Anti-her2 and trop2 bispecific single-domain antibody and use thereof

Abstract

The present invention relates to an anti-Her2 single-domain antibody, and a bispecific antibody constructed on the basis of the anti-Her2 single-domain antibody, a humanized antibody thereof, and an anti-Trop2 single-domain antibody. Further provided is an antibody-drug conjugate comprising the foregoing antibody. The anti-Her2 single-domain antibody exhibits cell-binding activity and endocytic activity significantly superior to those of anti-Her2 monoclonal antibodies, and the constructed bispecific antibody demonstrates a synergistic anti-tumor effect of dual targets, thereby having favorable prospects for tumor treatment use.

Description

Anti-Her2 and Trop2 bispecific single-domain antibodies and their applications Technical Field

[0001] This invention relates to the fields of biomedical or biopharmaceutical technology, and more specifically to single-domain antibodies against Her2 and Trop2, conjugates containing said single-domain antibodies, and their applications. Background Technology

[0002] Therapies that utilize the immune system to recognize, attack, and eliminate tumor cells have become one of the most effective cancer treatment strategies. Antibody therapy, immune checkpoint inhibitors, CAR-T cell therapy, and cancer vaccines are common cancer immunotherapies. Immunotherapy uses tumor-associated antigens (TAAs) as targets, activating and enhancing the body's immune system response to recognize, attack, and eliminate tumor cells. Several monoclonal antibody drugs targeting TAAs are already on the market, including antibodies targeting Her2, CD20, Trop2, PDL1, and EGFR. However, specific treatment strategies and outcomes are influenced by various factors (such as tumor type and individual differences), so detailed individualized assessments and treatment planning are usually required when selecting and applying immunotherapy.

[0003] HER2 is a tyrosine kinase receptor that is overexpressed in many tumors, such as breast and gastric cancer, with particularly high expression levels in breast cancer. This makes HER2 antibodies an important therapeutic and diagnostic tool for HER2-positive tumors. HER2-targeting antibodies (such as trastuzumab) have been widely used in the diagnosis and treatment of HER2-positive tumors and have been shown to significantly improve patient survival and treatment response.

[0004] Trop2 is a trophoblast surface glycoprotein that is highly expressed in various solid tumors, particularly in cervical cancer, triple-negative breast cancer, urothelial carcinoma, and prostate cancer. Trop2 can promote tumor cell proliferation, invasion, metastasis, and spread, and its high expression is closely related to shortened survival and poor prognosis in cancer patients. The Trop2-targeting antibody Sacituzumab has been approved for the treatment of unresectable locally advanced or metastatic triple-negative breast cancer.

[0005] However, single-target therapy still has some limitations, such as responsiveness and drug resistance. The development of bispecific antibodies can effectively enhance the specific binding of tumor cells and reduce the possibility of attacking normal tissue cells, thereby reducing side effects and improving safety. Currently, there are no bispecific single-domain antibody drugs targeting Her2 and Trop2 in clinical research, but the use of such bispecific antibodies for clinical diagnosis and treatment may have good application prospects. Summary of the Invention

[0006] The purpose of this invention is to provide an anti-Her2 single-domain antibody, an anti-Her2 and Trop2 bispecific single-domain antibody, a conjugate containing the aforementioned antibodies, and their applications.

[0007] In a first aspect of the invention, an anti-Her2 single-domain antibody or an antigen-binding fragment thereof is provided, wherein the VHH chain of the single-domain antibody or the antigen-binding fragment thereof comprises the following complementarity-determining region (CDR):

[0008] CDR1 as shown in SEQ ID NO:2, CDR2 as shown in SEQ ID NO:3, and CDR3 as shown in SEQ ID NO:4.

[0009] In another preferred embodiment, any of the amino acid sequences described above further includes a derived sequence that has optionally been added, deleted, modified, and / or substituted at least one amino acid and is capable of retaining Her2 binding affinity.

[0010] In another preferred embodiment, the VHH chain further includes a frame region (FR).

[0011] In another preferred embodiment, the frame region (FR) is selected from camel-derived FR and human-derived FR.

[0012] In another preferred embodiment, the frame region is selected from the group consisting of:

[0013] (Z1) FR1 as shown in SEQ ID NO:5, FR2 as shown in SEQ ID NO:6, FR3 as shown in SEQ ID NO:7, and FR4 as shown in SEQ ID NO:8; or

[0014] (Z2) FR1 as shown in SEQ ID NO:10, FR2 as shown in SEQ ID NO:6, FR3 as shown in SEQ ID NO:7, and FR4 as shown in SEQ ID NO:11.

[0015] In another preferred embodiment, the single-domain antibody includes a camel-derived single-domain antibody, a chimeric single-domain antibody, or a humanized single-domain antibody.

[0016] In another preferred embodiment, the VHH chain of the single-domain antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence having at least 80% (preferably at least 85%, more preferably at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence shown in SEQ ID NO:1.

[0017] In another preferred embodiment, the VHH chain of the single-domain antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:9, or an amino acid sequence having at least 80% (preferably at least 85%, more preferably at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence shown in SEQ ID NO:9.

[0018] In another preferred embodiment, the VHH chain of the single-domain antibody or its antigen-binding fragment is as shown in SEQ ID NO:1.

[0019] In another preferred embodiment, the VHH chain of the single-domain antibody or its antigen-binding fragment is as shown in SEQ ID NO:9.

[0020] In another preferred embodiment, the single-domain antibody includes monomers and multivalents (multivalent antibodies), such as bivalents (bivalent antibodies) and tetravalents (tetravalent antibodies).

[0021] In another preferred embodiment, the single-domain antibody comprises one or more (e.g., two) VHH chains having an amino acid sequence as shown in SEQ ID NO:1 or 9 or a derivative thereof.

[0022] In another preferred embodiment, the single-domain antibody or its antigen-binding fragment differs from the recognition epitope of Trastuzumab.

[0023] In another preferred embodiment, the single-domain antibody or its antigen-binding fragment differs from the recognition epitope of Pertuzumab.

[0024] In another preferred embodiment, the affinity KD value of the single-domain antibody or its antigen-binding fragment to the Her2 protein is <10. -10 M, preferably <10 -11 M, more preferably ≤10 -12 M.

[0025] In another preferred embodiment, the EC50 value of the single-domain antibody or its antigen-binding fragment binding to the Her2 protein expressed on the cell surface is <10 nM, preferably <5 nM, and more preferably <2 nM.

[0026] In a second aspect of the invention, a fusion protein is provided, the structure of which from the N-terminus to the C-terminus is shown in formula Ia or Ib:

[0027] ALB(Ia);

[0028] BLA(Ib);

[0029] in,

[0030] A is an anti-Her2 single-domain antibody or its antigen-binding fragment as described in the first aspect of the present invention;

[0031] B is the Fc fragment of IgG; and

[0032] L represents no or flexible joints.

[0033] In another preferred embodiment, the flexible connector is a peptide connector.

[0034] In another preferred embodiment, the peptide linker has 1-50 amino acids, more preferably 1-20 amino acids.

[0035] In another preferred embodiment, the peptide linker is selected from (GS)n, (GGS)n, (GGGS)n, and (GGGGS)n, where n is a positive integer from 1 to 5.

[0036] In another preferred embodiment, the Fc fragment of the IgG comprises the Fc fragment of human IgG.

[0037] In another preferred embodiment, the Fc fragment of the IgG is selected from the group consisting of the Fc fragments of IgG1, IgG2, IgG3, IgG4, or combinations thereof.

[0038] In a third aspect of the invention, a multispecific antibody is provided, the multispecific antibody comprising an anti-Her2 single-domain antibody or an antigen-binding fragment thereof as described in the first aspect of the invention.

[0039] In another preferred embodiment, the multispecific antibody includes bispecific antibodies, trispecific antibodies, etc.

[0040] In a fourth aspect of the invention, a bispecific antibody is provided, the bispecific antibody comprising an anti-Her2 single-domain antibody or an antigen-binding fragment thereof as described in the first aspect of the invention, and an anti-Trop2 single-domain antibody or an antigen-binding fragment thereof.

[0041] In another preferred embodiment, the VHH chain of the anti-Trop2 single-domain antibody or its antigen-binding fragment comprises the following complementarity-determining region (CDR):

[0042] CDR1 as shown in SEQ ID NO:13, CDR2 as shown in SEQ ID NO:14, and CDR3 as shown in SEQ ID NO:15.

[0043] In another preferred embodiment, the anti-Trop2 single-domain antibody includes a camel-derived single-domain antibody, a chimeric single-domain antibody, or a humanized single-domain antibody.

[0044] In another preferred embodiment, the anti-Trop2 single-domain antibody is a humanized single-domain antibody.

[0045] In another preferred embodiment, the VHH chain of the anti-Trop2 single-domain antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:12, or an amino acid sequence having at least 80% (preferably at least 85%, more preferably at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the amino acid sequence shown in SEQ ID NO:12.

[0046] In another preferred embodiment, the VHH chain of the anti-Trop2 single-domain antibody or its antigen-binding fragment is as shown in SEQ ID NO:12.

[0047] In another preferred embodiment, the bispecific antibody has a structure from the N-terminus to the C-terminus as shown in formula II(a) or II(b):

[0048] H-L1-TP II(a)

[0049] T-L1-HP II(b)

[0050] In the formula, each "-" represents a peptide bond independently;

[0051] H represents an anti-Her2 single-domain antibody or its antigen-binding fragment;

[0052] T represents an anti-Trop2 single-domain antibody or its antigen-binding fragment;

[0053] L1 is a linker peptide;

[0054] P is a peptide with no or no C-terminal linker.

[0055] In another preferred embodiment, the bispecific antibody has the structure shown in Formula II(a) from the N-terminus to the C-terminus.

[0056] In another preferred embodiment, the amino acid sequence of H, i.e., the anti-Her2 single-domain antibody, is shown in SEQ ID NO:9.

[0057] In another preferred embodiment, the amino acid sequence of T, i.e., the anti-Trop2 single-domain antibody, is shown in SEQ ID NO:12.

[0058] In another preferred embodiment, the amino acid sequence of L1 is selected from: (G4S)n, where n is a positive integer from 1 to 4; (SG)2 or (SG)3; (EAAAK)n, where n is a positive integer from 1 to 3.

[0059] In another preferred embodiment, the amino acid sequence of L1 is shown in SEQ ID NO:16.

[0060] In another preferred embodiment, P is a C-terminal linker peptide, which causes the C-terminus of the bispecific antibody to contain free cysteine ​​for site-specific conjugation of drug loading.

[0061] In another preferred embodiment, the C-terminal linker peptide is selected from: GGGSCSA (SEQ ID NO:17), GSC, GGC, GGGGC, GGGGCGGGGC, EEEEGC, DDDGC, or combinations thereof.

[0062] In another preferred embodiment, the amino acid sequence of the C-terminal linker peptide is shown in SEQ ID NO:17.

[0063] In another preferred embodiment, the bispecific antibody has the amino acid sequence shown in SEQ ID NO:18.

[0064] In a fifth aspect of the invention, a recombinant protein is provided, the recombinant protein having:

[0065] (i) an anti-Her2 single-domain antibody or its antigen-binding fragment as described in the first aspect of the invention, a multispecific antibody as described in the third aspect of the invention, or a bispecific antibody as described in the fourth aspect of the invention; and

[0066] (ii) Optional tag sequences to assist in expression and / or purification.

[0067] In another preferred embodiment, the tag sequence includes an Fc tag, an HA tag, and a His tag.

[0068] In a sixth aspect of the invention, a polynucleotide is provided, the polynucleotide encoding a protein selected from the group consisting of: an anti-Her2 single-domain antibody or an antigen-binding fragment thereof as described in the first aspect of the invention, a fusion protein as described in the second aspect of the invention, a multispecific antibody as described in the third aspect of the invention, a bispecific antibody as described in the fourth aspect of the invention, or a recombinant protein as described in the fifth aspect of the invention.

[0069] In another preferred embodiment, the polynucleotide includes DNA and RNA.

[0070] In another preferred embodiment, the polynucleotide encodes a bispecific antibody as described in the fifth aspect of the invention.

[0071] In another preferred embodiment, the polynucleotide encodes a bispecific antibody with an amino acid sequence as shown in SEQ ID NO:18; preferably, the sequence of the polynucleotide is shown in SEQ ID NO:19.

[0072] In a seventh aspect of the invention, an expression vector is provided, the expression vector comprising the polynucleotide as described in the sixth aspect of the invention.

[0073] In another preferred embodiment, the expression vector includes a eukaryotic expression vector and a prokaryotic expression vector.

[0074] 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.

[0075] 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 integrated with polynucleotides as described in the sixth aspect of the invention.

[0076] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0077] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0078] In a ninth aspect of the invention, a method is provided for generating an anti-Her2 single-domain antibody or an antigen-binding fragment thereof as described in the first aspect of the invention, or a bispecific antibody as described in the fourth aspect of the invention, comprising the steps of:

[0079] (i) Under suitable conditions, the host cells described in the eighth aspect of the present invention are cultured to obtain a culture containing the anti-Her2 single-domain antibody or its antigen-binding fragment or the bispecific antibody; and

[0080] (ii) The culture obtained in step (i) is purified and / or separated to obtain the anti-Her2 single-domain antibody or its antigen-binding fragment or bispecific antibody.

[0081] In another preferred embodiment, the purification can be performed by purifying and separating the target antibody using a protein A affinity column.

[0082] In a tenth aspect of the present invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0083] (a) an anti-Her2 single-domain antibody or its antigen-binding fragment as described in the first aspect of the present invention, a fusion protein as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, a bispecific antibody 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

[0084] (b) The conjugate selected from the following groups: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, gold nanoparticles / nanorobars, viral capsid proteins or VLPs, or combinations thereof.

[0085] In another preferred embodiment, the coupling portion is a drug or toxin.

[0086] In another preferred embodiment, the drug is a cytotoxic drug, such as: anti-tubulin drug, DNA minor groove binding agent, DNA replication inhibitor, alkylating agent, antibiotic, folic acid antagonist, antimetabolite, chemosensitizer, topoisomerase inhibitor, vinca alkaloid, or a combination thereof.

[0087] In another preferred embodiment, the coupling portion is monomethylolpropionate E (MMAE).

[0088] In another preferred embodiment, the coupling portion is a detectable marker, such as a fluorescent or luminescent marker.

[0089] In another preferred embodiment, the coupling portion is a radionuclide, which includes therapeutic and diagnostic radionuclides.

[0090] In an eleventh aspect of the invention, the use of an anti-Her2 single-domain antibody or antigen-binding fragment thereof as described in the first aspect of the invention, a fusion protein as described in the second aspect of the invention, a recombinant protein as described in the fifth aspect of the invention, or an immunoconjugate as described in the tenth aspect of the invention is provided for the preparation of: (a) a reagent for detecting Her2 molecules; or (b) a medicament for treating or preventing Her2-positive tumors.

[0091] In another preferred embodiment, the detection is an in vivo detection or an in vitro detection.

[0092] In another preferred embodiment, the detection includes flow cytometry and cell immunofluorescence detection.

[0093] In another preferred embodiment, the Her2-positive tumors include, but are not limited to, breast cancer, cervical cancer, ovarian cancer, gastric cancer, prostate cancer, urothelial carcinoma, colon cancer, pancreatic cancer, non-small cell carcinoma, bladder cancer, endometrial cancer, glioblastoma, salivary gland cancer, peritoneal cancer, penile cancer, intrahepatic bile duct cancer, thyroid cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, lung adenocarcinoma, esophageal adenocarcinoma, neuroendocrine tumors, pia mater cancer, gastrointestinal cancers, melanoma, ductal carcinoma in situ, serous uterine carcinoma, biliary tract cancer, and skin cancer.

[0094] In a twelfth aspect of the invention, the use of a bispecific antibody as described in a fourth aspect of the invention, a recombinant protein as described in a fifth aspect of the invention, or an immunoconjugate as described in a tenth aspect of the invention, is provided for the preparation of: (a) a reagent for detecting Her2 and / or Trop2 molecules; or (b) a medicament for treating or preventing Her2 and / or Trop2 positive tumors.

[0095] In another preferred embodiment, the detection is either in vivo or in vitro.

[0096] In another preferred embodiment, the detection includes flow cytometry and cell immunofluorescence detection.

[0097] In another preferred embodiment, the tumor includes, but is not limited to, breast cancer, cervical cancer, ovarian cancer, gastric cancer, prostate cancer, urothelial carcinoma, colon cancer, pancreatic cancer, non-small cell carcinoma, bladder cancer, endometrial cancer, glioblastoma, salivary gland cancer, peritoneal cancer, penile cancer, intrahepatic bile duct cancer, thyroid cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, lung adenocarcinoma, esophageal adenocarcinoma, neuroendocrine tumors, pia mater cancer, gastrointestinal cancers, melanoma, ductal carcinoma in situ, serous uterine carcinoma, biliary tract cancer, and skin cancer.

[0098] In a thirteenth aspect of the present invention, a pharmaceutical composition is provided comprising: an anti-Her2 single-domain antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, a fusion protein as described in the second aspect of the present invention, a multispecific antibody as described in the third aspect of the present invention, a bispecific antibody as described in the fourth aspect of the present invention, a recombinant protein as described in the fifth aspect of the present invention, or an immunoconjugate as described in the tenth aspect of the present invention; and a pharmaceutically acceptable carrier.

[0099] In another preferred embodiment, the pharmaceutical composition is used to treat Her2-positive tumors.

[0100] In another preferred embodiment, the pharmaceutical composition is used to treat Her2 and Trop2 positive tumors.

[0101] In another preferred embodiment, the Her2-positive tumors include, but are not limited to, breast cancer, cervical cancer, ovarian cancer, gastric cancer, prostate cancer, urothelial carcinoma, colon cancer, pancreatic cancer, non-small cell carcinoma, bladder cancer, endometrial cancer, glioblastoma, salivary gland cancer, peritoneal cancer, penile cancer, intrahepatic bile duct cancer, thyroid cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, lung adenocarcinoma, esophageal adenocarcinoma, neuroendocrine tumors, pia mater cancer, gastrointestinal cancers, melanoma, ductal carcinoma in situ, serous uterine carcinoma, biliary tract cancer, and skin cancer.

[0102] In another preferred embodiment, the Her2 and Trop2 positive tumors include, but are not limited to, breast cancer, cervical cancer, ovarian cancer, gastric cancer, prostate cancer, urothelial carcinoma, colon cancer, pancreatic cancer, non-small cell carcinoma, bladder cancer, endometrial cancer, glioblastoma, salivary gland cancer, peritoneal cancer, penile cancer, intrahepatic bile duct cancer, thyroid cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, lung adenocarcinoma, esophageal adenocarcinoma, neuroendocrine tumors, leptomeningeal carcinoma, gastrointestinal cancers, melanoma, ductal carcinoma in situ, serous uterine carcinoma, biliary tract cancer, and skin cancer.

[0103] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0104] In another preferred embodiment, the pharmaceutical composition further comprises other drugs for treating tumors.

[0105] In a fourteenth aspect of the invention, a Her2 detection reagent is provided, the detection reagent comprising an immunoconjugate as described in a tenth aspect of the invention, and a detection-acceptable carrier.

[0106] In a fifteenth aspect of the present invention, a detection kit for detecting Her2 molecules is provided, the kit comprising an immunoconjugate as described in a tenth aspect of the present invention or a detection reagent as described in a fifteenth aspect of the present invention, and an instruction manual.

[0107] In another preferred embodiment, the instruction manual states that the kit is used for non-invasive detection of Her2 expression in a test subject.

[0108] In another preferred embodiment, the kit is used for the detection of tumors expressing Her2 (i.e., Her2 positive).

[0109] In a sixteenth aspect of the invention, a method for treating a tumor is provided, the method comprising administering to a subject in need an anti-Her2 single-domain antibody or an antigen-binding fragment thereof as described in a first aspect of the invention, a fusion protein as described in a second aspect of the invention, a multispecific antibody as described in a third aspect of the invention, a bispecific antibody as described in a fourth aspect of the invention, a recombinant protein as described in a fifth aspect of the invention, an immunoconjugate as described in a tenth aspect of the invention, or a pharmaceutical composition as described in a thirteenth aspect of the invention.

[0110] In another preferred embodiment, the object includes a human or a non-human mammal.

[0111] In another preferred embodiment, the tumor includes Her2-positive tumors and Her2 and Trop2-positive tumors.

[0112] In another preferred embodiment, the tumor includes, but is not limited to, breast cancer, cervical cancer, ovarian cancer, gastric cancer, prostate cancer, urothelial carcinoma, colon cancer, pancreatic cancer, non-small cell carcinoma, bladder cancer, endometrial cancer, glioblastoma, salivary gland cancer, peritoneal cancer, penile cancer, intrahepatic bile duct cancer, thyroid cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, lung adenocarcinoma, esophageal adenocarcinoma, neuroendocrine tumors, pia mater cancer, gastrointestinal cancers, melanoma, ductal carcinoma in situ, serous uterine carcinoma, biliary tract cancer, and skin cancer.

[0113] 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

[0114] Figure 1 shows the results of flow cytometry analysis of the binding activity of Her2 single-domain antibody to MDA-MB-453 cells.

[0115] Figure 2 shows the results of flow cytometry analysis of the binding activity of Her2 monoclonal antibody to MDA-MB-453 cells.

[0116] Figure 3 shows the results of flow cytometry analysis of the binding activity of Her2 single-domain antibody to N87 cells.

[0117] Figure 4 shows the results of flow cytometry analysis of the binding activity of Her2 monoclonal antibody to N87 cells.

[0118] Figure 5 shows the results of flow cytometry analysis of the endocytic activity of Her2 single-domain antibody in N87 cells.

[0119] Figure 6 shows the results of ELISA detection of the antigen recognition epitopes of Her2 single-domain antibody and Trastuzumab.

[0120] Figure 7 shows the results of ELISA detection of the antigen recognition epitopes of Her2 single-domain antibody and Pertuzumab.

[0121] Figure 8 is a schematic diagram of the structure of the constructed Her2 / Trop2 bispecific antibody.

[0122] Figure 9 shows the results of flow cytometry analysis of the binding activity of Her2 / Trop2 bispecific antibody to BT474 cells.

[0123] Figure 10 shows the results of flow cytometry analysis of the binding activity of Her2 / Trop2 bispecific antibody to SK-OV-3 cells.

[0124] Figure 11 shows the results of flow cytometry analysis of the binding activity of Her2 / Trop2 bispecific antibody to N87 cells.

[0125] Figure 12 shows the results of flow cytometry analysis of the endocytic activity of Her2 / Trop2 bispecific antibody in SK-OV-3 cells.

[0126] Figure 13 shows the changes in tumor volume in a mouse model of N87 tumors after administration of Her2 / Trop2 bispecific antibody. Detailed Implementation

[0127] Through extensive and in-depth research, the inventors isolated a single-domain antibody gene from camel peripheral blood lymphocytes immunized with the HER2 antigen protein. After extensive screening using phage display technology, a novel antigen-specific Her2 single-domain antibody was obtained. After humanizing the screened Her2 single-domain antibody, it was combined with a humanized Trop2 single-domain antibody to construct a bispecific single-domain antibody against both Her2 and Trop2. Experimental results show that the Her2 single-domain antibody of this invention exhibits excellent cell-binding and endocytic activity, with significantly superior binding activity compared to existing Her2 monoclonal antibodies. Furthermore, the bispecific antibody constructed in this invention demonstrates significantly superior binding and endocytic activity compared to single-target antibodies, exhibiting a synergistic anti-tumor effect targeting both targets. Based on these findings, this invention was completed.

[0128] the term

[0129] To better understand this invention, the following terms are defined.

[0130] Unless the context clearly indicates otherwise, the term “about” includes values ​​within the standard deviation range of the stated value.

[0131] Unless the context clearly requires otherwise, throughout the specification and claims, the words “comprising,” “having,” “including,” etc., should be understood as encompassing rather than exclusive or exhaustive; that is, meaning “including but not limited to.” Unless otherwise stated, “comprising” includes “consisting of.”

[0132] In this invention, the "subject" or "patient" is an animal, including human patients requiring Her2 and / or Trop2-targeted disease treatment. In some aspects, the invention can also be applied in veterinary practice to any mammal or other animal requiring such Her2 and / or Trop2-targeted treatment. This may include, for example, non-human primates, dogs, felines, pigs, horses, and any other animals requiring Her2 and / or Trop2 treatment.

[0133] As used herein, the terms "Her2 single-domain antibody of the present invention", "anti-Her2 single-domain antibody of the present invention", "anti-Her2 single-domain antibody", "Her2 single-domain antibody", "single-domain antibody targeting Her2", "anti-Her2 nanobody", and "Her2 nanobody" have the same meaning and can be used interchangeably, all referring to the antibody containing a VHH chain that specifically recognizes and binds to the Her2 protein, screened and prepared according to the present invention.

[0134] As used herein, the terms "bispecific antibody of the present invention," "anti-Her2 and Trop2 bispecific antibody," "Her2 / Trop2 bispecific antibody," "Her2 / Trop2 bispecific single-domain antibody," and "anti-Her2 and Trop2 bispecific single-domain antibody" have the same meaning and can be used interchangeably. They all refer to antibodies constructed according to the present invention that simultaneously recognize and bind to both Her2 and Trop2 proteins, including the anti-Her2 single-domain antibody VHH chain and the anti-Trop2 single-domain antibody VHH chain of the present invention.

[0135] 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.

[0136] As used herein, the terms "nanobody" and "single domain antibody (sdAb)" have the same meaning and are used interchangeably. They refer to the cloning of the variable region of the antibody heavy chain to construct a single-domain antibody consisting of only one heavy chain variable region (VHH). 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 consisting of only one heavy chain variable region.

[0137] 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 framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet 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.

[0138] As used in this article, the terms “heavy chain variable region” and “VH” are used interchangeably.

[0139] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0140] 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., antibody-drug conjugate 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.

[0141] 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 complementarity-determining regions (CDRs). These regions are divided into four frame regions (FRs). 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 β-sheets formed by the FRs. 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 antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0142] The variable regions of the heavy chains of the 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 variable regions of antibody heavy chains with CDRs, provided that their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein.

[0143] 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.

[0144] 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.

[0145] The term "antibody" in this invention refers to a polypeptide containing the aforementioned CDR region and possessing Her2 and / or Trop2 binding activity. This term also includes variants of polypeptides containing the aforementioned CDR region that have the same function as the antibodies of this 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 this invention.

[0146] 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.

[0147] The present invention also provides other polypeptides, such as fusion proteins comprising a single-domain antibody or a fragment thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the single-domain antibody of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, 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.

[0148] 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 A.

[0149] Table A

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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 are hybridizable 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%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] The 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 parts, or any combination of the above substances.

[0163] 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.

[0164] Therapeutic agents that can bind to or conjugate with the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines; 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.

[0165] Anti-Her2 single-domain antibody

[0166] Through extensive screening, this invention provides a single-domain antibody specifically targeting the Her2 protein with high binding affinity and improved endocytosis efficiency. Furthermore, through humanization, a corresponding humanized anti-Her2 single-domain antibody is provided.

[0167] The camel-derived anti-Her2 single-domain antibody obtained in this invention was named Nb2, and humanized based on Nb2 to obtain the humanized anti-Her2 single-domain antibody Nb2. The VHH chain of the Nb2 antibody before and after humanization contains CDR1 as shown in SEQ ID NO:2, CDR2 as shown in SEQ ID NO:3, and CDR3 as shown in SEQ ID NO:4, divided according to the IMGT rule.

[0168] The VHH chain of the anti-Her2 single-domain antibody Nb2 of the present invention has the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the VHH chain has an amino acid sequence that is at least 80% (preferably at least 85%, more preferably at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence shown in SEQ ID NO:1.

[0169] In one embodiment of the invention, the VHH chain of the humanized anti-Her2 single-domain antibody Nb2 has an amino acid sequence as shown in SEQ ID NO:9. In some embodiments, the VHH chain has an amino acid sequence that is at least 80% (preferably at least 85%, more preferably at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence shown in SEQ ID NO:9.

[0170] Anti-Her2 and Trop2 bispecific antibodies

[0171] The present invention also provides a bispecific antibody comprising the anti-Her2 single-domain antibody and the anti-Trop2 single-domain antibody described herein. In one embodiment of the present invention, the VHH chain of the anti-Trop2 single-domain antibody comprises CDR1 as shown in SEQ ID NO:13, CDR2 as shown in SEQ ID NO:14, and CDR3 as shown in SEQ ID NO:15. In a preferred embodiment of the present invention, the anti-Her2 and Trop2 bispecific antibody comprises the VHH chain of the humanized Her2 single-domain antibody Nb2 and the humanized anti-Trop2 single-domain antibody VHH chain; specifically, it comprises the VHH chain of the humanized Her2 single-domain antibody Nb2 as shown in SEQ ID NO:9 and the humanized anti-Trop2 single-domain antibody VHH chain as shown in SEQ ID NO:12.

[0172] The anti-Her2 and Trop2 bispecific antibody of the present invention is obtained by linking an anti-Her2 single-domain antibody VHH chain and an anti-Trop2 single-domain antibody via a linker peptide. Available linker peptides include flexible and rigid linker peptides commonly used in the art, such as classic GS linker peptides like G4S, (G4S)2, (G4S)3, and (G4S)4; SG repeat sequences like SGSG or SGSGSG; and EAAAK repeat sequences like (EAAAK)3. Furthermore, a short peptide sequence can be linked to the C-terminus of the bispecific antibody, so that the C-terminus of the antibody contains a free cysteine ​​residue for site-specific conjugation of drug delivery (e.g., drugs, toxins, radionuclides, etc.). Available C-terminal linker peptides include GGGSCSA, GSC, GGC, GGGGC, GGGGCGGGGC, EEEEGC, and DDDGC.

[0173] In one specific embodiment of the present invention, a bispecific antibody against Her2 and Trop2 with the structure shown in FIG8 was constructed. Specifically, the VHH chain of the humanized Her2 single-domain antibody Nb2 as shown in SEQ ID NO:9 and the VHH chain of the humanized anti-Trop2 single-domain antibody as shown in SEQ ID NO:12 were linked by a (G4S)4 linker peptide as shown in SEQ ID NO:16, and a short peptide GGGSCSA was linked to the C-terminus. Further, the constructed bispecific antibody was site-conjugated with a drug such as MMAE via a free cysteine ​​residue at the C-terminus to obtain a bispecific antibody-drug conjugate (conjugate).

[0174] Pharmaceutical Composition

[0175] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above-described antibody or its active fragment, or its fusion protein, or its antibody-drug conjugate, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 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.

[0176] The pharmaceutical compositions of the present invention can be directly used to bind Her2 and / or Trop2 protein molecules, and are therefore used to treat Her2 and / or Trop2-mediated diseases, such as cancers or tumors, including Her2-positive tumors (e.g., breast cancer, gastric cancer), Trop2-positive tumors (e.g., breast cancer, cervical cancer, prostate cancer, urothelial carcinoma), and Her2 and Trop2-double-positive tumors. Furthermore, other therapeutic agents can be used concurrently.

[0177] 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 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0178] In one embodiment of the invention, when using the pharmaceutical composition, a safe and effective amount of the antibody or antibody-drug conjugate of the invention is administered to a mammal, wherein the safe and effective amount is typically 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 between about 10 micrograms per kilogram of body weight and 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 a skilled physician's expertise.

[0179] Reagent kits and detection methods

[0180] The present invention also provides a kit containing the antibody (or fragment thereof) or detection reagent of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0181] This invention also provides a detection kit for detecting Her2 levels. The kit includes an antibody that recognizes the Her2 protein (the anti-Her2 single-domain antibody described in this invention), 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.

[0182] The present invention also relates to a method for detecting Her2 protein. 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.

[0183] 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.

[0184] application

[0185] As described above, the Her2 single-domain antibody of the present invention has broad biological and clinical application value, encompassing multiple fields related to Her2, particularly the diagnosis and treatment of Her2-positive tumors, basic medical research, and biological research. The Her2 / Trop2 bispecific antibody of the present invention can be used for the clinical treatment and basic medical research of Her2 and / or Trop2-positive tumors. A preferred application is the treatment of Her2 and Trop2 double-positive tumors, such as Her2 and Trop2 double-positive breast cancer, gastric cancer, and ovarian cancer.

[0186] The main advantages of this invention include:

[0187] (1) The Her2 single-domain antibody obtained in this invention has better cell binding activity than commercially available products with the same target.

[0188] (2) The Her2 single-domain antibody obtained in this invention has a different antigen recognition epitope from commercially available antibodies targeting the same target.

[0189] (3) The Her2 / Trop2 bispecific antibody obtained in this invention can bind to a variety of cancer cells and has significantly better cell binding activity and endocytosis effect than single-target antibodies.

[0190] (4) The Her2 single-domain antibody and the Her2 / Trop2 bispecific antibody obtained in this invention have excellent anti-tumor effects, and the Her2 / Trop2 bispecific antibody exhibits a synergistic anti-tumor effect targeting two targets.

[0191] The present invention is 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 and Russell et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001), CSHL Press), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0192] Example 1: Screening and expression of Her2 single-domain antibodies

[0193] High-purity recombinant Her2 protein was mixed with an immune adjuvant and injected into Bactrian camels. Peripheral blood was collected from the camels weekly for seven weeks. Total RNA was prepared from isolated lymphocytes, and the VHH gene for single-domain antibody amplification was performed. These fragments were cloned into a phage vector and transformed into *E. coli* TG1 cells to construct a phage display library. Specific phages were repeatedly panned and enriched from the library using recombinant Her2 protein. Multiple clones were randomly selected for periplasmic ELISA identification, and positive clones were sequenced. Single-domain antibodies with different family sequences were expressed using *E. coli*.

[0194] Example 2: Detection of cell binding activity of Her2 single-domain antibody

[0195] The binding activity of candidate single-domain antibodies to Her2-overexpressing cell lines was detected by flow cytometry. The method is briefly described below: Her2-overexpressing cell lines MDA-MB-453 and N87 were seeded at 2E5 cells per well in 96-well plates. Serially diluted candidate Her2 single-domain antibodies were incubated with MDA-MB-453 and N87 cells at 4°C for 30 min. After washing the cells with PBS, APC anti-HA antibody was added and incubated at 4°C for 30 min. After centrifugation, the cells were washed to remove the supernatant, and the cells were resuspended in PBS. The APC signal of each sample was then detected by flow cytometry.

[0196] The results are shown in Figures 1 and 2. All five Her2 single-domain antibodies showed good binding activity with MDA-MB-453 cells, with Nb1 and Nb2 exhibiting better cell binding activity than the control monoclonal antibody.

[0197] As shown in Figures 3 and 4, all five Her2 single-domain antibodies exhibited good binding activity with N87 cells, and the cell binding activity of Nb1, Nb2, Nb3, and Nb5 was superior to that of the control monoclonal antibody.

[0198] Example 3: Endocytotic activity of Her2 single-domain antibody

[0199] Flow cytometry was used to detect the endocytic activity of candidate single-domain antibodies on Her2-overexpressing cells. The method is briefly described below: N87 cells were seeded at 5E5 cells per well in 96-well plates. Serially diluted candidate Her2 single-domain antibodies were incubated with cells at 4°C for 30 min. After washing the cells with PBS, APC anti-HA antibody was added and incubated at 4°C for 30 min. After washing the cells, medium containing 1% FBS was added to the non-endocytosis (0 h) experimental group and the maximum endocytosis (0 h) experimental group. The cells were resuspended and placed at 4°C. Simultaneously, medium containing 1% FBS was added to the endocytosis (0.25 h, 0.5 h, 1 h, and 2 h) experimental groups, and the plates were incubated at 37°C in a CO2 incubator. Samples were collected at 0.25 h, 0.5 h, 1 h, and 2 h and placed at 4°C. Finally, all samples were centrifuged, the supernatant was removed, and stripping buffer was added and incubated at 4°C. After washing the cells, PBS was added and the APC signal of each sample was detected by flow cytometry.

[0200] As shown in Figure 5, all five Her2 single-domain antibodies exhibited good endocytic activity.

[0201] Example 4: Epitope Analysis of Her2 Single-Domain Antibody

[0202] The binding of five Her2 single-domain antibodies to Her2 antigen protein was detected by ELISA after co-incubation with Trastuzumab or Pertuzumab for epitope analysis.

[0203] Recombinant Her2 protein was coated onto 96-well plates overnight at 4°C. After blocking with 3% milk at room temperature for 2 hours, serially diluted Her2 single-domain antibody and a certain amount of biotin-Trastuzumab or biotin-Pertuzumab were added. After incubation at room temperature for 1 hour, diluted SA-HRP was added and incubated at 37°C for 30 minutes. TMB was added for color development and the reaction was terminated with sulfuric acid. The absorbance at 450 nm was detected by microplate.

[0204] The results of co-incubation with Trastuzumab are shown in Figure 6. The results show that Nb5 has similar antigen recognition epitopes to Trastuzumab, while the epitopes of the other four antibodies are different from those of Trastuzumab.

[0205] The results of co-incubation with Pertuzumab are shown in Figure 7. The results show that all five single-domain antibodies have different antigen recognition epitopes from Pertuzumab.

[0206] Example 5: Humanized design of Her2 single-domain antibody and construction and expression of bispecific single-domain antibody

[0207] 5.1 Humanization Design of Her2 Single-Domain Antibodies

[0208] First, the Her2 single-domain antibody Nb2 (amino acid sequence shown in SEQ ID NO:1) was humanized. The CDR was kept unchanged by CDR grafting, and only the FR region was humanized by mutation. The amino acid sequence of the mutated antibody is shown in SEQ ID NO:9.

[0209] The humanized and unhumanized Her2 single-domain antibodies Nb2 were labeled with biotin, and their binding activity to Her2-overexpressing cells was identified by flow cytometry. 2E5 cells were seeded in each well of a 96-well plate, and serially diluted biotinylated humanized and unhumanized single-domain antibodies were incubated with BT474 cells at 4°C for 30 min. After washing the cells with PBS, diluted SA-PE was added and incubated at 4°C for 30 min. After centrifugation, the cells were washed again to remove the supernatant, and the cells were resuspended in PBS. PE signal was detected for each sample by flow cytometry. The results are shown in Table 1 below. The humanized Her2 single-domain antibodies maintained good biological activity.

[0210] Table 1

[0211] 5.2 Construction of Bispecific Single-Domain Antibodies

[0212] The humanized Her2 single-domain antibody Nb2 was fused with a Trop2 single-domain antibody (SEQ ID NO:12, antibody sequence derived from the HuNb1 sequence in PCT application WO2024051383A1). The two single-domain antibodies were linked via an amino acid linker (SEQ ID NO:16) and expressed using Pichia pastoris cells. Simultaneously, a short amino acid sequence was designed to be added to the C-terminus to maintain a free cysteine ​​residue (sequence shown in SEQ ID NO:17) for site-specific conjugation of other substances (such as drugs or radionuclides). The constructed bispecific antibody structure is shown in Figure 8.

[0213] The designed amino acid sequence (as shown in SEQ ID NO:18) was synthesized into the pPICZaA vector according to the Pichia pastoris codon-optimized base sequence (as shown in SEQ IN NO:19). The linearized vector was then electroporated into X-33 yeast competent cells, and the electroporated cells were plated in bleomycin-resistant agar plates and grown for 3-4 days. Single colonies were picked for induction culture, and the antibody expression level in the culture supernatant was detected. High-purity antibody protein was then obtained by protein A affinity chromatography.

[0214] Example 6: Detection of cell binding activity of bispecific single-domain antibodies

[0215] The binding activity of the bispecific single-domain antibody constructed in Example 5 to the highly expressed cell line was detected by flow cytometry. The method is briefly described below: The highly expressed cell line was seeded at 2E5 cells per well in a 96-well plate. Serially diluted Her2 / Trop2 bispecific single-domain antibody was added to N87, Bt474, and SKOV3 cells and incubated at 4°C for 30 min. After washing the cells with PBS, goat anti-VHH antibody-biotin was added and incubated at 4°C for 30 min. After centrifugation, the cells were washed and the supernatant was removed. Diluted SA-PE was added and incubated at 4°C for 30 min. After centrifugation again, the cells were washed and the supernatant was removed. The cells were resuspended in PBS, and the PE signal of each sample was detected by flow cytometry.

[0216] The results are shown in Figures 9, 10 and 11. The candidate Her2 / Trop2 bispecific single-domain antibody showed good binding activity with different types of tumor cell lines, and the bispecific antibody showed more significant binding activity than the single antibody.

[0217] Example 7: Endocytotic activity of bispecific single-domain antibodies

[0218] Flow cytometry was used to detect the endocytic activity of bispecific single-domain antibodies on cells with high expression. The method is briefly described below: BT474 cells were seeded at 5E5 cells per well in 96-well plates. Serially diluted biotinylated single-domain antibodies were incubated with the cells at 4°C for 30 min. After washing the cells with PBS, SA-PE was added and incubated at 4°C for 30 min. After washing the cells, medium containing 1% FBS was added to the non-endocytosis (0 h) experimental group and the maximum endocytosis (0 h) experimental group. The cells were resuspended and placed at 4°C. Simultaneously, medium containing 1% FBS was added to the endocytosis (0.5 h, 1 h, and 2 h) experimental groups, and the plates were incubated at 37°C in a CO2 incubator. Samples were collected at 0.5 h, 1 h, and 2 h and placed in a 4°C freezer. Finally, the samples were centrifuged, the supernatant was removed, and stripping buffer was added and incubated at 4°C. After washing the cells, PBS was added and the PE signal of each sample was detected by flow cytometry.

[0219] The results are shown in Figure 12 and Table 2. The candidate bispecific single-domain antibody has a better endocytosis effect compared with the single-target single-domain antibody.

[0220] Table 2

[0221] Example 8: Antitumor activity of bispecific single-domain antibodies

[0222] Monoclonal antibodies expressing C-terminal free cysteine ​​and bispecific single-domain antibodies were conjugated to MMAE. The method is briefly described below: The antibody was incubated overnight at 4°C with PBS buffer containing TCEP. After removing excess TCEP, it was reacted with an equimolar ratio of Mc-Vc-PBC-MMAE (VcMMAE) at room temperature for 2 hours. The reaction was then terminated by adding excess acetylcysteine, thus obtaining the antibody-MMAE conjugate.

[0223] The in vivo antitumor efficacy of candidate bispecific single-domain antibodies was evaluated using a cell line xenograft model. N87 cells were mixed with Matrix and subcutaneously injected into 6-week-old female BALB / c nude mice. Tumor volume reached 200 mm². 3 At approximately 10:00 AM, mice were randomly divided into 5 groups of 6 mice each. The positive control group received 10 mg / kg 5-FU (5-fluorouracil), and the negative control group received PBS. The experimental groups received 5 mg / kg Her2 Nb2-MMAE, 5 mg / kg Trop2 Nb-MMAE, 5 mg / kg Her2 Nb2-Trop Nb-MMAE, and 10 mg / kg Her2Nb2-Trop Nb-MMAE, respectively. All groups were administered the medication every other day for a total of 6 doses. Tumor size in individual mice was recorded and measured periodically. Finally, all mice were euthanized, and the tumor tissue was excised and weighed.

[0224] The results are shown in Figure 13 and Table 3. Compared with the positive drug group, the tumor volume of mice in each dose group of monoclonal antibody and bispecific antibody drugs decreased significantly. Moreover, the anti-tumor effect of bispecific antibody drugs was better than that of monoclonal antibody drugs, showing a synergistic anti-tumor effect.

[0225] Table 3

[0226] Sequence information of the present invention:

[0227] 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 single domain antibody or an antigen-binding fragment thereof, characterized in that, The VHH chain of the single-domain antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): a CDR1 as set forth in SEQ ID NO: 2, a CDR2 as set forth in SEQ ID NO: 3, and a CDR3 as set forth in SEQ ID NO:

4.

2. The anti-Her2 single domain antibody or an antigen-binding fragment thereof of claim 1, wherein, The VHH chain of the single-domain antibody or antigen-binding fragment thereof has an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO:

1.

3. The anti-Her2 single domain antibody or an antigen-binding fragment thereof of claim 1, wherein, The VHH chain of the single-domain antibody or antigen-binding fragment thereof has an amino acid sequence as set forth in SEQ ID NO: 9, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO:

9.

4. A fusion protein, characterized in that, The fusion protein has a structure from N-terminus to C-terminus as set forth in Formula Ia or Ib: A-L-B (Ia); B-L-A (Ib); wherein, A is the anti-Her2 single-domain antibody or antigen-binding fragment thereof according to any one of claims 1-3; B is the Fc fragment of IgG; and L is null or a flexible linker.

5. A multispecific antibody, characterized in that The multispecific antibody comprises the anti-Her2 single-domain antibody or antigen-binding fragment thereof according to any one of claims 1-3.

6. A bispecific antibody, characterized in that The bispecific antibody comprises the anti-Her2 single-domain antibody or antigen-binding fragment thereof according to any one of claims 1-3, and an anti-Trop2 single-domain antibody or antigen-binding fragment thereof.

7. The bispecific antibody of claim 6, wherein The VHH chain of the anti-Trop2 single-domain antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): a CDR1 as set forth in SEQ ID NO: 13, a CDR2 as set forth in SEQ ID NO: 14, and a CDR3 as set forth in SEQ ID NO:

15.

8. The bispecific antibody as described in claim 7, characterized in that, The VHH chain of the anti-Trop2 single-domain antibody or antigen-binding fragment thereof has an amino acid sequence as set forth in SEQ ID NO: 12, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO:

12.

9. The bispecific antibody of any one of claims 6-8, wherein, The bispecific antibody has a structure from N-terminus to C-terminus as set forth in Formula II(a) or II(b): H-L1-T-P II(a) T-L1-H-P II(b) wherein each of “-” is independently a peptide bond; H is the anti-Her2 single-domain antibody or antigen-binding fragment thereof; T is the anti-Trop2 single-domain antibody or antigen-binding fragment thereof; L1 is a connecting peptide; P is null or a C-terminal connecting peptide.

10. The bispecific antibody as described in claim 9, characterized in that, The P is a C-terminal connecting peptide, which makes the C-terminus of the bispecific antibody contain a free cysteine for site-directed conjugation of a drug load.

11. A recombinant protein, characterized in that, The recombinant protein has: (i) the anti-Her2 single-domain antibody or antigen-binding fragment thereof according to any one of claims claims 1-3, the multispecific antibody according to claim 5, or the bispecific antibody according to any one of claims 6-10; and (ii) an optional tag sequence for facilitating expression and / or purification.

12. A polynucleotide encoding a protein selected from the group consisting of the anti-Her2 single domain antibody or antigen-binding fragment thereof of any one of claims 1-3, the fusion protein of claim 4, the multispecific antibody of claim 5, the bispecific antibody of any one of claims 6-10, or the recombinant protein of claim 11.

13. An expression vector comprising the polynucleotide of claim 12.

14. An immunoconjugate, comprising, The immunoconjugate comprises: (a) the anti-Her2 single domain antibody or antigen-binding fragment thereof of any one of claims 1-3, the fusion protein of claim 4, the multispecific antibody of claim 5, the bispecific antibody of any one of claims 6-10, or the recombinant protein of claim 11; and (b) a conjugating moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a viral coat protein or VLP, or a combination thereof.

15. Use of the anti-Her2 single domain antibody or antigen-binding fragment thereof of any one of claims 1-3, the fusion protein of claim 4, the recombinant protein of claim 11, or the immunoconjugate of claim 14, for the manufacture of: (a) a reagent for detecting Her2 molecules; or (b) a medicament for treating or preventing Her2-positive tumors.

16. Use of the bispecific antibody of any one of claims 6-10, the recombinant protein of claim 11, or the immunoconjugate of claim 14, for the manufacture of: (a) a reagent for detecting Her2 and / or Trop2 molecules; or (b) a medicament for treating or preventing Her2 and / or Trop2-positive tumors. ​

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