Trop2-binding nanobody, product and application thereof

Abstract

The invention provides a Trop2-combined nano antibody as well as a product and application thereof, and relates to the technical field of antibodies. The invention discloses a nano antibody specifically binding to Trop2 protein, and a VHH sequence and a corresponding CDR sequence of the nano antibody. The nano antibody has the capability of high-affinity binding to target protein, and the nano antibody subjected to humanized modification still keeps the high-affinity binding characteristic.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202411472453.4 (22) Application Date 2024.10.21 (71) Applicant Beijing Hexin Biotechnology Co., Ltd. Address 102604 No. 312, 3rd Floor, Building E2, Free Trade Innovation Service Center, Daxing Airport Area, Beijing Free Trade Zone, No. 1 Yuanping North Road, Lixian Town, Daxing District, Beijing Applicant Beijing Hexin Xunming Pharmaceutical Technology Co., Ltd. (72) Inventors Zhang Peng, Wu Peiwen, Huangfu Zhenyuan, Jin Guang, Wang Ximei, Zuo Conglin (74) Patent Agency Beijing Jingjinshi Intellectual Property Agency Co., Ltd. 11470 Patent Attorney Zhao Xiaoyan (51) Int.Cl. C07K 16 / 30 (2006.01) C12N 15 / 13 (2006.01) A61K 39 / 395 (2006.01) A61K 47 / 68(2017.01) A61P 35 / 00(2006.01) G01N 33 / 68(2006.01) G01N 33 / 57515(2026.01) (54) Invention Title: Trop2-Binding Nanobody, Its Products and Applications (57) Abstract: This invention provides a Trop2-binding nanobody, its products and applications, relating to the field of antibody technology. This invention discloses a nanobody that specifically binds to the Trop2 protein, along with its VHH sequence and corresponding CDR sequence. This nanobody possesses high affinity for binding target proteins, and the humanized nanobody retains its high affinity binding properties. Claims (2 pages), Description (12 pages), Sequence Listing (electronic publication), Drawings (4 pages) CN 121949556 A 2026.05.01 CN 1 21 94 95 56 A 1. A nanobody, characterized in that the nanobody can specifically bind to Trop2 protein, and the complementarity-determining region (CDR) of the VHH chain in the nanobody is as follows: CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.3. 2. The nanobody according to claim 1, characterized in that the VHH chain of the nanobody further includes a framework region (FR), the framework region (FR) being as follows: FR1 shown in SEQ ID NO.4, FR2 shown in SEQ ID NO.5, FR3 shown in SEQ ID NO.6, and FR4 shown in SEQ ID NO.7, or FR1 shown in SEQ ID NO.8, FR2 shown in SEQ ID NO.9, FR3 shown in SEQ ID NO.10, and FR4 shown in SEQ ID NO.11.3. The nanobody according to claim 1 or 2, characterized in that the amino acid sequence of the VHH chain of the nanobody is as shown in SEQ ID NO. 12 or SEQ ID NO. 13. 4. The nanobody according to claim 3, characterized in that the amino acid sequence of the VHH chain of the nanobody has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to SEQ ID NO. 12 and the CDR region is the same, or the amino acid sequence of the VHH chain of the nanobody has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to SEQ ID NO. 13 and the CDR region is the same. 5. A conjugate, characterized in that the conjugate is prepared by conjugating a nanobody according to any one of claims 1-4 with a conjugating element. 6. The conjugate according to claim 5, characterized in that the conjugating element comprises: a fluorescent or luminescent marker, a radionuclide, a contrast agent for magnetic resonance imaging or computed tomography, an enzyme, a cytokine, an antibody VHH fragment, an antibody Fc fragment, an antibody scFv fragment, a viral particle, a liposome, a prodrug activating enzyme, a chemotherapeutic agent, or nanoparticles. 7. The conjugate according to claim 6, characterized in that the antibody Fc fragment is the human IgG1 heavy chain constant region, and the sequence of the human IgG1 heavy chain constant region is SEQ ID NO. 14. 8. An antibody formulation, characterized in that the antibody formulation comprises a nanobody according to any one of claims 1-4 and at least one pharmaceutically acceptable carrier. 9. A nucleic acid molecule, characterized in that the nucleic acid molecule encodes a nanobody according to any one of claims 1-4. 10. The nucleic acid molecule according to claim 9, wherein the nucleic acid molecule sequence is SEQ ID NO. 15 or SEQ ID NO. 16. 11. An expression vector, wherein the expression vector comprises the nucleic acid molecule according to any one of claims 9-10. 12. The expression vector according to claim 11, wherein the expression vector comprises a prokaryotic expression vector and a eukaryotic expression vector. 13. A host cell, wherein the host cell comprises the expression vector according to claim 11 or 12, or the host cell genome comprises the nucleic acid molecule according to claim 9 or 10. 14. The host cell according to claim 13, wherein the host cell comprises bacterial or mammalian cells, wherein the bacteria comprise *Escherichia coli*, and the mammalian cells comprise HEK293 cells and CHO cells.15. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the nanobody of any one of claims 1-4, the conjugate of any one of claims 5-7, the antibody preparation of claim 8, the nucleic acid molecule of any one of claims 9-10, the expression vector of any one of claims 11-12, or the host cell of any one of claims 13-14. 16. A method for detecting Trop2 protein, characterized in that the detection is performed using the nanobody of any one of claims 1-4, the conjugate of any one of claims 5-7, the antibody preparation of claim 8, the nucleic acid molecule of any one of claims 9-10, the expression vector of any one of claims 11-12, or the host cell of any one of claims 13-14, wherein the detection method is a non-disease diagnosis or treatment method. 17. The use of the nanobody according to any one of claims 1-4 in the preparation of a medicament for the prevention and / or treatment of cancer. 18. The use according to claim 17, characterized in that the cancer is breast cancer, triple-negative breast cancer, lung cancer, gastric cancer, pancreatic cancer, or prostate cancer. 19. A product comprising the nanobody according to any one of claims 1-4, the conjugate according to any one of claims 5-7, the antibody preparation according to claim 8, the nucleic acid molecule according to any one of claims 9-10, the expression vector according to any one of claims 11-12, or the host cell according to any one of claims 13-14, characterized in that the product is a drug or a kit. 20. The product according to claim 19, characterized in that the drug is an antitumor drug, and the kit is a Trop2 protein detection kit. Claims 2 / 2 Page 3 CN 121949556 A Trop2-bound nanobody and its products and applications Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to Trop2-bound nanobodies and their products and applications. Background Art

[0002] Trop-2 (human trophoblast cell surface glycoprotein antigen 2) is a type I transmembrane glycoprotein highly expressed in various tumors, especially in triple-negative breast cancer, and its expression level is closely related to tumor invasiveness and prognosis. Trop-2 consists of 323 amino acid residues, including an extracellular region, a transmembrane region, and a cytoplasmic tail, and participates in intercellular and cell-matrix interactions.

[0003] As a member of the GA733 gene family, Trop-2's structural features enable it to participate in various intercellular interactions. Trop2 is expressed in trophoblast cells and is crucial for cell proliferation and differentiation. Trop-2 regulates processes such as apoptosis, angiogenesis, and tumor metastasis through a complex signaling network.Specific mechanisms include binding to IGF-1 to inhibit the IGF-1R signaling pathway, downregulating AP-1 levels through RAS-MAPK / ERK, binding to β-catenin to promote the transcription of tumor-related genes, activating the β1 integrin-RACK1-FAK-Src signaling axis to regulate cell adhesion, and mediating Ca2+ release to affect the cell cycle.

[0004] The high expression and low normal tissue expression of Trop-2 in breast cancer make it an ideal target for cancer treatment. In addition, Trop-2 is also one of the markers of breast cancer stem cells, which have strong self-renewal and tumor-forming capabilities.

[0005] Nanobody (Nb), also known as single-domain antibody (sdAbs), VHH antibody or camelid antibody, is derived from natural heavy chain antibodies (HCAbs) of camel family and some cartilaginous fish. Its characteristic is the absence of light chain, and its molecular weight is about one-tenth of that of traditional antibodies (12-14kDa), which is the smallest complete antigen-binding fragment.

[0006] Nanobodies are structurally similar to ordinary antibodies (VH), but the FR2 region of VHH contains hydrophilic amino acid substitutions, enhancing water solubility; simultaneously, its CDR3 is longer, forming a convex structure, improving its ability to recognize hidden antigenic epitopes. Nanobodies are diverse, including monovalent, bivalent, bispecific, multivalent, and fusion types, and their monomeric structures are easy to modify. Nanobodies have advantages such as simple preparation, high stability, excellent tissue permeability, and ease of humanization, and are receiving increasing attention in the field of anti-tumor drug development.

[0007] Antibodies, antibody-drug conjugates, and combination therapies targeting Trop2 are under development. The efficacy of anti-Trop2 antibodies conjugated with other chemotherapeutic drugs has been confirmed in various preclinical studies. The antibody-drug conjugate IMMU-132 for the treatment of Trop2-overexpressing epithelial malignancies is in phase II / III clinical trials. IMMU-132 targets Trop2 and is formulated by conjugating the humanized antibody hRS7 as a targeting vector to the active metabolite SN38 of irinotecan. It can be used to treat various epithelial malignancies such as breast cancer (triple-negative breast cancer), ovarian cancer, and small cell lung cancer. Furthermore, other humanized anti-Trop2 IgG-SN-38 conjugates, such as the anti-Trop2 hRS7-CL2A-SN-38 antibody-drug conjugate, have been shown to have significant specific anticancer effects in xenograft models of various tumor cell lines (Calu-3, Capan-1, BxPC-3, and COLO-205).Studies have found that, based on the previously constructed Trop2-Fab antibody, a Trop2-IgG antibody was constructed using a eukaryotic expression system, and in vitro and in vivo experiments demonstrated that this novel human monoclonal Trop2-IgG antibody has an inhibitory effect on tumors. Trop2 promotes the growth and metastasis of glioblastoma cells by activating the JAK2 / STAT3 pathway, which provides a basis for further understanding the role of Trop2 in glioblastoma, indicating that Trop2 is a potential biomarker and therapeutic target for glioblastoma patients. Specification 1 / 12 pages 4 CN 121949556 A

[0008] Prior art CN116406424A provides a heavy chain antibody that specifically binds to human Trop2, or its antigen-binding moiety. It also provides a nucleic acid molecule encoding the antibody or its antigen-binding moiety, an expression vector, host cell, and method for expressing the antibody or its antigen-binding moiety. It also provides an immunoconjugate and pharmaceutical composition containing the antibody or its antigen-binding moiety, and a treatment method using the Trop2 antibody or its antigen-binding moiety.

[0009] Existing technology CN111518212B discloses a group of nanobodies specifically targeting Trop2, disclosing the amino acid sequences of the framework region FR and complementarity-determining region CDR of its VHH chain, and also disclosing the gene sequence encoding the nanobody or its VHH chain. The anti-Trop2 nanobodies provided by this technology can effectively bind to Trop2 antigens, providing a research foundation for Trop2-targeted research, including the development of antibody-drug conjugates and multispecific antibody drugs.

[0010] To date, no nanobody drugs targeting Trop2 have been published on the market. There is a need in the art to develop an anti-Trop2 nanobody, especially an anti-Trop2 nanobody with good Trop2 antigen binding ability and strong functional activity. Developing new nanobodies that recognize Trop2 targets, and further preparing monoclonal antibody drugs and antibody-drug conjugates (such as conjugated small molecule toxins or radioactive metal elements) for the treatment of various malignant tumors or for combination therapy with other drugs or therapies, is of practical significance for improving patient survival.

[0011] The present invention addresses the problems existing in the prior art by providing a nanobody that binds to Trop2, its products, and applications. This nanobody can specifically bind to the Trop2 protein with strong binding ability; its VHH sequence and corresponding CDR sequence are clear and novel.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] In one aspect, the present invention provides a nanobody that can specifically bind to the Trop2 protein.

[0014] Nanobody (Nb), also known as single-domain antibody (sdAbs), VHH antibody, or camelid antibody, is an artificially designed antibody molecule with unique structural and functional properties. Nanobody is a single-domain antibody containing only the heavy chain variable region (VHH), and its molecular weight is much smaller than that of traditional antibodies, hence the name nanobody. Nanobody was initially discovered in camels such as alpacas and dromedaries, as well as cartilaginous fish such as sharks and rays. Nanobody is encoded by a single gene and contains only the heavy chain variable region (VHH), lacking light chains and other heavy chain constant regions. Its crystal structure is elliptical, with a size of approximately 4.0 nm × 2.5 nm × 3.0 nm, and a molecular weight between 12 and 14 kDa, only about 1 / 10 the molecular weight of traditional antibodies.

[0015] CDR stands for Complementarity-determining region, a key part of the antibody molecule that plays a decisive role in the antibody's specificity and affinity. The antibody CDR (Complementarity Determinant Region) is the region in an antibody molecule responsible for binding to the antigen, also known as the hypervariable region (HVR) or complementarity-determining region. The highly variable amino acid sequence of the CDR region allows antibodies to recognize and bind to a variety of different antigens. This high sequence variability and spatial conformational diversity are the basis for antibody specificity and affinity. CDR1 and CDR2 are relatively short and do not vary much, while CDR3 is the most variable and significantly different in length within the CDR region. The length of CDR3 varies not only between different antibodies but can also differ significantly between different antibodies of the same species. There are several rules for dividing the antibody complementarity-determining region (CDR), such as Kabat, Chothia, IMGT, and AbM. The Kabat scheme, proposed by Edelman et al. in the 1970s based on antibody sequence alignment, is one of the earliest antibody numbering systems. This scheme defines residue numbers based on the location of highly variable regions between sequences of different domain types. These schemes differ in their definition of the CDR, but they are generally consistent in numbering most conserved regions. Therefore, the results of different numbering schemes can usually be converted between each other when performing sequence alignment or analysis. Instructions 2 / 12 pages 5 CN 121949556 A

[0016] The framework region of the VHH chain is divided into four parts, namely FR1, FR2, FR3 and FR4. Each framework region is composed of specific amino acid sequences, which are crucial for maintaining the overall structure and function of the VHH chain.

[0017] The frame region and complementarity-determining region (CDR) together constitute the antigen-binding domain of the VHH chain, wherein the CDR is responsible for directly binding to the antigen, while the frame region plays a role in supporting and stabilizing the CDR.

[0018] Preferably, the VHH chain in the nanobody includes a complementarity-determining region (CDR) and a frame region (FR).

[0019] Preferably, the VHH chain in the nanobody includes a complementarity-determining region (CDR) as shown below:

[0020] CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.3;

[0021] SEQ ID NO.1: SYNMA;

[0022] SEQ ID NO.2: AIKWSGDSTLYADSVKG;

[0023] SEQ ID NO.3: DQGSWYLERREYDY.

[0024] Preferably, the framework region FR of the VHH chain of the nanobody is as follows:

[0025] FR1 shown in SEQ ID NO. 4, FR2 shown in SEQ ID NO. 5, FR3 shown in SEQ ID NO. 6 and FR4 shown in SEQ ID NO. 7, or FR1 shown in SEQ ID NO. 8, FR2 shown in SEQ ID NO. 9, FR3 shown in SEQ ID NO. 10 and FR4 shown in SEQ ID NO. 11;

[0026] SEQ ID NO. 4: QVQLVESGGGLVQAGGSLRLSCAASGRMFS;

[0027] SEQ ID NO. 5: WFRQAPGKEREFVA;

[0028] SEQ ID NO. 6: RFTISKDNAKNTMYLQMNTLKPEDTAVYYCAA;

[0029] SEQ ID NO. 7: WGQGTQVTVSS;

[0030] SEQ ID NO.8: EVQLVESGGGLVQPGGSLRLSCAASGRMFS;

[0031] SEQ ID NO.9: WFRQAPGKEREFVA;

[0032] SEQ ID NO.10: RFTISKDNSKNTLYLQMNSLRAEDTAVYYCAA;

[0033] SEQ ID NO.11: WGQGTQVTVSS.

[0034] Preferably, the amino acid sequence of the VHH chain of the nanobody is as shown in SEQ ID NO.12 or SEQ ID NO.13;

[0035] SEQ ID NO.12:

[0036] QVQLVESGGGLVQAGGSLRLSCAASGRMFSSYNMAWFRQAPGKEREFV AAIKWSGDSTLYADSVKGR FTISKDNAKNTMYLQMNTLKPEDTAVYYCAAD QGSWYLERREYDYWGQGTQVTVSS;

[0037] SEQ ID NO.13:

[0038] EVQLVESGGGLVQPGGSLRLSCAASGRMFSSYNMAWFRQAPGKEREFV AAIKWSGDSTLYADSVKGR FTISKDNSKNTLYLQMNSLRAEDTAVYYCAAD QGSWYLERREYDYWGQGTQVTVSS.

[0039] In some embodiments, the amino acid sequence of the VHH chain of the nanobody differs from the amino acid sequence of SEQ ID

[0040] NO.12 or SEQ ID NO.13 by at least one amino acid.

[0041] In some embodiments, the amino acid sequence of the VHH chain of the nanobody has at least 85% sequence similarity to SEQ ID

[0042] NO.12 or SEQ ID NO.13 and the CDR region is the same.

[0043] In some embodiments, the amino acid sequence of the VHH chain of the nanobody has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,

[0045] 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to SEQ ID

[0044] NO.12 and the CDR region is the same.

[0046] In some embodiments, the amino acid sequence of the VHH chain of the nanobody has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to SEQ ID

[0047] NO.13 and the CDR region is the same.

[0049] On the other hand, the present invention provides a conjugate prepared by conjugating the above-mentioned nanobody with a conjugation element.

[0050] Preferably, the conjugating element comprises: a fluorescent or luminescent marker, a radionuclide, a contrast agent for magnetic resonance imaging or computed tomography, an enzyme, a cytokine, an antibody VHH fragment, an antibody Fc fragment, an antibody scFv fragment, a viral particle, a liposome, a prodrug activating enzyme, a chemotherapeutic agent, or nanoparticles.

[0051] Antibody-drug conjugates (ADCs) are an innovative drug form that combines the advantages of antibodies and small molecule toxins. ADCs are produced by conjugating a biologically active small molecule drug (usually a cytotoxic drug) to a monoclonal antibody (mAb) via a specific linker. This drug design aims to utilize the targeting ability of antibodies and the highly efficient killing effect of cytotoxic drugs to achieve precise targeting of tumor cells. The monoclonal antibody here can be an antibody molecule targeting the same target or multiple targets, and the antibody-binding fragment can be in the form of Fab, scFv, VHH, or a combination thereof. Small molecule toxins are diverse and can be mainly divided into two categories according to their mechanism of action: microtubule inhibitors and DNA damaging agents. For example, MMAE (monomethylaurestatin E), MMAF (monomethylaurestatin F), maytansinoids such as DM1 and DM4, calicacins (CLM) such as oxazolidin and calicacin, and topoisomerase inhibitors such as camptothecin, hydroxycamptothecin, irinotecan, topotecan, etoposide, and teniposide.

[0052] Antibody radionuclide conjugates (ARC) are an innovative form of drug that conjugates a radionuclide to an antibody through a specific linker arm and chelating agent, thereby combining the advantages of both. This type of drug utilizes the targeting specificity of antibodies to precisely deliver radionuclides to lesion sites, performing various functions such as diagnosis and treatment within the body. Antibody-drug conjugates (ADCs) achieve targeted drug delivery by binding antibodies to specific antigens on the surface of target cells. Simultaneously, the radiation released during the decay of the radionuclide (such as alpha particles and beta rays) can directly act on target cells, causing DNA damage and inducing apoptosis or necrosis, thus achieving the goal of treating diseases such as tumors. Furthermore, by changing different radionuclides, ARC can also achieve integrated diagnosis and treatment.Commonly used radionuclides in medicine include technetium-99m (Tc-99m), gallium-68 (Ga-68), zirconium-89 (Zr-89), fluorine-18 (F-18), indium-111 (In-111), iodine-131 (I-131), yttrium-90 (Y-90), and lutetium-177 (Lu-177). Newer radionuclides include copper-64 (Cu-64), actinium-225 (Ac-225), and lead-212 (Pb-212).

[0053] Preferably, the nanobody is coupled to the coupling element via a chemical bond.

[0054] In some embodiments, the coupling element is an antibody Fc fragment.

[0055] The antibody Fc is an important component of the antibody molecule and belongs to the constant region (C region) of the antibody. Specifically, the Fc segment of an antibody is located on the two heavy chains of the antibody molecule and mainly includes the CH2 and CH3 domains (in some cases, it may also include other domains, but these two are the most important). The Fc segment is named for its ability to crystallize. The Fc segment is mainly responsible for mediating the interaction between the antibody and immune cells, such as binding to Fc receptors (FcRs), thereby triggering a series of immune responses. These responses include, but are not limited to, phagocytosis, extracellular killing, antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP). The Fc segment of IgG antibodies can bind to Fcγ receptors (such as FcγRI, FcγRIIa, FcγRIIIc, and FcγRIIIIa) on the surface of immune cells, thereby mediating immune responses such as phagocytosis and extracellular killing. There are four subtypes of human IgG antibodies: IgG1, IgG2, IgG3, and IgG4.

[0056] In some embodiments, the antibody Fc fragment is a human IgG antibody, comprising IgG1, IgG2, IgG3 and IgG4 (page 4 / 12 of specification, CN 121949556 A).

[0057] Specifically, the antibody Fc fragment is the human IgG1 heavy chain constant region, the sequence of which is SEQ ID NO.14, and the antibody Fc fragment is coupled to the above-mentioned nanobody via peptide bonds;

[0058] SEQ ID NO.14:

[0059] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK.

[0060] On the other hand, the present invention provides an antibody formulation comprising the above-described nanobody and at least one pharmaceutically acceptable carrier.

[0061] A carrier (also called a transporter, excipient, or excipient) refers to any inactive substance added in addition to the active pharmaceutical ingredient (API) when preparing a pharmaceutical formulation. These carriers play a crucial role in pharmaceutical formulations, such as improving the stability, solubility, taste, and appearance of the drug, while ensuring that the drug is accepted by the patient in an appropriate form and exerts its effect. These carriers play a crucial role in pharmaceutical formulations and can significantly improve the efficacy and safety of the drug. There are many pharmaceutically acceptable carriers, mainly including the following categories:

[0062] 1. Sugars: such as sucrose, glucose, etc., often used to increase the solubility and stability of drugs;

[0063] 2. Polymers: such as polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), etc., used to improve the release characteristics and bioavailability of drugs;

[0064] 3. Liposomes: composed of lipid bilayers such as phospholipids, which can encapsulate drugs and improve their targeting and biocompatibility;

[0065] 4. Microspheres: small spherical carriers made of polymer materials, used to control the release rate of drugs;

[0066] 5. Surfactants: such as polysorbates, Tween, etc., used to increase the solubility and stability of poorly soluble drugs;

[0067] 6. Other excipients: such as preservatives, antioxidants, buffers, etc., used to ensure the stability and safety of drug formulations.

[0068] On the other hand, the present invention provides a nucleic acid molecule, which encodes the above-mentioned nanobody.

[0069] Preferably, the nucleic acid molecule sequence is the sequence specifically shown in SEQ ID NO.15 or SEQ ID NO.16;

[0070] SEQ ID NO.15:

[0071] CAAGTGCAGTTGGTGGAAAGTGGGGGTGGACTTGTACAAGCAGGCGGGTCACTGCGGCTGTCCTGCGC AGCTTCTGGGCGGATGTTTTCCAGCTACAACATGGCTTGGTTCAGACAGGCCCCAGGAAAAGAGCGGGAATTCGTG GCCGCTATCAAGTGGTCTGGCGATTCTACCCTGTACGCAGATTCTGTGAAGGGGCGCTTCACTATTTCCAAGGACA ACGCTAAGAACACCATGTACTTGCAGATGAACACGCTCAAACCGGAGGATACGGCCGTCTACTACTGCGCTGCTGA TCAGGGAAGCTGGTACTTGGAGAGGAGGGAATACGATTATTGGGGTCAGGGGGACCCAAGTGACTGTCAGCAGT;

[0072] SEQ ID NO.16:

[0073] GAAGTGCAGTTGGTAGAATCAGGTGGAGGGCTGGTACAGCCTGGAGGGTCCCTGAGATTGAGTTGTGC CGCCAGTGGAAGGATGTTTTCTTCCTACAACATGGCATGGTTTCGCCAGGCTCCCGGAAAGGAAAGGGAGTTCGTA GCTGCCATCAAGTGGTCTGGAGACTCAACACTTTATGCCGATTCTGTGAAAGGACGCTTCACAATTTCCAAAGACA ACAGCAAGAACACCCTTTATCTTCAGATGAATAGTCTGCGCGCTGAAGATACTGCTGTGTATTATTGTGCAGCTGA TCAGGGCTCATGGTACCTCGAACGCCGGGAATACGACTATTGGGGACAAGGTACCCAGGTGACTGTTTCCTCT.

[0074] On the other hand, the present invention provides an expression vector comprising the above-mentioned nucleic acid molecule. Specification 5 / 12 pages 8 CN 121949556 A

[0075] Preferably, the expression vector comprises a prokaryotic expression vector and a eukaryotic expression vector.

[0076] Expression vectors in genetic engineering are constructed by adding expression elements (such as promoters, terminators, etc.) to cloning vectors, enabling the target gene to be expressed in recipient cells. Expression vectors typically consist of a target gene, promoter, terminator, and marker gene. Commonly used expression vectors include plasmids, bacteriophages, and artificial chromosomes, each with its own characteristics. For example, plasmids are suitable for rapid expression and screening, bacteriophage vectors have high capacity and high stability, and artificial chromosomes can carry larger DNA fragments. When constructing expression vectors, strategies such as codon optimization need to be considered to improve expression efficiency. Plasmid expression vectors are divided into prokaryotic expression vectors and eukaryotic expression vectors. Prokaryotic expression vectors, such as E. coli expression vectors, have a basic backbone containing a plasmid replication origin and an antibiotic resistance gene, and carry regulatory sequences that enable gene expression, such as promoters and terminators. Eukaryotic expression vectors include yeast expression vectors and mammalian cell expression vectors, which also contain a target gene, promoter, terminator, and marker gene, but their regulatory sequences and replication mechanisms are more complex to adapt to the transcription and translation processes of eukaryotic cells.

[0077] On the other hand, the present invention provides a host cell, wherein the host cell comprises the above-mentioned expression vector or the host cell genome contains the above-mentioned nucleic acid molecules.

[0078] Preferably, the host cell is a bacterial or mammalian cell, wherein the bacteria comprises Escherichia coli, and the mammalian cells comprise HEK293 cells and CHO cells.

[0079] Specifically, the bacteria is Escherichia coli TG1, and the mammalian cells are HEK293F cells.

[0080] On the other hand, the present invention provides a pharmaceutical composition comprising the above-mentioned nanobody, conjugate, antibody preparation, nucleic acid molecule, expression vector, and host cell.

[0081] On the other hand, the present invention provides a method for producing a nanobody bound to Trop2 protein, the method comprising the following steps: immunizing alpacas with human Trop2 protein, separating peripheral blood to establish a VHH phage library, panning with human Trop2 protein, constructing VHH-Fc for expression and purification, measuring the protein binding characteristics and cell binding characteristics of VHH-Fc, and humanizing VHH.

[0082] Phage libraries are a technique used to screen and identify specific antigen-associated binding molecules (such as antibodies or peptides). They utilize bacteriophages as genetic vectors, inserting a large number of DNA or cDNA fragments (corresponding to the heavy or light chain gene sequences of antibodies) into the phage genome, forming a phage population containing a large number of different recombinant DNA molecules. Phage library technology is widely used in antibody screening, protein-protein interaction and co-existence studies, and other fields. It enables rapid acquisition of antigen-specific molecules through high-throughput and diverse screening, providing a powerful tool for biological research and drug development.In addition, phage libraries are also important materials used in genetic engineering for preserving and isolating target genes. They can store the genetic information of the entire genome of an organism in a stable recombinant that can be preserved for a long time.

[0083] Humanization of antibodies refers to the modification of mouse or other non-human monoclonal antibodies into human form through genetic engineering technology to reduce their immunogenicity and retain or improve their specificity and affinity for antigens. Such antibodies are structurally closer to naturally produced antibodies in the human body, thus reducing immune responses and improving safety and efficacy in treatment.

[0084] On the other hand, the present invention provides a method for detecting Trop2 protein, which uses the above-mentioned nanobodies, conjugates, antibody preparations, nucleic acid molecules, expression vectors or host cells for detection. The detection method is not a disease diagnosis or treatment method.

[0085] On the other hand, the present invention provides the application of the above-mentioned nanobodies in the preparation of drugs for the prevention and / or treatment of cancer.

[0086] Preferably, the drug prepared from the nanobody is used to prevent or treat cancers including breast cancer, colorectal cancer, gastric adenocarcinoma, esophageal cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, ovarian epithelial cancer, prostate cancer, pancreatic ductal adenocarcinoma, head and neck cancer, squamous cell carcinoma, renal cell carcinoma, bladder tumor, cervical cancer, endometrial cancer, follicular thyroid carcinoma, and glioblastoma multiforme.

[0087] Preferably, the drug prepared from the nanobody can be used to prevent or treat breast cancer, triple-negative breast cancer, lung cancer, gastric cancer, pancreatic cancer, or prostate cancer.

[0088] On the other hand, the present invention provides a product comprising the above-mentioned nanobody, conjugate, antibody preparation, nucleic acid molecule, expression vector, or host cell.

[0089] Preferably, the product is a drug or a reagent kit.

[0090] Preferably, the drug is an anti-tumor drug, and the reagent kit is a Trop2 protein detection kit.

[0091] Compared with the prior art, the present invention has the following beneficial effects:

[0092] The present invention obtained camel-derived Trop2 protein nanobodies with high affinity for human Trop2 protein, and obtained the VHH-Fc of the antibody, and then obtained humanized Trop2 protein nanobodies. Verification showed that both the nanobodies before and after humanization had high Trop2 protein affinity, and the antibody sequences were clear and novel. Brief Description of the Drawings

[0093] Figure 1: ELISA results of serum titer determination after immunization of alpacas.

[0094] Figure 2: FACS results of serum titer determination after immunization of alpacas.

[0095] Figure 3: Results of determining the library capacity using the limiting dilution method.

[0096] Figure 4: Results of Trop2 protein binding assay using P4-64 clone phage supernatant.

[0097] Figure 5 shows the binding assay of different concentrations of P4-64 antibody with human Trop2 protein.

[0098] Figure 6 shows the binding assay of different concentrations of P4-64 antibody with monkey Trop2 protein.

[0099] Figure 7 shows the binding assay of different concentrations of P4-64 antibody with MDA-MB-468 cells.

[0100] Figure 8 shows the SPR spectrum of the binding affinity of P4-64 antibody with human Trop2 protein.

[0101] Figure 9 shows the SPR spectrum of the binding affinity of P4-64 antibody with monkey Trop2 protein.

[0102] Figure 10 shows the multi-concentration affinity assay of P4-64 molecules before humanization.

[0103] Figure 11 shows the multi-concentration affinity assay of P4-64 molecules after humanization. Detailed Description

[0104] To ensure easier understanding of this application, some terms are defined first. Other definitions are given throughout the specific description.

[0105] "Affinity" refers to the strength of the binding between the antibody binding site and the antigen epitope. This binding strength determines the antibody's ability to recognize and bind to antigens and is one of the important parameters of antibody function. Affinity describes the tightness of the interaction between the antibody and the antigen, reflecting the strength of the binding force between them. When an antibody binds to an antigen, if the interaction force between them is strong, then the affinity of this binding is high; conversely, if the interaction force is weak, then the affinity is low. The affinity of an antibody can be measured by various methods, such as biomembrane interference (BLI) technology and surface plasmon resonance (SPR) technology. These methods can quantitatively assess the binding strength between the antibody and the antigen, providing an important basis for the research and development and application of antibody drugs.

[0106] "Diagnosis" refers to the analysis and judgment made by a doctor after obtaining the patient's medical history, symptoms, signs, and auxiliary examination results regarding the patient's disease or health status. In the field of cancer diagnosis, it refers to the process by which doctors collect information such as the patient's medical history, symptoms, and signs, and combine it with the results of various clinical and laboratory examinations to comprehensively determine whether the patient has cancer, and further determine the type, stage, and extent of the cancer. Radiological diagnosis in the field of cancer refers to the use of radioactive materials such as X-rays and radioactive isotopes or related imaging techniques (such as X-ray computed tomography, i.e., CT; magnetic resonance imaging, i.e. MRI; digital subtraction angiography, i.e., DSA, etc.) to scan and image the inside of the human body, thereby observing and analyzing the morphological structure, functional changes, and pathological changes of human tissues and organs, in order to diagnose cancer and its lesions.

[0107] "Treatment" refers to the process by which doctors take a series of intervention measures for the patient's disease or health condition in order to eliminate the cause, relieve symptoms, improve function, prevent complications, and improve the quality of life.Typically, medications are used to treat diseases by directly targeting the pathogens, pathological processes, or symptoms, aiming to eliminate the cause, alleviate symptoms, or cure the disease. In some cases, medication is also used to maintain health, such as supplementing nutrients that the body cannot synthesize on its own, or preventing certain diseases.

[0108] "Combination therapy" refers to the method of treating cancer patients by using two or more drugs simultaneously or sequentially. This method aims to improve the therapeutic effect through the synergistic effect between different drugs, reduce the dosage and toxicity of single-drug use, and increase the killing power against tumor cells and the ability to overcome drug resistance. In single-drug therapy, the drug dosage often needs to reach a high level to achieve a good therapeutic effect, but this also increases the risk of adverse reactions. In combination therapy, due to the synergistic effect between drugs, the therapeutic effect can be maintained or improved while reducing the dosage of each drug, thereby reducing the occurrence of adverse reactions. In combination therapy, due to the different mechanisms of action between drugs, drug resistance can be addressed more effectively. Even if one drug develops resistance, the other drugs can still continue to play a role. The application of combination therapy in cancer treatment involves a variety of drugs and quantities that are not fixed, but rather determined by factors such as the patient's specific condition, tumor type, stage, molecular subtyping, and synergistic effects of the drugs.

[0109] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. Unless otherwise specified in the embodiments, the conditions are performed according to conventional conditions or the manufacturer's recommendations. All reagents or instruments without a specified manufacturer are commercially available conventional products. Numerous specific details are given in the following detailed embodiments to better illustrate the present invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the present invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the present invention. Such structures and techniques are also described in many publications, such as *Molecular Cloning: A Laboratory Manual (Fourth Edition)* (Cold Spring Harbor Laboratory Science Press), Ausubel, F.M. et al., *Current Protocols in Molecular Biology*, Greene Publishing Assoc., and Wiley-Interscience.

[0110] The present invention uses a screening method that is relatively conventional in the field for antibody screening. This part is not the inventive point of the present invention. In addition, for the screening method, the single screening result may have multiple nanobodies. In order to ensure the singleness of the technical solution of the present invention, other screening results have been filed separately.

[0111] Basic Example 1: Construction of Trop2 Nanobody Library

[0112] 1. Animal Immunization

[0113] (1) In order to stimulate the production of specific nanobodies against human Trop2 protein antigen by B cells in alpacas, the following immunization procedure was adopted in this application: 400 micrograms of human Trop2 protein antigen (catalog number CU64) purchased from Suzhou Nearshore Protein Technology Co., Ltd. were mixed with an equal volume of Freund's adjuvant, and then alpacas were immunized four times at two-week intervals.

[0114] (2) After the four immunization cycles were completed, alpaca serum was detected and the effect was evaluated by ELISA technology or direct cell-based flow cytometry (FACS).

[0115] The specific operation for detecting the serum titer of anti-human Trop2 antigen by ELISA is as follows: Instruction manual, page 8 / 12, CN 121949556 A

[0116] First, human Trop2 antigen protein is coated onto the detection plate at a concentration of 1 μg / mL and incubated at 25°C for 2 hours, followed by washing once with 0.05% PBST solution. 200 μL of blocking buffer is added to each well, and incubation is continued at 25°C for 1 hour, followed by washing again with 0.05% PBST.

[0117] Next, 100 μL of diluted alpaca serum is added to each well, and incubation is carried out at 25°C for 1 hour, followed by thorough washing five times with 0.05% PBST.

[0118] Subsequently, goat anti-alpaca IgG (H+L) horseradish peroxidase (Jackson, catalog number 128-035-160) diluted 1:10000 was added as a secondary antibody, and the cells were incubated at 25 degrees Celsius for 1 hour, followed by washing eight times with 0.05% PBST.

[0119] Finally, 100 μL of TMB substrate was added to each well, and the cells were incubated at 25 degrees Celsius in the dark for 3 minutes. After adding 50 μL of stop buffer, the absorbance value (OD450nm) was measured at a wavelength of 450 nm. The results are shown in Figure 1.

[0120] The specific steps for detecting the serum binding titer of MDA-MB-468 cells by FACS method are as follows:

[0121] MDA-MB-468 cells (ATCC, catalog number HTB-132) were pre-cultured, and 1.5 x 10⁵ cells were seeded in each well. Alpaca serum was serially diluted, with the first well being diluted 1 / 100, the next well 1 / 1000, and then serially diluted twofold. 50 μL of diluted serum was added to each well of a 96-well plate, mixed, and incubated at 4°C for 1 hour.

[0122] Subsequently, 100 μL of Alexa 488 anti-alpaca IgG (H+L) diluted 1:1000 was added to each well, and the plate was incubated at 4°C for another 30 minutes. Flow cytometry analysis was then performed, and the results are shown in Figure 2.

[0123] After four immunizations, the test results showed that the serum titer against the human Trop2 protein antigen was as high as 512,000, while the serum titer against MDA-MB-468 cells was 128,000. In summary, this immunization protocol successfully induced a strong immune response of alpacas to human Trop2 protein, laying a solid foundation for the subsequent construction of nanobody libraries.

[0124] 2. Construction of human Trop2 nanobody library

[0125] (1) Lymphocytes were isolated from 100 ml of camel peripheral blood, and total RNA was extracted from these cells. Subsequently, cDNA was synthesized using these RNAs as templates, and the VHH (heavy chain variable region) gene fragment was further amplified by nested PCR technology.

[0126] (2) The amplified VHH fragment was precisely inserted into the phage display vector using molecular biology methods to ensure the efficiency and accuracy of the ligation.

[0127] (3) The above-mentioned ligation product was introduced into electroporated competent cells TG1 by electroporation to construct a nanobody library targeting human Trop2. Subsequently, the library volume was measured, and the results (see Figure 3) showed that its volume reached 1.0 × 10⁸ CFU (colony forming units), indicating that the library was large in scale and rich in diversity.

[0128] In addition, in order to verify the construction quality of the library, 48 clones were randomly selected for colony PCR detection. The detection results showed that all the detected clones successfully inserted the VHH fragment, that is, the insertion rate of the library was as high as 100%, which further proved the high efficiency and reliability of the constructed human Trop2 nanobody library.

[0129] Basic Example 2: Screening and Identification of Trop2 Nanobodies

[0130] 1. Antibody Panning Process

[0131] In order to enrich specific nanobodies against human Trop2 from the constructed library, solid-phase panning technology was used. The specific steps are as follows:

[0132] 5 μg / mL of human Trop2 protein was dissolved in a buffer containing 100 mmol / L NaHCO3 and pH 8.2. Then, it was coated onto a NUNC microplate and incubated overnight at 4°C to ensure strong binding of the protein.

[0133] The next day, 300 μL of 4% BSA solution was added to the microplate and blocked at room temperature for 2 hours to reduce non-specific binding.

[0134] After blocking, 100 μL of phage suspension was added to each well and incubated at room temperature for 1 hour to allow the phage to fully bind to the human Trop2 protein.

[0135] The ELISA plate was washed 5 times with 0.05% PBS + Tween-20 solution to remove unbound phages.

[0136] Phages that specifically bind to human Trop2 protein were dissociated using a 100 mmol / L triethanolamine solution and then infected with E. coli TG1 cells in the logarithmic growth phase. After culturing at 37°C for 1 hour, the phages were collected and purified for the next round of screening. This screening process was repeated for 3 rounds to gradually improve the specificity of the phages.

[0137] 2. Phage ELISA screening for specific positive clones

[0138] For the phage clones obtained in each round of screening, they were cultured in a shaker incubator to the logarithmic growth phase, and helper phage M13KO7 was added. The cells were induced overnight at 30°C to produce more phage particles.

[0139] The next day, the phage supernatant was collected by centrifugation and screened by ELISA according to the following steps:

[0140] First, human Trop2 protein and monkey Trop2 protein (from Suzhou Nearshore Protein Technology Co., Ltd., catalog number C08B) were coated onto the detection plate at a concentration of 1 μg / mL and incubated at 25°C for 2 hours. Then, the plate was washed once with 0.05% PBST solution.

[0141] Next, 200 μL of blocking buffer was added to each well and incubated at 25°C for 1 hour to block non-specific binding sites. After washing once again with 0.05% PBST, 100 μL of phage supernatant diluted 1:1 with blocking buffer was added to each well and incubated at 25°C for 1 hour.

[0142] After incubation, the plate was thoroughly washed 5 times with 0.05% PBST to remove unbound phage. Then, anti-M13-HRP antibody diluted 1:5000 (purchased from Beijing Sinocare Co., Ltd., catalog number 11973-MM05T-H) was added to each well and incubated at 25°C for 1 hour. Afterwards, the sample was washed 8 times with 0.05% PBST.

[0143] Finally, 100 μL of ABTS substrate (purchased from Thermo Fisher Scientific, catalog number 00-2024) was added to each well and incubated at 25°C for 15 minutes. After incubation, the absorbance value of each well at a wavelength of 415 nm (OD415nm) was measured to identify positive clones. After multiple rounds of screening, several positive clones were successfully obtained, with the ELISA results for the target clone P4-64 being particularly significant (see Figure 4).

[0144] Gene sequencing analysis was performed on the P4-64 clone, and the nucleic acid sequence of VHH of the P4-64 clone is shown in SEQ ID NO.15, and the amino acid sequence is shown in SEQ ID NO.12.

[0145] Example 1: Expression and purification of nanobodies in mammalian cells HEK293F

[0146] The P4-64 nanobody fragment (sequence shown in SEQ ID NO. 12) and the human IgG1 Fc fragment (sequence shown in SEQ ID NO. 14) were fused using genetic engineering technology and constructed into an expression vector suitable for eukaryotes. Subsequently, HEK293F cells were transfected using this vector to achieve the expression of the P4-64 fusion antibody. After expression, the antibody was purified to obtain the target product with high purity.

[0147] To evaluate the performance of the purified antibody, various affinity assays, including ELISA, FACS, and SPR, were performed.

[0148] 1. ELISA binding assay

[0149] Antigen coating: First, human Trop2 protein and monkey Trop2 protein at a concentration of 1 μg / mL were uniformly coated on the experimental plate, and then incubated at 25°C for 2 hours to ensure that the antigens were firmly bound to the experimental plate. Then, wash gently once with 0.05% PBST solution to remove unbound protein.

[0150] Blocking treatment: Next, add 200 μL of blocking buffer to each well and continue incubation at 25°C for 1 hour to block non-specific binding sites on the experimental plate. After incubation, wash again with 0.05% PBST.

[0151] Antibody incubation: Subsequently, dilute the P4-64 fusion antibody with an initial concentration of 100 nM in a 5 to 6-fold gradient and add 100 μL of diluted antibody solution to each well. Incubate at 25°C for 1 hour to allow sufficient reaction between antigen and antibody. After incubation, wash thoroughly three times with 0.05% PBST to remove unbound antibody.

[0152] Secondary antibody incubation: Next, an appropriate amount of goat anti-human IgG-Fc (HRP) antibody was added to each well, and incubation was continued at 25°C for 30 minutes to detect specifically bound antibodies. After incubation, the cells were washed five times with 0.05% PBST to ensure the removal of unbound secondary antibodies and improve the accuracy of the detection results.

[0153] Colorimetric reaction and result determination: Finally, 100 μL of ABTS substrate solution was added to each well, and incubation was carried out at 25°C for 15 minutes to initiate the colorimetric reaction. After colorimetric reaction, the absorbance of each well was measured at a wavelength of 415 nm (OD415nm) using a spectrophotometer. By comparing the absorbance values ​​of antibodies at different concentration gradients, the binding strength between the antigen and antibody can be analyzed, thereby assessing the affinity of the P4-64 fusion antibody. The results are shown in Figures 5 and 6.

[0154] 2. Cell Binding Assay

[0155] First, MDA-MB-468 cells were evenly seeded on an experimental plate at a density of 1.5 × 10⁵ cells per well. Next, a 100 nM P4-64 antibody solution was introduced into each well of a 96-well plate and serially diluted 5-fold. The plate was then gently shaken to ensure thorough mixing of the antibody solution with the cells. The plate was then incubated at 4°C for 1 hour. This step was designed to promote effective binding of the P4-64 antibody to the surface antigen of MDA-MB-468 cells.

[0156] After incubation, 100 μL of pre-diluted (1:1000) anti-goat anti-human Ig fluorescent antibody (purchased from Jackson, catalog number BYJS-109-605-170) was added to each well. The plate was then incubated again at 4°C for 30 minutes. This process was used to label those P4-64 antibodies that had successfully bound to the cell surface, causing them to emit a fluorescent signal.

[0157] Finally, the treated cells were analyzed using flow cytometry. The results are shown in Figure 7. Flow cytometry analysis can accurately assess the binding of P4-64 antibody to the MDA-MB-468 cell surface antigen, including the binding ratio, strength, and possible binding modes.

[0158] 3. SPR Affinity Assay

[0159] In the SPR (surface plasmon resonance) experiment, the first step was to immobilize human Trop2 protein (concentration of 0.5 μg / mL) or monkey Trop2 protein (concentration of 2 μg / mL) in the running buffer (i.e., HBS-EP+Buffer) at a constant flow rate of 10 μL / min, ensuring that the capture volume was maintained in the range of approximately 247 to 1383 RU. Next, the system was set up at a flow rate of 30 μL / min to conduct binding and dissociation experiments of different concentrations of P4-64 antibody (concentration range from 3.125 to 100 nM). The antibody-protein binding phase lasted 120 seconds, while the dissociation phase could be selected as 200 or 300 seconds as needed to observe the dynamic interaction between the antigen and antibody in detail. The binding affinity assay results of P4-64 antibody with Trop2 protein are shown in Table 1. The SPR spectra of binding affinity between P4-64 antibody and human Trop2 protein and monkey Trop2 protein are shown in Figures 8 and 9, respectively.

[0160] Table 1 Results of binding affinity determination between P4-64 antibody and Trop2 protein

[0161] Analyte RU2 ka (1 / Ms) kd (1 / s) KD (M) Human Trop2 protein P4-64 1.16E-01 3.04E+05 6.11E-04 2.01E-09 Monkey Trop2 protein P4-64 1.45E-01 3.76E+05 7.71E-04 2.05E-09

[0162] Example 2: Humanization of P4-64 antibody molecules

[0163] The amino acid sequence of P4-64 nanobody (SEQ ID) NO.12) The structure was placed in the structure database for homology search. The structure with high sequence homology was selected and compared. Based on the crystal structure resolution and the constructed phylogenetic tree, multi-template homology modeling of the target nanobody sequence was performed. Then, for the optimal modeled structure, the solvent accessibility of the residues was calculated using the ProtSA server. The optimal modeled structure and P4-64 were sequence compared. The corresponding residues exposed to the solvent were replaced, and the necessary reversion mutations were introduced. Finally, a humanized P4-64 nanobody was determined. Its nucleic acid sequence is shown in SEQ ID NO.16, and its amino acid sequence is shown in SEQ ID NO.13.

[0164] Subsequently, the antibody molecules before and after modification were fused with human IgG1 Fc fragments to construct plasmids suitable for eukaryotic expression systems. The expression system was used for expression, and the expression product was purified to obtain high-purity target protein. To evaluate the binding properties of the modified protein, biolayer interference (BLI) technology was used to determine the binding affinity. The binding affinity of P4-64 antibody before and after humanization to human Trop2 protein is shown in Table 2. The SPR spectra of binding affinity of P4-64 antibody before and after humanization to human Trop2 protein are shown in Figures 10 and 11, respectively. The experimental results show that the humanized P4-64 antibody molecule maintains a similar level of affinity as the original molecule before modification.

[0165] Table 2 Affinity determination results of P4-64 molecule before and after humanization

[0166]

[0167] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.Instruction Manual 12 / 12 Page 15 CN 121949556 A Figure 1 Figure 2 Figure 3 Instruction Manual Drawings 1 / 4 Page 16 CN 121949556 A Figure 4 Figure 5 Figure 6 Instruction Manual Drawings 2 / 4 Page 17 CN 121949556 A Figure 7 Figure 8 Figure 9 Instruction Manual Drawings 3 / 4 Page 18 CN 121949556 A Figure 10 Figure 11 Instruction Manual Drawings 4 / 4 Page 19 CN 121949556 A Title of Invention: Trop2-Binding Nanobody, Product and Application Thereof ABSTRACT The present invention provides a Trop2-Binding Nanobody, product and application thereof, relating to the field of antibody technology. The present invention discloses a nanobody specifically binding to Trop2 protein, as well as its VHH sequence and corresponding CDR sequence. The nanobody has the ability to bind to the target protein with high affinity, and the humanized nanobody still retains high affinity binding properties.

Claims

1. A nanobody, characterized in that, The nanobody can specifically bind to the Trop2 protein, and the complementarity-determining region (CDR) of the VHH chain in the nanobody is shown below: CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.

3.

2. The nanobody according to claim 1, characterized in that, The VHH chain of the nanobody also includes a framework region FR, which is shown below: FR1 shown in SEQ ID NO.4, FR2 shown in SEQ ID NO.5, FR3 shown in SEQ ID NO.6 and FR4 shown in SEQ ID NO.7, or FR1 shown in SEQ ID NO.8, FR2 shown in SEQ ID NO.9, FR3 shown in SEQ ID NO.10 and FR4 shown in SEQ ID NO.

11.

3. The nanobody according to claim 1 or 2, characterized in that, The amino acid sequence of the VHH chain of the nanobody is shown in SEQ ID NO.12 or SEQ ID NO.

13.

4. The nanobody according to claim 3, characterized in that, The amino acid sequence of the VHH chain of the nanobody has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to SEQ ID NO. 12 and the CDR region is the same; or the amino acid sequence of the VHH chain of the nanobody has 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to SEQ ID NO. 13 and the CDR region is the same.

5. A coupling agent, characterized in that, The conjugate is prepared by conjugating the nanobody according to any one of claims 1-4 with a conjugate element.

6. The coupling according to claim 5, characterized in that, The coupling element comprises: a fluorescent or luminescent marker, a radionuclide, a contrast agent for magnetic resonance imaging or computed tomography, an enzyme, a cytokine, an antibody VHH fragment, an antibody Fc fragment, an antibody scFv fragment, a viral particle, a liposome, a prodrug-activating enzyme, a chemotherapeutic agent, or a nanoparticle.

7. The coupling according to claim 6, characterized in that, The antibody Fc fragment is the human IgG1 heavy chain constant region, and the sequence of the human IgG1 heavy chain constant region is SEQ ID NO.

14.

8. An antibody preparation, characterized in that, The antibody formulation comprises the nanobody as described in any one of claims 1-4 and at least one pharmaceutically acceptable carrier.

9. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the nanobody according to any one of claims 1-4.

10. The nucleic acid molecule according to claim 9, characterized in that, The nucleic acid molecule sequence is SEQ ID NO.15 or SEQ ID NO.

16.

11. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule according to any one of claims 9-10.

12. The expression vector according to claim 11, characterized in that, The expression vectors include prokaryotic expression vectors and eukaryotic expression vectors.

13. A host cell, characterized in that, The host cell contains the expression vector of claim 11 or 12 or the host cell genome contains the nucleic acid molecule of claim 9 or 10.

14. The host cell according to claim 13, characterized in that, The host cell comprises bacterial or mammalian cells, wherein the bacteria comprise Escherichia coli, and the mammalian cells comprise HEK293 cells and CHO cells.

15. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the nanobody of any one of claims 1-4, the conjugate of any one of claims 5-7, the antibody preparation of claim 8, the nucleic acid molecule of any one of claims 9-10, the expression vector of any one of claims 11-12, or the host cell of any one of claims 13-14.

16. A method for detecting Trop2 protein, characterized in that, The detection is performed using the nanobody according to any one of claims 1-4, the conjugate according to any one of claims 5-7, the antibody preparation according to claim 8, the nucleic acid molecule according to any one of claims 9-10, the expression vector according to any one of claims 11-12, or the host cell according to any one of claims 13-14, wherein the detection method is a non-disease diagnosis or treatment method.

17. Use of nanobodies according to any one of claims 1-4 in the preparation of medicaments for the prevention and / or treatment of cancer.

18. The application according to claim 17, characterized in that, The cancers mentioned are breast cancer, triple-negative breast cancer, lung cancer, stomach cancer, pancreatic cancer, or prostate cancer.

19. A product comprising the nanobody according to any one of claims 1-4, the conjugate according to any one of claims 5-7, the antibody preparation according to claim 8, the nucleic acid molecule according to any one of claims 9-10, the expression vector according to any one of claims 11-12, or the host cell according to any one of claims 13-14, characterized in that, The product in question is a drug or a reagent kit.

20. The product according to claim 19, characterized in that, The drug is an anti-tumor drug, and the kit is a Trop2 protein detection kit.

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