobodies expressed on SKBR3 cells (HER2 nanobodies cellular affinity assay). Figure 8 This involves HER2-2, HER2-3, HER2-8, HER2-11, HER2-38, HER2-5, HER2-10, and HER2-41. Figure 9 This involves HER2-1, HER2-4, HER2-16, HER2-32, HER2-34, HER2-37, and HER2-40.

[0182] Figure 10 The purified humanized nanobodies were identified by non-reduced SDS-PAGE gel electrophoresis. 1: HER2-3-hFc; 2: HER2-8-hFc; 3: HER2-10-hFc; 4: HER2-11-hFc; 5: HER2-38-hFc; 6: Protein molecular weight standard.

[0183] Figure 11 The images show the identification of purified antibody proteins by SDS-PAGE gel electrophoresis. 1: Trastuzumab non-reduced SDS-PAGE, 2: Protein molecular weight standard, 3: Trastuzumab reduced SDS-PAGE.

[0184] Figure 12 The images show the identification of purified antibody proteins by SDS-PAGE gel electrophoresis. 1: Pertuzumab non-reduced SDS-PAGE, 2: Protein molecular weight standard, 3: Pertuzumab reduced SDS-PAGE.

[0185] Figure 13 The binding activity of five purified nanobody Fc fusion proteins and the positive control trastuzumab to HER2 protein was shown by ELISA (affinity identification of nanobody Fc fusion proteins).

[0186] Figure 14 The results of affinity assays for the Fc fusion protein nanobody at the cellular level are shown.

[0187] Figure 15 The results show the specificity detection of the HER2 nanobody Fc fusion protein.

[0188] Figure 16 Epitope validation of nanobodies and trastuzumab was demonstrated.

[0189] Figure 17 Epitope validation of nanobodies and pertuzumab was demonstrated.

[0190] Figure 18 The endocytosis rate of nanobodies in SKBR3 cells was shown. Detailed Implementation

[0191] Through extensive and in-depth research and numerous screenings, the inventors of this invention have unexpectedly obtained, for the first time, an anti-HER2 nanobody with high affinity and high specificity. Specifically, this invention designed and constructed a corresponding vector containing the HER2 extracellular region gene, obtained the HER2 extracellular region protein, and used this protein to immunize healthy adult alpacas, thereby obtaining high-quality 10 11 An immune library was developed. Phage display technology was used to screen the immune library, resulting in a class of HER2-specific nanobodies. The anti-HER2 nanobodies of this invention exhibit high affinity, simple structure, and ease of preparation. Furthermore, unlike trastuzumab and pertuzumab, they bind to different antigenic epitopes, making them promising for applications in tumor therapy and immune detection. For example, the HER2-targeting nanobodies developed in this invention can serve as novel therapeutic agents for targeted treatment of breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small bowel cancer, large bowel cancer, cholangiocarcinoma, cervical cancer, lymphoma, and esophageal cancer. This invention was completed based on these findings.

[0192] the term

[0193] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0194] Definitions of abbreviations and key terms

[0195] Abbreviations (full English name, full Chinese name)

[0196] AHC anti-human IgG(Fc)capture antibody

[0197] CDR complementary determining region

[0198] EC 50 Concentration for 50% of maximal effect (half-maximal effect concentration)

[0199] FBS fetal bovine serum

[0200] Fc fragment of crystallization (antibody Fc region)

[0201] FITC fluorescein isothiocyanate (fluorescein thiocyanate)

[0202] HRP horseradish peroxidase

[0203] IC 50 half maximal inhibitory concentration

[0204] IFN interferon

[0205] IgG immunoglobulin G

[0206] kD kilodalton kilodalton

[0207] mAb monoclonal antibody

[0208] MFI median fluorescent intensity

[0209] NK Nature Killer

[0210] nM nanomole

[0211] OD optical density value

[0212] PBS (phosphate-buffered saline)

[0213] PCR (polymerase chain reaction)

[0214] PEG (polyethylene glycol)

[0215] SDS sodium dodecyl sulfacetate

[0216] SPR (Surface Plasmon Resonance)

[0217] TMB 3,3',5,5'-tetramethylbenzidine 3,3',5,5'-tetramethylbenzidine

[0218] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0219] The term “administration” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0220] The terms "nanobody", "single-domain antibody", "antibody of the present invention", and "nanobody of the present invention" are used interchangeably and all refer to antibodies as described in the first aspect of the present invention that specifically recognize and bind to HER2 proteins (including human HER2 proteins).

[0221] The antibody numbers and corresponding sequence numbers of the nanobodies of this invention are shown in Table A below.

[0222] Table A

[0223]

[0224]

[0225] Note: Each value in the table represents a sequence number, i.e., "1" represents "SEQ ID NO:1". The sequence numbers of CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4 shown in the table are the sequence numbers of their amino acid sequences.

[0226] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0227] As used herein, the terms "single-domain antibody," "VHH," "nanobody," and "single-domain antibody (sdAb, or nanobody)" have the same meaning and are used interchangeably. They refer to the cloning of the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting of only one variable region of the heavy chain. It is the smallest antigen-binding fragment with complete function. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one variable region of the heavy chain.

[0228] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called backbone regions (FRs). The variable regions of the native heavy and light chains each contain four FR regions, which are generally β-sheeted and linked by three CDRs forming a linking loop, and in some cases, partially β-sheeted structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0229] As those skilled in the art will know, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens bound to the described HER2-targeting nanobodies or fragments thereof.

[0230] As used in this article, the terms “hypervariant region”, “highly variable region”, “complementarity determining region”, and “complementarity determining region (CDR)” are used interchangeably.

[0231] In a preferred embodiment of the present invention, the heavy chain variable region of the nanobody or antibody includes three complementarity-determining regions CDR1, CDR2, and CDR3.

[0232] In a preferred embodiment of the present invention, the heavy chain of the nanobody or antibody includes the aforementioned heavy chain variable region and heavy chain constant region.

[0233] In this invention, the terms "nanobody of the present invention," "antibody of the present invention," "protein of the present invention," or "peptide of the present invention" are used interchangeably and all refer to peptides that specifically bind to the HER2 protein, such as proteins or peptides having a heavy chain variable region. They may or may not contain an initiating methionine.

[0234] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0235] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four backbone regions (FR). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through the β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of similar antibodies can be compared to determine which amino acids constitute the FR or CDR regions.

[0236] The heavy chain variable regions of nanobodies or antibodies of the present invention are of particular interest because at least a portion of them are involved in binding antigens. Therefore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, provided that their CDRs have at least 90% (preferably at least 95%, most preferably at least 98%) homology to the CDRs identified herein.

[0237] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0238] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0239] The term "antibody of the present invention" refers to a polypeptide containing the aforementioned CDR region that has HER2 protein-binding activity. This term also includes variants of polypeptides containing the aforementioned CDR region that have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibodies of the present invention.

[0240] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0241] The present invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids of the antibody of the present invention.

[0242] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table B.

[0243] Table B

[0244] The initial residues Representative substitution Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0245] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0246] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0247] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0248] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0249] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0250] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0251] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0252] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0253] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0254] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0255] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0256] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0257] The nanobodies or antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified portions, or any combination of the above substances.

[0258] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0259] Therapeutic agents that can bind to or conjugate with the antibodies of this invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-cardiac flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0260] HER2 protein

[0261] Human epidermal growth factor receptor 2 (HER2, ERBB2) is a tyrosine kinase receptor membrane glycoprotein encoded by the ErbB gene, belonging to the epidermal growth factor receptor family. In humans, this gene is located on chromosome 17q21 and is a proto-oncogene. Its encoded product, HER2 protein, is a 185kD transmembrane protamine, abbreviated as p185, composed of 1255 amino acids, with positions 720-987 belonging to the tyrosine kinase domain. The HER2 protein structure consists of three parts: the extracellular binding domain (ECD), a transmembrane domain (TM), and an intracellular domain (ICD). The extracellular domain can be divided into four subdomains (I-IV). Subdomains I and III are ligand binding sites, while subdomains II and IV contain abundant cysteine ​​residues, allowing for the formation of homodimers or heterodimers. The transmembrane domain has an α-helix structure. The intracellular region contains several important ring structures that constitute the active sites of tyrosine kinases.

[0262] The HER2 protein primarily binds to its ligands by forming heterodimers with other members of its family, including HER1 (EGFR), HER3, and HER4. HER2 is often the preferred chaperone for these heterodimers and its activity is typically stronger than that of other heterodimers. Once HER2 binds to its ligand, it activates tyrosine kinase activity mainly by inducing receptor dimerization and autophosphorylation of the cytoplasmic tyrosine kinase domain. HER2-mediated signal transduction pathways include the RAS / RAF / mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol 3-hydroxykinase (PI3K) / AKT pathway, the signal transduction and activation of transcription (STAT) pathway, and the phosphatase C (PLC) pathway. Variations in HER2 include overexpression, mutation, and amplification.

[0263] By targeting the overexpression of the HER2 protein, the anti-HER2 nanobody of the present invention can be used for targeted therapy of diseases with high HER2 expression, including but not limited to: breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small bowel cancer, large bowel cancer, bile duct cancer, cervical cancer, lymphoma, esophageal cancer, or combinations thereof.

[0264] Anti-HER2 nanobody

[0265] In this invention, the anti-HER2 nanobody comprises a monomer, a bivalent (bivalent antibody), a tetravalent (tetravalent antibody), and / or a multivalent (multivalent antibody).

[0266] In a preferred embodiment of the present invention, the amino acid sequence of the VHH chain of the anti-HER2 nanobody is selected from one or more of SEQ ID NO:6-20.

[0267] Labeled antibodies

[0268] In a preferred embodiment of the invention, the nanobody or antibody may carry a detectable marker. More preferably, the marker is selected from the group consisting of isotopes, colloidal gold markers, colored markers, or fluorescent markers.

[0269] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, the anti-HER2 nanobody or antibody is labeled with colloidal gold to obtain a colloidal gold-labeled antibody. The anti-HER2 nanobody or antibody of the present invention can effectively bind to the HER2 protein.

[0270] Detection methods

[0271] The present invention also relates to a method for detecting HER2 protein or fragments thereof. The method comprises the following steps: obtaining cell and / or tissue samples; dissolving the samples in a medium; and detecting the level of HER2 protein in the dissolved samples.

[0272] In the detection method of the present invention, there are no particular limitations on the samples used; a representative example is a cell-containing sample present in a cell preservation solution.

[0273] Reagent test kit

[0274] The present invention also provides a kit containing the anti-HER2 nanobody or antibody (or fragment thereof) or detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0275] This invention also provides a detection kit for detecting HER2 protein levels. The kit includes an antibody that recognizes the HER2 protein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0276] Pharmaceutical Composition

[0277] The present invention also provides a pharmaceutical composition comprising, as an active ingredient, an anti-HER2 nanobody as described in the first aspect of the present invention, an anti-HER2 antibody as described in the second aspect of the present invention, a chimeric antigen receptor as described in the third aspect of the present invention, a fusion protein as described in the fourth aspect of the present invention, a recombinant protein as described in the fifth aspect of the present invention, a host cell as described in the eighth aspect of the present invention, an engineered immune cell as described in the ninth aspect of the present invention, an immunoconjugate as described in the eleventh aspect of the present invention, or a combination thereof, and a pharmaceutically acceptable carrier.

[0278] These substances are typically formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, with a pH usually around 5-8, preferably around 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intraperitoneal, intravenous, or local administration.

[0279] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody (or conjugate thereof) described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0280] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals, wherein this safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight, preferably about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0281] application

[0282] As described above, the nanobodies and antibodies of the present invention have broad biological and clinical application value, and their applications involve multiple fields such as the diagnosis and treatment of HER2 protein-related diseases, basic medical research, and biological research. A preferred application is for the clinical diagnosis, prevention, and treatment of HER2 protein.

[0283] The present invention also provides a method for stimulating T-cell-mediated immune responses targeting mammalian tumor cell populations or tissues, comprising the following steps: administering the CAR-T cells of the present invention to a mammal.

[0284] In one embodiment, the present invention includes a type of cell therapy in which patient-associated (or allogeneic) T cells are isolated, activated, and genetically modified to produce CAR-T cells, which are then injected into the same patient. This approach results in an extremely low probability of graft-versus-host disease, and the antigen is recognized by the T cells in an MHC-free manner. Furthermore, a single CAR-T cell can treat all cancers expressing that antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-lasting, durable antibodies that lead to sustained tumor control.

[0285] In one embodiment, the CAR-T cells of the present invention can undergo stable in vivo expansion and persist for months to years. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-T cells can induce a specific immune response against tumor cells that highly express antigens recognized by the CAR antigen-binding domain. For example, the CAR-T cells of the present invention elicit a specific immune response against tumor cells that highly express HER2.

[0286] Treatable cancers include tumors that are not vascularized or are substantially not vascularized, as well as vascularized tumors. Types of cancer treated with the CAR of this invention include, but are not limited to: breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small bowel cancer, large bowel cancer, bile duct cancer, cervical cancer, lymphoma, esophageal cancer, etc.

[0287] Typically, activated and expanded cells, as described herein, can be used to treat and prevent diseases such as tumors. Therefore, this invention provides a method for treating cancer, comprising administering a therapeutically effective amount of the CAR-T cells of this invention to a subject in need of them.

[0288] The CAR-T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2, IL-17, or other cytokines or cell populations. In short, the pharmaceutical compositions of the present invention may include target cell populations as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0289] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the treatment (or prevention) of a disease. The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, or may be determined by clinical trials.

[0290] When referring to "immunologically effective dose," "antitumor effective dose," "tumor-inhibitory effective dose," or "therapeutic dose," the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disease condition. Pharmaceutical compositions including T cells described herein can be administered in doses of 10... 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 7 Administered at a dose of cells per kg body weight (inclusive of all integer values ​​within the range). The T-cell composition may also be administered multiple times at these doses. Cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a specific patient can be readily determined by a medical professional by monitoring the patient's signs of disease and adjusting the treatment accordingly.

[0291] The composition can be administered in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the T-cell composition of the present invention is administered to a patient via intradermal or subcutaneous injection. In another embodiment, the T-cell composition of the present invention is preferably administered via intravenous injection. The T-cell composition can be injected directly into the tumor, lymph node, or site of infection.

[0292] In some embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination with any number of relevant treatment modalities (e.g., before, simultaneously with, or after), including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or nastatinumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell composition of the invention is administered to a patient in combination with bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiotherapy (XRT), or cyclophosphamide (e.g., before, simultaneously with, or after). For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an injection of the expanded immune cells of the present invention after transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0293] The dosage of the above treatments administered to patients will vary depending on the precise nature of the condition being treated and the recipient of the treatment. The dosage ratios administered to individuals can be implemented according to accepted practices in the field. Typically, 1 × 10⁻⁶ ppm can be administered per treatment or per course of treatment. 5 One to 1×10 10 The modified T cells of this invention are administered to a patient, for example, via intravenous infusion.

[0294] Main advantages of the invention

[0295] 1. The anti-HER2 nanobody of the present invention has high affinity, simple structure, and is easy to prepare. Moreover, it binds to different antigen epitopes than trastuzumab and pertuzumab, and has broad application prospects in the fields of tumor treatment and immune detection.

[0296] 2. This invention also utilizes the constructed anti-HER2 nanobody and cytokines (such as IL-12 protein) to prepare a fusion protein. This fusion protein can target tumor lesions, precisely release cytokines, and stimulate immune cells to exert their immune-killing function. The aforementioned fusion protein can locally and precisely increase the effective concentration of cytokines at the tumor site, thereby avoiding systemic toxicity.

[0297] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0298] sequence list

[0299]

[0300]

[0301]

[0302]

[0303]

[0304] Example 1: Screening of heavy chain single-domain antibodies against HER2

[0305] 1.1 Preparation of Immunogens

[0306] The extracellular domain of the HER2 antigen (23-652) was synthesized by gene synthesis, and a 6-histidine tag was added to the C-terminus. The gene was then subcloned into the eukaryotic expression vector pcDNA3.1 to construct an expression vector for the recombinant HER2 protein (HER2-His). The amino acid sequence is shown below:

[0307] (SEQ ID NO:1)

[0308] The constructed HER2-His plasmid was extracted, and after correct sequencing, it was transfected into a well-functioning FreeStyle transfection system. TM 293-F cells were cultured in shake flasks at 37℃, 120 rpm, and 5% CO2 for 3–4 days. The expression supernatant was collected by centrifugation, filtered through a 0.45 μm filter membrane, and subjected to HisTrap. TM Proteins were purified by affinity FF, eluted with 500 mM imidazole, collected, and then concentrated by buffer replacement in an ultrafiltration tube. Protein purity was identified by SDS-PAGE, and protein concentration was quantified by spectrophotometer.

[0309] The results are as follows Figure 1 As shown, the electrophoretic positions of the antigen proteins were as expected, the bands were clear, and the purity was high. No broken or degraded bands were observed in the HER2 protein. This indicates that the purified antigen proteins obtained can be used in subsequent immunization procedures.

[0310] 1.2 Library Construction

[0311] A healthy male alpaca (1.5 years old, weighing approximately 160 catties, raised and immunized by Qingdao Kangda Biotechnology Co., Ltd.) was selected for immunization, with multiple subcutaneous injections in the neck and shoulder. After 5 immunizations, 100 ml of peripheral blood lymphocytes were extracted from the alpaca. The PBMCs were isolated using Alpaca Peripheral Blood Lymphocyte Separation Solution KIT from Tianjin Haoyang Biotechnology Co., Ltd. RNA was extracted using the Novizan RNA Keeper Tissue Atabilizer extraction kit. The extracted RNA was reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (Novizan, R312-01). Nested PCR was used to amplify the nucleic acid fragments of the variable region of the heavy chain antibody.

[0312] First round of PCR:

[0313] Upstream primer F1: GTCCTGGCTGCTCTTCTACAAGG (SEQ ID NO:2)

[0314] Downstream primer R1: GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO:3)

[0315] Fab of heavy chain antibody was obtained by PCR amplification using cDNA as a template. The reaction procedure is shown in Table 1 below:

[0316] Table 1

[0317]

[0318] Electrophoresis was performed using a 2% agarose gel. Bands of approximately 700 bp were cut, and the target gene was recovered using a gel extraction kit (Novizan) according to the instructions.

[0319] Second round of PCR:

[0320] Using the first-round PCR product as a template, nanobodies were obtained by PCR amplification again. The reaction system and reaction procedure are shown in the table.

[0321] Upstream primer F2: ATGGCCCAGGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO:4)

[0322] Downstream primer R2: GTGGTGTGAGGAGACGGTGACCTGGGT (SEQ ID NO:5)

[0323] The reaction system is shown in Table 2 below:

[0324] Table 2

[0325]

[0326] The PCR reaction procedure is shown in Table 3 below:

[0327] Table 3

[0328]

[0329] The target heavy chain single-domain antibody nucleic acid fragment was recovered and cloned into the phage display vector pMES4 using restriction endonucleases (NEB) PstⅠ and BsteⅡ. Fresh competent cells TG1 were prepared, and the product was electroporated into competent cells TG1. The conversion efficiency and accuracy of electroporation were determined. The library size was calculated by serial dilution plating. 11 Twenty clones were randomly selected for colony PCR and sequencing verification. The results showed that the insertion rate was 100%, and the heavy chain single-domain antibody phage display library targeting HER2 was successfully constructed.

[0330] 1.3 Screening of HER2 heavy chain single-domain antibodies

[0331] The HER2-His fusion protein was coated onto the immunotube at 10 μg / ml and incubated overnight at 4°C. The next day, the immunotube was blocked with 1 ml of PBS milk (PBS containing 2% skim milk powder) at 37°C for 1 hour. At the same time, 100 μl of phage library (from the heavy chain single-domain antibody phage display library constructed in Example 1.2) was blocked.

[0332] Add the blocked phage antibody library to the immunotherapy tube and incubate overnight at 4°C. Discard the liquid in the immunotherapy tube and wash with PBS, PBST and PBS+NaCl, respectively, with the washing intensity increasing in each round.

[0333] After three rounds of washing, the liquid in the immunoassay tube was discarded, and 1 ml of 0.2 mol / L glycine-hydrochloric acid (pH 2.2) was added for elution. The solution was then neutralized to pH 7.4 with 1 mol / L Tris. Logarithmically growing Escherichia coli TG1 was added to the immunoassay tube for infection. The eluted infected bacterial solution was resuspended and evenly spread on 2YT-AG (A: ampicillin sodium; G: glucose) plates. After incubation at 37°C overnight, all colonies were collected.

[0334] Add an appropriate amount of bacterial culture to 100 ml of 2YT-AG liquid medium and incubate until the absorbance (A) at 600 nm is 0.7. Add helper phage M13KO7 at an infection coefficient (MOI) of 50:1, incubate at room temperature for 30 min, then incubate at 37°C and 150 rpm for 1 h. Add IPTG to a final concentration of 0.15 mmol / L and incubate at 30°C and 200 rpm for 10 h. Precipitate and recover the phage antibody using PEG8000, and use a certain amount for the next round of screening.

[0335] After three rounds of screening, positive clones were finally enriched, achieving the goal of screening for HER2-specific antibodies using phage display technology.

[0336] 1.4 Screening for positive clones using phage enzyme-linked immunosorbent assay (ELISA).

[0337] Selected single colonies from the screening were incubated overnight at 37°C and 220 rpm in 96-well plates containing 2×YT-AG medium until saturation. The saturated bacterial culture was transferred to an OD600 of approximately 0.5, and helper phage M13KO7 was added at an infection rate (MOI) of 50:1. After standing at room temperature for 30 min, the culture was incubated at 37°C and 150 rpm for 1 h. Kanamycin was added to a final concentration of 20 μg / ml, and IPTG was added to a final concentration of 0.15 mM. Induction was then performed at 30°C and 200 rpm for 12 h.

[0338] The supernatant was collected by centrifugation and used as phage antibody. This antibody was coated with the target antigen (HER2-His) and control antigen (BSA) and incubated overnight at 4°C. The phage antibody then bound at 37°C for 1 hour. After washing, anti-M13 antibody (anti-M13 phage polyclonal antibody (HRP), secondary antibody) bound at 37°C for 45 minutes. After washing, TMB single-component chromogenic buffer was added. Absorption peaks were measured at 450 nm. Sample wells with an OD value greater than three times that of control wells were identified as positive monoclonal colonies and sent for sequencing to obtain the variable region gene of the candidate antibody. The antibody sequence information is as follows:

[0339] HER2-1:

[0340] QVQLQESGGGLVQAEGSLRLSCAAS GRTFSTYD IGWFRQAPGKEREFVAR ITRYGTGP

[0341] LYAGSVKGRFTISRDNAKNTVYLQMNGLKPEDTAVYYC AVQSGIGRVYDYRASVSYTY WGQGTQVTVSS(SEQ ID NO:6)

[0342] HER2-2:

[0343] QVQLQESGGGLVQAGDSLRLSCAAS GLTFSTVG MGWFRQLLGKEREPVAA ISWTGNVI GYGDSVKGRFTISRDSAKNTVYLQMNSLKPEDTAVYYC AARRRGTSSYDY WGQGTQVTVSS(SEQ ID NO:7)

[0344] HER2-3:

[0345] QVQLQESGGGLVQAGDSLRLSCAAS GLTFSTVG MGWFRQVLGMEREPVAG ISWTGNVI GYAESVTGRFTISRDSAKNTVYLQMNSLKPEDTAVYYC AARRRGMSSYDY WGQGTQVTVSS(SEQ ID NO:8)

[0346] HER2-4:

[0347] QVQLQESGGGLVPAEGSLRLSCAAS GRTFIPYD IGWFRQAPGKEREFVAH INRLGIGA LYAGSVEGRVTISRDNVKNTVYLQMNGLKPEDTAVYYC AAQSGIGAIYDYRASVSYTY WGQGTQVTVSS(SEQ ID NO:9)

[0348] HER2-5:

[0349] QVQLQESGGGLVQAGDSLRLSCAAS ERTFARYV MGWFRQAPGKDREFVAH IYSSGST AYEGSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC AARDRDSTWNQGTSH WGQGTQVTVSS(SEQ ID NO:10)

[0350] HER2-8:

[0351] QVQLQESGGGLVQAGGSLRLSCAAS GRTFSGAG VGWFRQDPEKEREFVAA IAWSGGST RYADSVKGRFTISRDNTENTVFLQMNNLRPEDTAVYYC AATRRFYSGLTYTQRDVYDN WGQGTQVTVSS(SEQ ID NO:11)

[0352] HER2-10:

[0353] QVQLQESGGGLVQPGGSLRLSCTAA RNIFGRNV MGWFRQAPGKQREFVAH ITGGSAV IYATSVKGRFTISRDNAKNTVYLQMNNLTLDDTAVYFC RAVNDDL WGQGTQVTVSS(SEQ ID NO:12)

[0354] HER2-11:

[0355] QVQLQESGGGLVQAGGSLRLSCAAS GHTVSRNM MGWFRQAPGKEREFVSA IAWNGEDT YYAESVEGRFTISKDNVKNTIYLQMNSLKPEDTAVYYC AASLFRLWNSATAGNNRVYHY WGQGTQVTVSS(SEQ ID NO:13)

[0356] HER2-16:

[0357] QVQLQESGGGLVQPGGSLRLSCAPS GSIFAFNA MGWYRQAPGKQRELVAT ITKEGNT NYVDSVKGRFTISRDNYKNTVDLHMTSLKPDDTAVYYC NARDTRKWVSGGYDY WGQGTQVTVSS(SEQ ID NO:14)

[0358] HER2-32:

[0359] QVQLQESGGGLVQAGGSLRLTCAAS GGTISTDV MGWFRQDPGKEREFVAA IQWSRDYT YYSDSVKGRFTGSRDNAKNTVYLQMNSLKPEDAAVYYC ARHWSGDIYASNSYNS WGQGTQVTVSS(SEQ ID NO:15)

[0360] HER2-34:

[0361] QVQLQESGGGLVQPGGSLNLSCAAS RSIFNALM GWYRQAPGNQREFVAHI TRGGST MYADSVKGRFTISRDNAKNTVYLQMNGLKPDDSAVYYC RDWGHDS WGQGTQVTVSS(SEQ ID NO:16)

[0362] HER2 - 37:

[0363] QVQLQESGGGLVQAGGSLRLSCAAS GRTFSSYD VAWFRQAPEKEREFVAG IRWRGSIA YYVDSVKGRFTISRDNAKNTVYLQMNGLKPEDTAVYYC AATVRTYYGENYDRDASAYGY WGQGTQVTVSS(SEQ ID NO:17)

[0364] HER2 - 38:

[0365] QLQESGGGLVQAGDSLRLSCAAS ERTFARYV MGWFRQAPGKDREFVAH IYSSGST AYEGSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYC AARPRDTVWTSSAS WGQGTQVTVSS(SEQ ID NO:18)

[0366] HER2 - 40:

[0367] RVQLQESGGGLVQAGGSMRLSCAAS GLTFSTVG MGWFRQLLGKEREPVAA ISWTGNVI GYGDSVKGRFTISRDSAKNTVYLQMNSLKPEDTAVYYC AARRRGTSSYDY WGQGTQVTVSS(SEQ ID NO:19)

[0368] HER2 - 41:

[0369] QVQLQESGGGLVQAGGSLRLSCAAS GRIESSYV VGWFRQPPGKEREFVTS IPWSGGAT AYAGSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYC AARTRDSVWTSSTS WGQGTQVTVSS(SEQ ID NO:20)

[0370] Example 2: Evaluation and identification of heavy chain single-domain antibodies against HER2

[0371] 2.1 Expression and purification of heavy - chain single - domain antibodies in Escherichia coli

[0372] After extracting plasmids from 15 single-domain antibody strains obtained from sequencing analysis, they were transformed into BL21(DE3) competent cells using a heat shock method. In a clean bench, 1 mL of positive clone culture was added to 100 mL of LB liquid medium (containing 100 μg / mL ampicillin) and cultured at 37°C with shaking until OD600nm = 0.8. Then, 1 mM IPTG was added, and the mixture was cultured overnight at 30°C with shaking.

[0373] The following day, the bacterial cells were collected by centrifugation at 8000 rpm for 10 min. The precipitate was resuspended in 1.5 mL of pre-chilled TES buffer and stirred on ice for 30 min. Then, 3.0 mL of TES / 4 (TES diluted 4-fold with pure water) was added, and stirring continued on ice for another 30 min. The cells were then centrifuged at 9000 rpm at 4°C for 10 min, and the supernatant (periplasmic extract) was collected for SDS-PAGE electrophoresis analysis. Further, affinity purification was performed using a nickel-packed column based on the protein purification system, followed by elution with 300 mM imidazole buffer. Antibody concentration was quantified using a spectrophotometer, and the purity of the purified antibody was identified by SDS-PAGE.

[0374] like Figure 2 As shown, the results indicate that the molecular weights of the 15 nanobodies (single-domain antibodies) expressed and purified in Escherichia coli all met expectations, with clear bands and high purity.

[0375] 2.2 Construction of Recombinant Antigen Protein

[0376] An expression vector for the recombinant protein HER2 and human Fc (HER2-Fc) was constructed. The HER2 gene and human Fc gene were cloned into the pcDNA3.1 vector. After correct sequencing, a FreeStyle transfection vector was used. TM 293-F cells were cultured in shake flasks at 37°C, 120 rpm, and 5% CO2 for 3.5 days. The supernatant was collected, purified using a Protein A column, eluted with citrate-sodium citrate buffer, and concentrated using ultrafiltration tubes. The purity of the HER2-Fc recombinant protein was determined by SDS-PAGE.

[0377] like Figure 3 As shown, the results indicate that the electrophoretic positions of the HER2-Fc recombinant protein are as expected, the bands are clear, and the purity is high. No broken or degraded bands were observed in the HER2-Fc recombinant protein.

[0378] Furthermore, the concentration of HER2-Fc recombinant protein was quantified using a spectrophotometer, with a protein concentration of 2 mg / mL.

[0379] 2.3 Detection of binding of candidate HER2 heavy chain single-domain antibodies to human HER2 protein

[0380] Human HER2-Fc fusion protein was coated onto plates at 200 ng / well and incubated overnight at 4°C. Subsequently, the heavy chain single-domain antibody obtained in Example 2.1 was serially diluted 3-fold from 10 μg / ml in 12 gradients, blocked at 37°C for 1 h, and 100 μl of the blocked whole antibody was added to each well, incubated at 37°C for 1 h. After washing, HRP-labeled anti-His-tagged antibody was added at 100 μl / well to the ELISA plate, and incubated at 37°C for 45 min. After washing, TMB substrate chromogenic solution was added at 100 μl / well, and incubated at room temperature for 10 min. 100 μl of 1M H₂SO₄ was added to terminate the reaction. The absorbance was measured at 450 nm using a microplate reader. Data processing and parameter fitting were performed using Graphd Prism Software 5.0 to calculate EC. 50 value.

[0381] The binding activity of the purified 15 nanobodies to the HER2-Fc protein was analyzed by ELISA. The results are as follows: Figure 4-5 As shown, HER2-1, HER2-2, HER2-3, HER2-4, HER2-5, HER2-8, HER2-10, HER2-11, HER2-16, HER2-32, HER2-34, HER2-37, HER2-38, HER2-40, and HER2-41 all exhibit high binding activity to the HER2-Fc recombinant protein and show typical dose-dependent characteristics.

[0382] EC 50 The results of the values ​​are shown in Tables 4 and 5 below:

[0383] Table 4

[0384] name HER2-1 HER2-2 HER2-3 HER2-4 HER2-5 HER2-8 HER2-10 HER2-11 <![CDATA[EC 50 ]]> 1.213 0.7233 0.01254 6.519 0.0803 0.01376 0.02574 0.02075

[0385] Table 5

[0386] name HER2-16 HER2-32 HER2-34 HER2-37 HER2-38 HER2-40 HER2-41 <![CDATA[EC 50 ]]> 0.4201 1.984 1.24 1.166 0.02955 0.3275 0.8707

[0387] 2.4 SPR method (i.e., BIcore) to identify the HER2 binding ability of HER2 single-domain antibody protein

[0388] A single-concentration affinity screening method was used, with all nanobodies tested at the same single concentration. Parallel comparisons of the nanobodies were conducted to detect differences in binding activity. The capture antibodies were conjugated to the CM5 chip surface using the Human Antibody Capture Kit, and the kinetic parameters of antibody-antigen interactions were determined using a multi-cycle kinetics method.

[0389] The purified HER2-His nanobodies were diluted to 1 μg / mL with HBS-EP buffer and captured and immobilized on the chip surface at 25℃ and a flow rate of 5 μl / min for 1 min, with a capture response value of approximately 400 RU. The nanobodies HER2-1, HER2-2, HER2-3, HER2-4, HER2-5, HER2-8, HER2-10, HER2-11, HER2-16, HER2-32, HER2-34, HER2-37, HER2-38, HER2-40, and HER2-41 were diluted to 1.5 μg / mL with HBS-EP buffer as the mobile phase. The testing conditions were 25℃, 30 μl / min, binding for 60 s, dissociation for 120 s, and regeneration conditions were 10 mM glycine hydrochloride buffer (pH 2.5), 30 μl / min for 90 s. The results were then analyzed.

[0390] The results are as follows Figure 6 As shown, the binding process between the antibody and the antigen, as well as the dissociation process of the antigen-antibody complex, can be clearly and intuitively observed through the fitted curve.

[0391] Antibody affinity is an important indicator for evaluating antibody molecules, and surface plasmon resonance (SPR) technology is the recognized gold standard for detecting antibody affinity. Furthermore, the inventors used the BIAcore T100 system and a single-concentration affinity screening method to screen HER2-1, HER2-2, HER2-3, HER2-4, HER2-5, HER2-8, HER2-10, HER2-11, HER2-16, HER2-32, HER2-34, HER2-37, HER2-38, HER2-40, and HER2-41, combined with activity parameters as follows: Figure 7 As shown.

[0392] 2.5 Detection of antibody binding activity to SKBR3 cells using FACS method

[0393] Purchase SKBR3 cells (Shanghai Cell Bank, Chinese Academy of Sciences) and culture them to the logarithmic growth phase. Resuspend the cells in FACS buffer and adjust the cell density to 3E6 / ml, seeding 100 μl per well. Then, dilute the heavy chain single-domain antibody obtained in Example 2.1 5-fold with FACS buffer to 30 μg / ml, resuspending the cells in 100 μl / well and incubate at 4°C for 60 min. Wash three times with PBS, then add the secondary antibody THE... TMHis Tag Antibody [FITC], mAb, and Mouse were used to resuspend cells, and incubated at 4°C for 60 min. Cells were washed three times with PBS, and then resuspended in 100-200 μl of PBS per well. MFI was detected using Beckman / CytoFLEX, and data processing and parameter fitting were performed using Graphd Prism Software 5.0 to calculate EC50. 50 value.

[0394] Analysis results as follows Figure 8-9 As shown, HER2-3, HER2-5, HER2-8, HER2-10, HER2-11, and HER2-38 exhibit high binding activity to the HER2 protein expressed in SKBR3 cells, and show typical dose-dependent characteristics.

[0395] EC 50 The results of the values ​​are shown in Tables 6 and 7 below:

[0396] Table 6

[0397] name HER2-2 HER2-3 HER2-8 HER2-11 HER2-38 HER2-5 HER2-10 HER2-41 <![CDATA[EC 50 ]]> 1.103 0.1041 0.05424 0.4848 0.295 0.4416 0.06086 0.7466

[0398] Table 7

[0399] name HER2-1 HER2-4 HER2-16 HER2-32 HER2-34 HER2-37 HER2-40 <![CDATA[EC 50 ]]> 16.37 33.49 3.748 23.68 0.7497 2.75 2.119

[0400] 2.6 Preparation of anti-HER2-Fc nanobody fusion protein using mammalian cells

[0401] Clones identified as positive single-chain antibodies were sequenced, and the sequencing results were analyzed. The positive single-chain variable region gene was amplified using primers HF (CCTTAAGGGCGTGCAGTGCCAGGTGCAGCTGCAGGAGTC, SEQ ID NO:21) and HR (GATTTGGGCTCGCTAGCTGAGGAGACGGTGACCTGGG, SEQ ID NO:22). The positive single-chain antibody gene was cloned into the vector pABG1-Fc (a pABG1 vector containing human Fc, available in the laboratory) via homologous recombination.

[0402] The amplification reaction system is shown in Table 8 below:

[0403] Table 8

[0404] Phagocytic template 0.5μL HF primers (10 μM) 1μL HR primers (10 μM) 1μL 2×Phanta Max Master Mix 15μL Sterilized deionized water 12.5μL sum 30μL

[0405] The amplification PCR reaction procedure is shown in Table 9 below:

[0406] Table 9

[0407]

[0408] The homologous recombination reaction system is shown in Table 10 below:

[0409] Table 10

[0410]

[0411]

[0412] The plasmid was extracted using an endotoxin-free plasmid extraction reagent, quantified spectrophotometer-based, and then sent for sequencing. FreeStyle samples were collected by centrifugation the day before transfection. TM 293-F cells, with the cell density adjusted to 1×10⁻⁶. 6 Transfection ratio: cells / mL (cells:medium = 20:1); Polyethylenimine Lineae (PEI) 2 μL / mL; Plasmid 1 μg / mL; Dissolve 40 μg of plasmid in 2 mL of Opti-MEM medium, mix gently, then add 80 μL of PEI transfection reagent, vortex for 10 s to mix thoroughly, and incubate at room temperature for 10-15 min; add 2 mL of the mixture to 40 mL of well-preserved FreeStyle medium. TM 293-F cells were gently mixed and cultured in shake flasks at 37°C, 120 rpm, and 5% CO2 for 3–4 days. The supernatant was collected for SDS-PAGE electrophoresis analysis. Protein A affinity purification was performed using the protein purification system, followed by elution with 0.1 M citrate-sodium citrate buffer.

[0413] The results are as follows Figure 10 As shown, the results indicate that the molecular weight of the humanized nanobody Fc as shown by protein electrophoresis is in line with expectations.

[0414] 2.7 Preparation of Trastuzumab

[0415] Trastuzumab light and heavy chain genes were synthesized via gene therapy:

[0416] Trastuzumab light chain:

[0417] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:23)

[0418] Trastuzumab heavy chain:

[0419] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQG TLVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:24)

[0420] Subcloning into the eukaryotic expression vector pcDNA3.1 resulted in a FreeStyle strain with good light and heavy chain co-transfection status. TM 293-F cells were cultured in shake flasks at 37°C, 120 rpm, and 5% CO2 for 3.5 days. The supernatant was collected, purified using a Protein A column, eluted with 0.1 M citrate-sodium citrate buffer, and concentrated using an ultrafiltration concentrator. The protein purity was then determined by SDS-PAGE.

[0421] like Figure 11 As shown, the results of trastuzumab protein identification are presented, indicating that the protein molecular weight is as expected and the purity is high.

[0422] Furthermore, the protein concentration was quantified using a spectrophotometer, and the protein concentration was 1 mg / mL.

[0423] 2.8 Preparation of Pertuzumab

[0424] Pertuzumab light and heavy chain genes were synthesized via gene synthesis:

[0425] Pertuzumab light chain:

[0426] DIQMTQSPSSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:25)

[0427] Pertuzumab heavy chain:

[0428] EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGT LVTVSSASTKGPSVFPLPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:26)

[0429] Subcloning into the eukaryotic expression vector pcDNA3.1 resulted in a FreeStyle strain with good light and heavy chain co-transfection status. TM 293-F cells were cultured in shake flasks at 37°C, 120 rpm, and 5% CO2 for 3.5 days. The supernatant was collected, purified using a Protein A column, eluted with 0.1 M citrate-sodium citrate buffer, and concentrated using an ultrafiltration concentrator. The protein purity was then determined by SDS-PAGE.

[0430] like Figure 12 As shown, the results of the pertuzumab protein identification are presented, indicating that the protein molecular weight is as expected and the purity is high.

[0431] Furthermore, the protein concentration was quantified using a spectrophotometer, and the protein concentration was 1 mg / mL.

[0432] 2.9 Detection of the binding of candidate anti-HER2-Fc nanobody fusion protein to human HER2 protein

[0433] Human HER2-His protein was coated onto plates at 200 ng / well and incubated overnight at 4°C. Then, the nanobody fusion protein obtained in Example 2.6 (positive control: Trastuzumab) was serially diluted 3-fold from 20 μg / ml in 12 gradients, blocked at 37°C for 1 h. 100 μl of the blocked whole antibody was added to each well, and the plates were incubated at 37°C for 1 h. After washing, 100 μl / well of HRP-labeled anti-human IgG-tagged antibody was added, and the plates were incubated at 37°C for 45 min. After washing, 100 μl / well of TMB substrate chromogenic solution was added, and the plates were incubated at room temperature for 10 min. 100 μl / well of 1M H2SO4 was added to terminate the reaction. The absorbance was measured at 450 nm using a microplate reader. Data processing and parameter fitting were performed using Graphd Prism Software 5.0 to calculate EC50. 50 value.

[0434] The binding activity of the five purified Fc nanobody fusion proteins to the HER2 protein was analyzed by ELISA. The results are as follows: Figure 13 As shown, HER2-3-hFc, HER2-8-hFc, HER2-10-hFc, and HER2-11-hFc exhibit better binding activity and typical dose-dependent characteristics compared to the control antibody Trastuzumab.

[0435] EC 50 The results of the values ​​are shown in Table 11 below:

[0436] Table 11

[0437] name HER2-3-hFc HER2-8-hFc HER2-10-hFc HER2-11-hFc HER2-38-hFc Trastuzumab <![CDATA[EC 50 ]]> 0.005864 0.006985 0.006238 0.009997 0.5382 0.03054

[0438] 2.10 Detection of the binding activity between HER2 nanobody Fc fusion protein and SKBR3 cells using FACS method

[0439] SKBR3 cells were cultured to the logarithmic growth phase, resuspended in FACS buffer, and the cell density was adjusted to 3E6 / ml, with 100 μl per well. Then, the nanobody Fc fusion protein obtained in Example 2.6 (positive control: Trastuzumab) was diluted 5-fold with FACS buffer to a 30 μg / ml primary antibody, and resuspended in 100 μl / well of cells, incubated at 4°C for 60 min. After washing three times with PBS, cells were resuspended in secondary antibody anti-Human IgG, Fc Fragment (Alexa Fluor 488-conjugated AffiniPure), and incubated at 4°C for 60 min. After washing three times with PBS, cells were resuspended in 100 μl / well of PBS. MFI was detected by Beckman / CytoFLEX, and data processing and parameter fitting were performed using GraphdPrism Software 5.0 to calculate EC50. 50 value.

[0440] The results are as follows Figure 14 As shown, HER2-3-hFc, HER2-8-hFc, HER2-10-hFc, and HER2-11-hFc exhibit better binding activity than Trastuzumab at the cellular level.

[0441] EC 50 The results of the values ​​are shown in Table 12 below:

[0442] Table 12

[0443] name Trastuzumab HER2-3-hFc HER2-8-hFc HER2-10-hFc HER2-11-hFc <![CDATA[EC 50 ]]> 0.7676 0.1788 0.2314 0.4685 0.1649

[0444] 2.11 Analysis of the specificity of HER2 nanobody Fc fusion protein binding to HER2 protein

[0445] Different proteins (200 ng / well) were coated with antigens including HER2, PDL1, BCMA, CD38, TSLP, IL-18, IL-15, IL-23, HAS, BSA, TNF-α, and IL-4. All proteins were from the inventor's laboratory. Coating was performed overnight at 4°C. 0.2 μg each of antibodies (HER2-3-Fc, HER2-8-Fc, HER2-10-Fc, and HER2-11-Fc) were added to replicates. The plates were incubated at 37°C for 1 hour. After washing, 100 μl of HRP-labeled anti-IgG tag antibody was added to each well. The plates were incubated at 37°C for 45 minutes. After washing, TMB substrate chromogenic buffer was added, and the chromogenic process was terminated with 1 M H2SO4. Absorbance was measured at 450 nm using a microplate reader. Data processing was performed using Graphd Prism Software 5.0.

[0446] As Figure 15The specific experimental results shown indicate that HER2-3-Fc, HER2-8-Fc, HER2-10-Fc, and HER2-11-Fc bind to human HER2 protein with high specific affinity, and no cross-reactivity with other detected proteins was found.

[0447] 2.12 Epitope Analysis of HER2 Nanobody with Trastuzumab and Pertuzumab

[0448] Capture antibodies were conjugated to the surface of a CM5 chip using a Human Antibody Capture Kit. The kinetic parameters of antibody-antigen-antibody interactions were determined using a multi-cycle kinetics method. Purified HER2-his and trastuzumab / pertuzumab were diluted to 1 μg / mL with HBS-EP buffer. Trastuzumab or pertuzumab was captured and immobilized on the chip surface at 25℃ and a flow rate of 5 μl / min for 1 min, with a capture response value of approximately 400 RU. HER2-his antigen bound for 100 s. Nanobodies HER2-3, HER2-8, HER2-10, and HER2-11 were diluted to 1.5 μg / mL with HBS-EP buffer as the mobile phase. The test conditions were 25℃, 30 μl / min, binding for 120 s, dissociation for 120 s, and regeneration conditions were 10 mM glycine hydrochloride buffer (pH 2.5), 30 μl / min for 90 s. The results were then analyzed.

[0449] Depend on Figure 16 , Figure 17 Epitope verification experiments of nanobodies and trastuzumab / pertuzumab showed that the epitopes of HER2-3-Fc, HER2-8-Fc, HER2-10-Fc, and HER2-11-Fc antibodies were different from those of trastuzumab and pertuzumab.

[0450] 2.13 Endocytosis test determination

[0451] For the endocytosis assay, seed SKBR3 cells / NCI-N87 2E5 per well (adjust cell density to 4E6, seed 100 μl / well, diluted with culture medium). Add 50 μl of antibody HER2-3, HER2-8, HER2-10, HER2-11, trastuzumab, and pertuzumab respectively, at 20 μg / ml, to 50 μl (i.e., 1 μg / well). Incubate at 4°C on ice for 30 min, then at 37°C for 0 h, 30 min, 1 h, 2 h, 4 h, 10 h, and 24 h. Centrifuge at 1500 rpm for 5 min, wash three times with PBS, and add 200 μl / well. Add secondary antibody 1 / 200 anti-Fc-FITC 100 μl / well (using FACS buffer: 2% serum), incubate at 4°C for 1 h, centrifuge at 1500 rpm for 5 min, wash three times with PBS, and add 200 μl / well. Finally, add 100 μl of the remaining antibody. FACS resuspension and flow cytometry to measure throughput at different time intervals.

[0452] Table 13

[0453]

[0454] The results are as follows Figure 18 As shown in Table 13, HER2-10-Fc exhibits a higher endocytosis rate and is the preferred sequence for ADC-conjugated drugs. HER2-3-Fc, HER2-8-Fc, and HER2-11-Fc have relatively low endocytosis efficiencies and can be used as target proteins for tumor drug therapy.

[0455] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-HER2 nanobody, characterized in that, The anti-HER2 Nanobody has complementarity determining regions (CDRs) selected from the group consisting of: (1) CDR1 of SEQ ID NO: 27, CDR2 of SEQ ID NO: 28, and CDR3 of SEQ ID NO: 80; or (2) CDR1 of SEQ ID NO: 27, CDR2 of SEQ ID NO: 28, and CDR3 of SEQ ID NO:

29.

2. The anti-HER2 nanobody of claim 1, wherein The anti-HER2 Nanobody further comprises framework regions (FRs) selected from the group consisting of: (1) FR1 of SEQ ID NO: 30, FR2 of SEQ ID NO: 81, FR3 of SEQ ID NO: 82, and FR4 of SEQ ID NO: 33; or (2) FR1 of SEQ ID NO: 30, FR2 of SEQ ID NO: 31, FR3 of SEQ ID NO: 32, and FR4 of SEQ ID NO: 33; or (3) FR1 of SEQ ID NO: 51, FR2 of SEQ ID NO: 31, FR3 of SEQ ID NO: 32, and FR4 of SEQ ID NO:

33.

3. The anti-HER2 nanobody of claim 1, wherein The VHH chain of the anti-HER2 Nanobody has an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 8, or 19.

4. The anti-HER2 nanobody of claim 1, wherein The anti-HER2 Nanobody is a humanized antibody, a camelid antibody, a chimeric antibody.

5. An anti-HER2 nanobody, characterized in that, The anti-HER2 Nanobody comprises one or more VHH chains of the anti-HER2 Nanobody of any one of claims 1-4.

6. The anti-HER2 nanobody of claim 5, wherein The VHH chain of the anti-HER2 Nanobody has an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 8, or 19.

7. The anti-HER2 nanobody as described in claim 5, characterized in that, The anti-HER2 Nanobody is a monomeric and / or multivalent antibody.

8. A chimeric antigen receptor (CAR), characterized in that, The CAR comprises an extracellular domain comprising the anti-HER2 Nanobody of any one of claims 1-4, or the anti-HER2 Nanobody of any one of claims 5-7.

9. The chimeric antigen receptor of claim 8, wherein, The extracellular domain further comprises a signal peptide.

10. The chimeric antigen receptor of claim 8, wherein The CAR further comprises an intracellular domain of an interleukin.

11. The chimeric antigen receptor of claim 8, wherein, The CAR has a structure of Formula la: L-Nb-H-TM-C-CD3ζ (la) wherein, L is nothing or a signal peptide sequence; Nb is a specific binding domain; H is nothing or a hinge region; TM is a transmembrane domain; C is a costimulatory signaling domain; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ; the “-” is a connecting peptide or a peptide bond.

12. A recombinant protein, characterized in that, The recombinant protein has: (i) the anti-HER2 Nanobody of any one of claims 1-4, the anti-HER2 Nanobody of any one of claims 5-7; and (ii) a tag sequence that facilitates expression and / or purification.

13. The recombinant protein of claim 12, wherein, The tag comprises an Fc tag, an HA tag, a GGGS sequence, a FLAG tag, a Myc tag, a 6His tag, or a combination thereof.

14. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-13. The polynucleotide encodes a protein selected from the group consisting of an anti-HER2 nanobody as claimed in any one of claims 1-4, an anti-HER2 nanobody as claimed in any one of claims 5-7, a chimeric antigen receptor as claimed in any one of claims 8-11, a recombinant protein as claimed in claim 12 or 13, or a combination thereof.

15. The polynucleotide of claim 14, wherein, The polynucleotide is RNA, DNA.

16. The polynucleotide of claim 14, wherein The polynucleotide is cDNA.

17. An expression vector comprising the nucleic acid of claim 16. The expression vector contains a polynucleotide as claimed in any one of claims 14-16.

18. The expression vector of claim 17, wherein, The expression vector is selected from the group consisting of DNA, RNA.

19. The expression vector of claim 17, wherein The expression vector is selected from the group consisting of a viral vector, a plasmid, a transposon, or a combination thereof.

20. The expression vector of claim 19, wherein, The viral vector is selected from the group consisting of a lentivirus, an adenovirus, an AAV virus, a retrovirus, or a combination thereof.

21. A host cell, characterized in that, The host cell contains an expression vector as claimed in any one of claims 17-20, or has integrated in its genome a polynucleotide as claimed in any one of claims 14-16.

22. The host cell of claim 21, wherein The host cell comprises a prokaryotic cell or a eukaryotic cell.

23. An engineered immune cell, comprising: The engineered immune cell contains an expression vector as claimed in any one of claims 17-20 or has integrated in its genome an exogenous polynucleotide as claimed in any one of claims 14-16, or expresses a chimeric antigen receptor as claimed in any one of claims 8-11.

24. The engineered immune cell of claim 23, wherein, The engineered immune cell is selected from the group consisting of: (i) a chimeric antigen receptor αβ T cell; (ii) a chimeric antigen receptor γδ T cell; (iii) a chimeric antigen receptor NKT cell; (iv) a chimeric antigen receptor NK cell.

25. A method of producing an anti-HER2 nanobody, characterized in that, The method comprises the steps of: (a) culturing a host cell as claimed in claim 21 or 22 under conditions suitable for the production of a nanobody, thereby obtaining a culture containing an anti-HER2 nanobody; (b) isolating and / or recovering the anti-HER2 nanobody from the culture; and (c) purifying and / or modifying the anti-HER2 nanobody obtained in step (b).

26. An immunoconjugate, comprising a monoclonal antibody of any one of claims 1-25 conjugated to a cytotoxic agent. The immunoconjugate contains: (a) an anti-HER2 nanobody as claimed in any one of claims 1-4, an anti-HER2 nanobody as claimed in any one of claims 5-7, or a recombinant protein as claimed in claim 12 or 13; and (b) a conjugating moiety selected from the group consisting of a radiolabel, an enzyme, a gold nanoparticle or nanorod, a nanomagnetic particle, a viral coat protein or VLP, or a combination thereof. The conjugating moiety is selected from the group consisting of a radionuclide, an enzyme capable of producing a detectable product.

27. The immunoconjugate of claim 26, wherein The conjugating moiety is a prodrug-activating enzyme.

28. The immunoconjugate of claim 26, wherein The prodrug-activating enzyme is DT-diaphorase or a diphenyl hydrolase-like protein.

29. The immunoconjugate of claim 28, wherein The immunoconjugate contains:

30. An immunoconjugate, comprising a monoclonal antibody of any one of claims 1-29 conjugated to a cytotoxic agent. (a) an anti-HER2 nanobody as claimed in any one of claims 1-4, an anti-HER2 nanobody as claimed in any one of claims 5-7, or a recombinant protein as claimed in claim 12 or 13; and (b) a detectable label. The conjugating moiety is selected from the group consisting of a fluorescent or luminescent label, a magnetic resonance imaging (MRI) or computed tomography (CT) contrast agent, a viral particle, a liposome. ​ 31. The immunoconjugate of claim 30, wherein ​ 32. A method for detecting HER2 protein or a fragment thereof in a sample in vitro other than for the purpose of diagnosing a disease, characterized by, The method comprises the steps of: (1) contacting, in vitro, the sample with the anti-HER2 nanobody of any one of claims 1-4, the anti-HER2 nanobody of any one of claims 5-7, the chimeric antigen receptor of any one of claims 8-11, the recombinant protein of claim 12 or 13, the host cell of claim 21 or 22, the engineered immune cell of claim 23 or 24, the immunoconjugate of any one of claims 26-31, or a combination thereof; (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of the HER2 protein or fragment thereof in the sample.

33. A pharmaceutical composition comprising, The pharmaceutical composition comprises: (i) the anti-HER2 nanobody of any one of claims 1-4, the anti-HER2 nanobody of any one of claims 5-7, the chimeric antigen receptor of any one of claims 8-11, the recombinant protein of claim 12 or 13, the host cell of claim 21 or 22, the engineered immune cell of claim 23 or 24, the immunoconjugate of any one of claims 26-31, or a combination thereof as an active ingredient; and (ii) a pharmaceutically acceptable carrier.

34. A kit comprising, The kit comprises: (1) a first container comprising the anti-HER2 nanobody of any one of claims 1-4, the anti-HER2 nanobody of any one of claims 5-7, the chimeric antigen receptor of any one of claims 8-11, the recombinant protein of claim 12 or 13, the host cell of claim 21 or 22, the engineered immune cell of claim 23 or 24, the immunoconjugate of any one of claims 26-31, or a combination thereof; or, The kit comprises a detection plate comprising: a substrate and a test strip comprising the anti-HER2 nanobody of any one of claims 1-4, the anti-HER2 nanobody of any one of claims 5-7, the recombinant protein of claim 12 or 13, the immunoconjugate of any one of claims 26-31, or a combination thereof.

35. The kit of claim 34, wherein The kit further comprises: (2) a second container comprising a secondary antibody against the contents of the first container.

36. A method of producing a recombinant polypeptide, said recombinant polypeptide being an anti-HER2 nanobody according to any one of claims 1 to 4, an anti-HER2 nanobody according to any one of claims 5 to 7, a chimeric antigen receptor according to any one of claims 8 to 11, a recombinant protein according to claim 12 or 13, or a combination thereof, characterized in that, The method comprises: (a) culturing the host cell of claim 21 or 22 under conditions suitable for expression; and (b) isolating the recombinant polypeptide from the culture.

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