Anti-trop2 nanobody, nanobody conjugate drug and application thereof

Abstract

The application provides an anti-TROP2 nanobody, an antibody conjugate drug and application thereof. The amino acid sequence of CDR1 of the heavy chain variable region of the anti-TROP2 nanobody is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of CDR3 is shown in SEQ ID NO:3. The application screens a nanobody with small molecular weight and high affinity, and the antibody conjugate drug developed based on the nanobody can specifically recognize and combine with human TROP2 protein on the cell surface, has specific cytotoxicity, and has important application prospect in the development of tumor treatment drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an anti-TROP2 nanobody, a nanobody-conjugated drug, and their applications. Background Technology

[0002] TROP2 is a cell surface glycoprotein encoded by the TACSTD2 (tumor-associated calcium signaling 2) gene in the 1p32 region of chromosome 1. It was first identified as a highly expressed protein in trophoblast cells in 1981. The TROP2 protein consists of 323 amino acids, has a molecular size of 36 kDa, and is a single-pass transmembrane surface glycoprotein. It mainly contains the following domains: a hydrophobic leader peptide (1-26), an extracellular domain (27-274), a transmembrane domain (275-297), and a hydrophobic cytoplasmic tail (298-323). The protein also contains four predicted N-linked glycosylation sites located at positions 33, 120, 168, and 208. TROP2 can bind to and interact with various proteins such as IGF-1, Claudin-1, Claudin-7, and β-catenin. Subsequently, through the action of protein kinase C (PKC), the intracellular tail serine residue (S303) of the TROP2 protein is phosphorylated, thereby regulating downstream calcium ion signaling pathways, cell cycle protein expression, and reducing fibronectin adhesion, further promoting cell growth, proliferation, and metastasis.

[0003] Under normal physiological conditions, TROP2 is mainly expressed in epithelial cells and plays a crucial role in embryonic development. TROP2 expression can be detected in normal tissues such as the skin, cornea, salivary glands, respiratory tract, and lungs, but at low levels. However, it is highly expressed in various human malignancies, including breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer, and the level of TROP2 expression is correlated with the malignancy of the disease. The reasons for TROP2 overexpression in various tumor cells are not fully understood. Current research suggests that a network of transcription factors, including TP63 / TP53, ERG, GRHL1 / Get-1, and HNF1A / TCF-1, can regulate TROP2 expression, leading to dysregulation at both transcriptional and post-transcriptional levels. Due to its expression characteristics and mechanism of action, TROP2 has become an excellent natural target for tumor therapy, and drug development targeting this target is ongoing.

[0004] Antibody-drug conjugates (ADCs) are composed of monoclonal antibodies that target tumor-specific antigens or tumor-associated antigens, linked to varying numbers of small-molecule cytotoxic payloads via linkers. Combining the high targeting specificity of monoclonal antibodies with the high activity of cytotoxic drugs in tumor tissues, ADCs are one of the fastest-growing drug classes in the field of targeted cancer therapy in recent years.

[0005] Currently, ADC drugs targeting TROP2 have achieved success in the treatment of various tumors. For example, goxatuzumab (Trodelvy®) has been approved for the treatment of advanced triple-negative breast cancer (TNBC) and advanced high-risk breast cancer (HRB). + / HER2 - Treatment of breast cancer. In addition, ADC drugs such as Dato-Dxd and SKB264 have shown good efficacy in other solid tumors such as non-small cell lung cancer. However, to date, no nanobody ADC drugs targeting TROP2 have been announced on the market. Nanobodies (Nb), or variable domain of heavy chain antibody (VHH), are derived from a naturally occurring heavy chain antibody (HCAb) lacking a light chain, found in camels. Cloning its variable region yields a single-domain antibody consisting of only a heavy chain variable region, which is currently the smallest functional, stable, and antigen-binding unit available. Nanobodies possess characteristics such as high stability, good water solubility, simple humanization, high targeting, strong penetration, and convenient modification. As an emerging force in next-generation antibody diagnostics and therapy, they play a significant role in immunological experiments, diagnosis, and treatment.

[0006] Therefore, there is a need in this field to develop an anti-TROP2 nanobody, especially an anti-TROP2 nanobody with good TROP2 antigen binding ability and strong functional activity, and to construct an ADC drug for anti-tumor applications based on this. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide an anti-TROP2 nanobody, a nanobody-conjugated drug, and their applications. This invention provides a research foundation for research based on the TROP2 target, including the development of antibody-conjugated drugs, antigen-chimeric antibody-cell therapies, and multispecific antibody drugs.

[0008] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides an anti-TROP2 nanobody, wherein the amino acid sequence of the heavy chain variable region CDR1 of the anti-TROP2 nanobody is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of CDR3 is shown in SEQ ID NO:3.

[0009] Preferably, the amino acid sequence of the heavy chain variable region of the anti-TROP2 nanobody is as shown in SEQ ID NO:4 or SEQ ID NO:6.

[0010] In this invention, the amino acid sequence shown in SEQ ID NO:4 is the amino acid sequence of nanobody NbA9, and the amino acid sequence shown in SEQ ID NO:6 is the amino acid sequence of nanobody NbA9(S126C). Nanobody NbA9(S126C) is a nanobody obtained by mutating the amino acid of nanobody NbA9 by changing the serine at position 126 to cysteine.

[0011] In a second aspect, the present invention provides a nucleotide molecule that encodes the anti-TROP2 nanobody described in the first aspect.

[0012] Preferably, the sequence of the nucleotide molecule is selected from SEQ ID NO:5 or SEQ ID NO:7.

[0013] Thirdly, the present invention provides an expression vector containing the nucleotide molecules described in the second aspect.

[0014] Fourthly, the present invention provides a method for preparing anti-TROP2 nanobodies, the method comprising the following steps: (S1) Prepare an expression vector containing nucleotide molecules encoding anti-TROP2 nanobodies; (S2) Transfect eukaryotic or prokaryotic host cells with the expression vector from step (S1); (S3) Culture step (S2) of transfected eukaryotic or prokaryotic host cells; (S4) Separate and purify to obtain the anti-TROP2 nanobody.

[0015] Fifthly, the present invention provides a nanobody-conjugated drug containing the anti-TROP2 nanobody described in the first aspect.

[0016] In a sixth aspect, the present invention provides a lentiviral expression vector containing a nucleotide molecule encoding the human TROP2 molecular membrane protein.

[0017] In a seventh aspect, the present invention provides a method for constructing a cell line expressing the human TROP2 molecular membrane protein, the method comprising the following steps: (S1) Prepare a lentiviral expression vector containing nucleotide molecules encoding the human TROP2 molecular membrane protein; (S2) 293T / FT cells were co-transfected with the lentiviral expression vector and lentiviral packaging plasmid from step (S1); (S3) Culture of 293T / FT cells transfected in step (S2); (S4) Centrifuge to collect the culture medium and obtain a lentivirus solution; (S5) Infect the cell line with the lentivirus solution obtained in step (S4) to obtain the cell line expressing the human TROP2 molecular membrane protein.

[0018] Eighthly, the present invention provides a chimeric antigen receptor cell, wherein the chimeric antigen receptor cell is a CAR-T cell, a CAR-NK cell, or a CAR-M cell; and the antigen-binding domain of the chimeric antigen receptor is the anti-TROP2 nanobody described in the first aspect.

[0019] In a ninth aspect, the present invention provides the use of the anti-TROP2 nanobody described in the first aspect, the nanobody-conjugated drug described in the fifth aspect, or the chimeric antigen receptor cell described in the eighth aspect in the preparation of tumor therapeutic drugs.

[0020] Preferably, the tumor is head and neck cancer, bladder cancer, pancreatic cancer, esophageal cancer, cervical cancer, lung cancer, ovarian cancer, or breast cancer, etc.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) The nanobody-conjugated drug of the present invention can specifically recognize and bind to human TROP2 protein on the cell surface.

[0022] (2) The nano-antibody conjugate of the present invention is different from the traditional monoclonal antibody conjugate. It has a small molecular weight, high affinity and novel sequence.

[0023] (3) The nanobody of the present invention provides a research and development foundation for the study of TROP2 target, as well as the development of other antibody-drug conjugates, chimeric antigen receptor cell drugs and multispecific antibody drugs related to TROP2 target.

[0024] (4) The nanobody of the present invention is suitable for prokaryotic and eukaryotic expression. The nanobody-conjugated drug has stable performance and is suitable for laboratory and industrial development and production. Attached Figure Description

[0025] Figure 1This is the SDS-PAGE electrophoresis result of recombinant human TROP2 protein.

[0026] Figure 2 This is the result of serum antibody titer testing.

[0027] Figure 3 These are the SDS-PAGE electrophoresis results of the nanobody.

[0028] Figure 4 This is the result of flow cytometry detection of human TROP2 protein expression on tumor cells.

[0029] Figure 5 This describes the binding of nanobodies to human TROP2 molecules on tumor cells.

[0030] Figure 6 This is a schematic diagram of the structure of a lentiviral expression vector.

[0031] Figure 7 The expression of ZsGreen and TROP2 molecules in cell lines was detected by flow cytometry.

[0032] Figure 8 The flow cytometry was used to detect the binding of candidate nanobodies to wild-type 4T1 and TROP2-overexpressing 4T1 cells.

[0033] Figure 9 This is the result of the intracellular integration level of nanobodies.

[0034] Figure 10 These are the purification results of the NbA9 nanobody.

[0035] Figure 11 This is the result of the affinity assay between the NbA9 nanobody and the recombinant human TROP2 protein.

[0036] Figure 12 This is the SDS-PAGE electrophoresis result of the NbA9(S126C)-His nanobody.

[0037] Figure 13 These are the UV-Vis test results for NbA9(S126C)-His and NbA9(S126C)-CL2A-SN-38.

[0038] Figure 14 The results show the affinity assay between the NbA9(S126C)-CL2A-SN-38 nanobody conjugate and recombinant human TROP2 protein.

[0039] Figure 15 The results show the cytotoxic effects of the NbA9(S126C)-CL2A-SN38 nanobody conjugate on 293T cells, MDA-MB-231 cells, and NCI-H596 cells. Detailed Implementation

[0040] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0041] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0042] The amino acid and nucleotide sequences of the antibody involved in this invention are shown below.

[0043] Anti-TROP2 nanobody NbA9.

[0044] NbA9_CDR1 (SEQ ID NO:1): GFSLEYWT.

[0045] NbA9_CDR2 (SEQ ID NO:2):ISSSDGST.

[0046] NbA9_CDR3 (SEQ ID NO:3):AAGSLGYCSGATPSHEYDY.

[0047] The amino acid sequence of NbA9 (SEQ ID NO:4).

[0048] DVQLQESGGGLVQPGGSLRLSCAASGFSLEYWTIGWFRQAPGQEREGVSCISSSDGSTNYADSVKGRFTISRDNAKSTVYLQMNSLKPEDTAVYYCAAGSLGYCSGATPSHEYDYWGQGTQVTVSS.

[0049] The nucleotide sequence of NbA9 (SEQ ID NO:5).

[0050] GATGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCTTTGGAGTATTGGACCATCGGCTGGTTCCGCCAGGCCCCAGGGCAGGAGCGTGAGGGGGTCTCATGTATTAGTAGTAGTGACGGTAGCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAGCACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCAGGTTCACTTGGCTACTGTTCAGGCGCTACCCCGTCCCATGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA。

[0051] Amino acid sequence of anti-TROP2 nanobody NbA9 (S126C) (SEQ ID NO: 6).

[0052] DVQLQESGGGLVQPGGSLRLSCAASGFSLEYWTIGWFRQAPGQEREGVSCISSSDGSTNYADSVKGRFTISRDNAKSTVYLQMNSLKPEDTAVYYCAAGSLGYCSGATPSHEYDYWGQGTQVTVSC。

[0053] Nucleotide sequence of anti-TROP2 nanobody NbA9 (S126C) (SEQ ID NO: 7).

[0054] GATGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCTCTTTGGAGTATTGGACCATCGGCTGGTTCCGCCAGGCCCCAGGGCAGGAGCGTGAGGGGGTCTCATGTATTAGTAGTAGTGACGGTAGCACAAACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAGCACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCAGGTTCACTTGGCTACTGTTCAGGCGCTACCCCGTCCCATGAGTATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTGT。

[0055] The amino acid sequence of NbA12 nanobody (SEQ ID NO: 8).

[0056] DVQLQESGGGLVQPGGSLRLSCAASGFTFSSAVMRWYRQAPGKGRELVAFIDKADDRTIYADSVKGRFTISRDNNKNTLYLRMNSLKPEDTAIYYCNAVVTPYAYWGQGTQVTVSS。

[0057] The amino acid sequence of NbC1 nanobody (SEQ ID NO: 9).

[0058] DVQLQESGGGLVQAGGSLRLSCATSGRTFSDATMGWFRQAPGKEREGVAQVSWSGHTTYYADSVKGRFTISRDSARNTVYLQMNSLKPEDTAVYYCAARATRRSLRPGVPTALDYGYWGQGTQVTVSS。

[0059] The amino acid sequence of NbF2 nanobody (SEQ ID NO: 10).

[0060] DVQLQESGGGWVQAGGSLRLSCAASERPFSSNAMGWFRQAPGKDREFVAAISWSGGSTAYADSLKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASTWIGVRARDPNQYDYWGQGTQVTVSA。

[0061] Example 1 Construction and eukaryotic expression of recombinant human TROP2 protein expression vector.

[0062] 1. Construction of protein expression vectors.

[0063] Human TACSTD2 (NM_002353) cDNA clone (vector pDONR223) was obtained from Youbao Biotechnology. Primers F (SEQ ID NO:12) and R (SEQ ID NO:13) were synthesized from Qingke Biotechnology for PCR amplification of the extracellular segment of the TROP2 gene. The PCR product was cloned into a pcDNA3.1(+) vector containing a 5' IL-2 signal peptide, a 3' (G4S)3-Linker, and an IgG1-Fc sequence using the NEB homologous recombination kit, thus obtaining the final expression vector for the TROP2-(G4S)3-Fc fusion protein gene. The amino acid sequence of this fusion gene is SEQ ID NO:14.

[0064] Primer F: 5'-GGCTACGGCCAGCGCCCACACGGCCGCGCAGGAC-3', SEQ ID NO: 12.

[0065] Primer R: 5'-cagagccgccgccgccGGTGAGGCGCTTCATGGAGAACTT-3', SEQ ID NO: 13.

[0066] The amino acid sequence of the fusion gene (SEQ ID NO:14).

[0067] MGWSCIILFLVATASAHTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKG DLSLRCDELVRTHHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFS MKRLTGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH.

[0068] 2. Expression and purification of recombinant human TROP2 protein.

[0069] Seven days after transfecting Freestyle-293F cells (Gibco™) with the obtained expression vector, the culture supernatant was collected, and recombinant human TROP2 protein was purified using Cytiva Ni Sepharose excel nickel affinity chromatography protein purification packing. SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining showed that the monomer size of the recombinant human TROP2 protein was approximately 70 kDa, and the dimer size was approximately 130 kDa. The results are as follows... Figure 1 As shown, Figure 1 In the text, lane M represents the protein molecular weight standard.

[0070] Example 2 Preparation of anti-human TROP2 protein nanobodies.

[0071] 1. Construction of VHH phage display library.

[0072] (1) Alpaca immunity.

[0073] Alpaca selection: Choose alpacas that are strong, in good condition, and of moderate size. Select alpacas with glossy coats and no injuries, diseases, or other signs of discomfort. Allow them to rest for one week beforehand to cull any unsuitable animals, ensuring the smooth progress of later experiments.

[0074] Immunization protocol: Ear tagging was performed before immunization, followed by three immunizations. The immunization protocol was as follows: D0, 10 mL of blood was collected before immunization as a negative serum control. 500 μL of TROP2-(G4S)3-Fc fusion protein antigen (0.5 mg dissolved in chlorine-free, magnesium-free DPBS) was emulsified with an equal volume of Freund's complete adjuvant at 4°C for 2-3 h by shaking. After complete emulsification and mixing, it was injected subcutaneously. D21, 10 mL of blood was collected, and 0.25 mg of fusion protein antigen was mixed with an equal volume of Freund's incomplete adjuvant and injected subcutaneously. D28, 10 mL of blood was collected, and 0.25 mg of fusion protein antigen was mixed with an equal volume of Freund's incomplete adjuvant and injected subcutaneously. D49, 50 mL of peripheral blood was collected to separate lymphocytes.

[0075] Serum antibody titer testing: (a) Dilute the antigen to 2 μg / mL with 0.05 M carbonate buffer (pH 9.6), and coat at 100 μL / well overnight at 4°C.

[0076] (b) Discard the coating solution, wash three times with PBST (pH 7.4) containing 0.05% Tween-20, then add 300 μL of 5% skim milk to each well and block at 37°C for 1 h.

[0077] (c) Wash 3 times with PBST, add 100 μL / well of serum diluent (start with serial dilution from 1:2000), and incubate at 37°C for 45 min.

[0078] (d) Wash three times with PBST, add horseradish peroxidase-labeled goat anti-Alpaca secondary antibody (AlpHcAbs) at a 1:10000 dilution, 100 μL / well, and incubate at 37℃ for 45 min.

[0079] (e) Wash the plate 5 times with PBST. Add TMB chromogenic buffer (Biohao), 50 μL / well, incubate at 37℃ for 5 min. Stop the reaction by adding stop solution, 50 μL / well. Measure OD450 using a microplate reader. Results are as follows: Figure 2 As shown.

[0080] (2) cDNA synthesis.

[0081] Total RNA extraction from PBMCs: Alpaca peripheral blood PBMCs were isolated, cells were lysed with TRIzol (Invitrogen), chloroform was added, the mixture was stirred, and centrifuged at 12000 g at 4℃. The supernatant containing RNA was collected. Isopropanol was then added to precipitate RNA, and after washing with ethanol, high purity and concentration of total cellular RNA were obtained.

[0082] Two-step reverse transcription of cDNA: First, primer CALL_GSP: SEQ ID NO:15 is mixed with total cellular RNA and primer-RNA annealing reaction is performed. Then, SuperScript™ IV Reverse Transcriptase is used to synthesize cDNA product.

[0083] Primer CALL_GSP: 5'-CCTGCGGCTCCCGGGTCTGCCCTTTGGCC-3', SEQ ID NO: 15.

[0084] (3) VHH gene amplification.

[0085] Amplification of the VHH1 gene fragment: Using primers CALL_001 (SEQ ID NO:16) and CALL_002 (SEQ ID NO:17) as a template, the VHH1 gene fragment was amplified by PCR. The DNA band of about 750 bp was recovered by the HiPure Gel Pure DNA Mini Kit (Magen).

[0086] Primer CALL_001: 5'-GTCCTGGCTGCTCTTCTACAAGG-3', SEQ ID NO:16.

[0087] Primer CALL_002: 5'-GGTACGTGCTGTTGAACTGTTCC-3', SEQ ID NO:17.

[0088] Amplification of the VHH2 gene fragment: Using primers VHH_For (SEQ ID NO:18) and VHH_Rev (SEQ ID NO:19) as a template, the recovered VHH1 gene fragment was amplified by PCR. The DNA band of about 450 bp was recovered by the HiPure Gel Pure DNA MiniKit kit (Magen).

[0089] Primer VHH_For (SEQ ID NO:18).

[0090] 5'-TACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCTGGRGGA-3'.

[0091] Primer VHH_Rev (SEQ ID NO:19).

[0092] 5'-GTGATGTGTTGGCCTCCTGAGGAGACGGTGACCTGG-3'.

[0093] (4) Construction of VHH library.

[0094] The pADL-23c and VHH2 gene fragments from the phagepocytokines vector were digested with sfi-I (NEB) and then ligated with T4 DNA ligase (NEB). After purification and desalting, the ligation products were used for electroporation of TG1 competent cells using an Eppendorf electroporator. 50 μL of bacterial culture was serially diluted with PBS, and 10 μL of each dilution was plated onto 2YT plates containing ampicillin and incubated overnight at 37°C. The colony count was calculated, and the size of the phage antibody library was determined to be 3.9E+08. The remaining electroporated bacteria were plated onto plates containing 100 μg / mL ampicillin, and colonies cultured overnight were collected using 20% ​​glycerol-2YT to obtain the VHH immunoglobulin, which was stored at -80°C.

[0095] A portion of the frozen VHH immunological library culture was inoculated into 200 mL of 2YT medium containing ampicillin at an inoculation density of 0.05 OD. The culture was incubated at 37°C and 220 rpm for approximately 2 hours. When the bacterial density reached 0.4-0.5 OD, 2 × 10⁻⁶ oz. of ampicillin was added. 11 PFU M13KO7 helper phage was infecting the bacteria at 37°C for 1 h. After centrifugation, the supernatant was removed, and the bacterial pellet was resuspended in 200 mL of fresh 2YT medium containing ampicillin and kanamycin. The culture was then incubated overnight at 28°C and 220 rpm. The next day, the bacterial culture was centrifuged at 10000 g, and the supernatant was collected. 1 / 4 volume of 20% PEG8000-NaCl solution was added to the supernatant, and the mixture was incubated on ice for 1 hour. Afterward, the mixture was centrifuged at 8000 g for 10 minutes, the supernatant was discarded, and the pellet was collected. The pellet was dissolved in 1 mL of PBS, and then 250 μL of 20% PEG8000-NaCl solution was added. The pellet was mixed again, and then dissolved in 1 mL of PBS. The mixture was filtered through a 0.45 μm filter to obtain the VHH phage display library. The OD260 of the phage display library was measured; one OD value is equivalent to 1.1 × 10⁻⁶. 12Each virus particle was aliquoted into 1.5 mL centrifuge tubes at a rate of 6 Od / tube and stored at -80°C.

[0096] 2. Select anti-TROP2 VHH antibodies.

[0097] (1) Biotinylation of recombinant human TROP2 protein.

[0098] Recombinant human TROP2 protein was biotinylated using the EZ-Link™ NHS-Biotinylation Kit (Thermo Scientific) according to the kit instructions.

[0099] (2) Blocking phage libraries and negative magnetic beads.

[0100] Take 1.1 × 10 12 PFU display phages were added to 1 mL StartingBlock™ (PBS) blocking buffer (Thermo Scientific) and blocked for 30 min. Then, 10 μg of biotinylated recombinant human TROP2 protein was added to the blocked phages and placed on a rotating shaker at 10 rpm at room temperature for 1 hour to bind.

[0101] Place 100 μL of streptavidin magnetic beads DynaBeads 280 (Invitrogen) on a Magrack magnetic rack, wash 5 times with PBS, then add 1 mL of StartingBlock™ (PBS) blocking buffer and block on a rotating shaker at 10 rpm for 30 min.

[0102] (3) Positive screening.

[0103] Resuspend the blocked streptavidin magnetic beads in 100 μL of blocking buffer, add them to the antigen-phage incubator after binding, and bind on a rotating shaker for 7 min. Next, the magnetic beads were washed three times with PBS solution containing 3% skim milk powder. Finally, the beads were resuspended in 1 mL of PBS solution containing 3% skim milk powder and transferred to a new EP tube. The supernatant was removed using a Magrack magnetic rack. The beads were then washed with 1 mL of PBST solution containing 0.1% Tween-20, followed by resuspending in 500 μL of 1 mg / mL trypsin solution. The tubes were incubated at room temperature for 25 min on a rotating shaker, then incubated at 37°C for 5 min. The EP tubes were placed on a magnetic rack, and the supernatant was collected and added to 10 mL of TG1 bacterial culture with an OD600 of approximately 0.5 for 30 min. After centrifugation at 4500 rpm for 15 min, the bacterial pellet was resuspended in 1 mL of fresh 2YT medium and plated onto a large plate containing 100 μg / mL ampicillin. Colonies cultured overnight were collected using 20% ​​glycerol-2YT to obtain the first round of VHH immunoglobulin, which was stored at -80°C. The next day, phages were harvested using the same method as for harvesting phage libraries, resulting in the first round of phage libraries.

[0104] (4) Repeat positive screening.

[0105] The above screening method was repeated once more to obtain the VHH immune library for the second round of screening and the phage library for the second round of screening.

[0106] (5) ELISA screening for positive antibodies.

[0107] During the second round of VHH immunological library panning, after infecting TG1 with the eluent from the positively panned samples, 100 μL of bacterial culture was serially diluted and plated onto ampicillin-2YT plates. After overnight incubation, 96 clones were picked from the plates and inoculated into 96-well deep-well plates containing 2YT medium, and incubated overnight at 37°C and 220 rpm. The next day, 5 μL of culture was inoculated into each well of a 96-well deep-well plate containing 100 μL of 2YT medium per well, and incubated for another 2 h at 37°C. 20 μL of 1E10 was added to each well of the culture. 10Inoculate M13K07 helper phage at 37°C for 30 min with pfu / mL. Centrifuge at 3000 rpm, remove supernatant, add 300 μL of 2YT medium containing ampicillin and kanamycin to each well, and incubate overnight at 28°C. Coat ELISA plates with 5 μg / mL neutralidin (biotin-binding protein), wash with PBST, incubate with biotinylated recombinant human TROP2 protein for 30 min, then block with 3% skim milk PBS solution for 1 h, wash with PBST, and add overnight phage supernatant. Finally, add anti-M13-HRP at a ratio of 1:5000, incubate for 30 min, add TMB substrate for color development, then add stop solution to stop color development, and measure OD450. Finally, select the clone with the highest OD450 reading for Sanger sequencing to obtain the antibody gene sequence of the corresponding positive clone.

[0108] 3. Nanobody expression and affinity screening.

[0109] Based on Sanger sequencing results, all positive clone antibody gene sequences were found to be enriched in 13 genes. The corresponding pADL-23c-VHH2 plasmid was extracted and transformed into the WK6 strain, and prokaryotic expression of nanobodies was induced by IPTG.

[0110] The purified nanobodies were obtained by affinity chromatography using Ni Sepharose 6 Fast Flow chromatography. The nanobodies were then subjected to SDS-PAGE electrophoresis followed by Coomassie Brilliant Blue staining to determine their purity. Results are as follows: Figure 3 As shown, Figure 3 In the diagram, lane M represents the protein molecular weight standard. The protein concentration was determined by the A280 method, and the corresponding molar concentration was calculated. All nanobodies were diluted to a concentration of 1 μM and subjected to BLI (Octet 96e, Sartorius) affinity testing, ultimately yielding four candidate nanobodies: NbA9, NbA12, NbC1, and NbF2.

[0111] Example 3 Flow cytometry was used to detect the expression of human TROP2 on tumor cells.

[0112] Collect cultured A549 cells, MDA-MB-231 cells, and NCI-H596 cells to prepare single-cell suspensions. Take three 1×10⁻³ aliquots of each cell type. 6Cells were transferred to 1.5 mL EP tubes and labeled as ctl (control), sacituzumab (goxatozumab), and datopotamab (datopotamab), respectively. 1 μL of DPBS, 1 mg / mL of sacituzumab, and 1 mg / mL of datopotamab were added to each of the three cell groups. The mixture was incubated at 4°C in the dark for 1 h. After incubation, the cells were washed once, and then 1 μL of 0.5 mg / mL anti-human IgG (H+L)-alexa flour647 (GenScript Biotechnology) was added to each cell tube. The mixture was incubated at 4°C in the dark for 30 min. After incubation, the cells were washed twice, and the alexa flour 647 positivity rate for each cell type was detected. The results are shown below. Figure 4 As shown.

[0113] from Figure 4 It is known that A549 cells do not express human TROP2 molecules, while MDA-MB-231 cells and NCI-H596 cells express human TROP2 molecules to varying degrees.

[0114] Example 4 Flow cytometry was used to detect the binding of nanobodies to human TROP2 molecules on tumor cells.

[0115] Collect cultured NCI-H596 cells and prepare single-cell suspensions. Take 50 aliquots (2×10⁶ cells / cells). 5 Cells were loaded into 96-well circular plates, divided into 5 groups of 10 wells each. Five biotinylated nanobodies (NbA9, NbA12, NbC1, NbF2, and ctl_Nbs (anti-SARS-Cov2 nanobody)) were then serially diluted 4-fold from a maximum concentration of 1.25 μg / mL, for a total of 9 dilutions. The diluted nanobodies were then added to their respective wells, with an equal volume of PBS added to the last well of each group. Cells were incubated at 4°C for 30 min, washed once by centrifugation, and then labeled with APC-Streptavidin (Biolegend) antibody for 15 min. Cells were washed twice by centrifugation. The proportion of APC-positive cells in each well was measured, and the mean fluorescence intensity (MFI) of the APC-positive cell population for each sample was calculated using FlowJo software. The results are shown below. Figure 5 As shown.

[0116] from Figure 5 It can be seen that all four candidate nanobodies can bind to NCI-H596 cells to varying degrees in a concentration-dependent manner, with the NbA9 nanobodies exhibiting significantly stronger binding ability than the other three candidate nanobodies and the control nanobodies.

[0117] Example 5 We prepared a 4T1 cell line overexpressing TROP2 and detected the binding of nanobodies to human TROP2 molecules on the cells.

[0118] To further verify the specific binding of the obtained nanobody to the TROP2 molecule, a structure was constructed as follows: Figure 6 The lentiviral expression vector shown was used, and lentivirus was prepared by packaging 293T cells. The 4T1 cell line is a mouse breast cancer cell line that does not express human TROP2. 4T1 cells were infected with lentivirus, and flow cytometry sorting was performed 72 h post-infection to obtain a 4T1 cell line overexpressing TROP2. Flow cytometry detection of ZsGreen and TROP2 expression in the cell line yielded the following results: Figure 7 As shown.

[0119] from Figure 7 It can be seen that the prepared TROP2-overexpressing 4T1 cells can stably and highly express the ZsGreen reporter gene and human TROP2 molecules.

[0120] Collect successfully constructed 4T1 cell lines overexpressing TROP2 and wild-type 4T1 cells from culture and prepare single-cell suspensions. Take 30 aliquots (2 × 10⁶ cells) of each cell type. 5 Cells were loaded into 96-well circular plates, divided into 5 groups of 6 wells each. Five biotinylated nanobodies (NbA9, NbA12, NbC1, NbF2, and ctl_Nbs (anti-SARS-Cov2 nanobody)) were then diluted to 0.5 μg / μL, 0.1 μg / μL, 0.05 μg / μL, 0.01 μg / μL, and 0.002 μg / μL, respectively. The diluted nanobodies were then added to their corresponding wells, with an equal volume of PBS added to the last well of each group. Cells were incubated at 4°C for 30 min, washed once by centrifugation, and then labeled with R-PE-Streptavidin (Biolegend) antibody for 15 min. Cells were washed twice by centrifugation. The proportion of R-PE positive cells in each well was measured, and the mean fluorescence intensity (MFI) of the R-PE positive cell population for each sample was calculated using FlowJo software. The results are shown below. Figure 8 As shown.

[0121] from Figure 8 It is known that only NbA9 and NbF2 nanobodies can specifically bind to TROP2-overexpressing 4T1 cells in a concentration-dependent manner, but do not specifically bind to TROP2-negative wild-type 4T1 cells; while NbA12, NbC1, and ctl_Nbs (anti-SARS-Cov2 nanobodies) do not bind to TROP2-overexpressing 4T1 cells or wild-type 4T1 cells.

[0122] Example 6 Flow cytometry detection of nanobody internalization.

[0123] Collect cultured MDA-MB-231 cells and prepare single-cell suspensions. Take 20 aliquots (2 × 10⁶ cells / mL). 5 Cells were transferred to 1.5 mL EP tubes and divided into 5 groups of 4 tubes each, labeled as 0 h, 0.25 h, 0.5 h, and 1 h, respectively. Then, five nanobodies—NbA9, NbA12, NbC1, NbF2, and ctl_Nbs (anti-SARS-Cov2 nanobody)—were added to their respective cell wells at a concentration of 1 μg / μL. Cells were incubated at 4°C for 30 min, centrifuged and washed once, and then all cells were transferred to a 37°C incubator. At 0 h, 0.25 h, 0.5 h, and 1 h, the labeled cell tubes were removed, centrifuged and washed once, and then Anti-His-Alexa Flour 674 (Biolegend) antibody was added to each tube for labeling at 4°C for 15 min. Cells were then centrifuged and washed twice. The proportion of Alexa Flour 674 positive cells in each well was measured using a flow juxtaposition method. The mean fluorescence intensity (MFI) of the Alexa Flour 674 positive cell population in each sample was calculated using FlowJo software. The decrease in the mean fluorescence intensity of the sample at each time point relative to the mean fluorescence intensity of the 0 h treatment group represents the cell internalization level of the nanobody. Results are as follows: Figure 9 As shown.

[0124] from Figure 9 It is known that only NbA9 and NbF2 can be effectively internalized in cells, while NbA12, NbC1 and ctl_Nbs (anti-SARS-Cov2 nanobody) cannot be effectively internalized in cells.

[0125] Example 7 SPR assay for affinity between NbA9 nanobodies and TROP2 antigen.

[0126] Primers A9-F (SEQ ID NO:20) and A9-R (SEQ ID NO:21) were synthesized for PCR of the NbA9 nanobody gene fragment. The NbA9 nanobody gene fragment was then inserted into the pADL-23c vector via sfi-I restriction enzyme digestion and ligation, constructing the pADL-23c-NbA9-His prokaryotic expression vector. The expressed NbA9-His was subjected to rapid protein liquid chromatography (Superdex 75 10 / 300 GL, Cytiva) to obtain nanobodies with a purity of over 95%. The purification results are shown below. Figure 10 As shown.

[0127] A9-F (SEQ ID NO:20).

[0128] 5'-CGCGGCCCAGCCGGCCATGGCCGATGTGCAGCTGCAGGAGTC-3'.

[0129] A9-R (SEQ ID NO:21).

[0130] 5'-GTTGGCCTCCCGGGCCGTTATTAGTGATGGTGATGGTGATGTGAGGAGACGGTGACCTGG-3'.

[0131] Following the Biacore X100 (Cytiva) instrument operation manual, the biotinylated recombinant human TROP2 protein antigen was immobilized onto the Capture Kit chip. Then, NbA9 nanobody purified by molecular sieves was passed through the chip for multi-cycle kinetic analysis. Finally, the affinity constant (KD value) between the NbA9 nanobody and the recombinant human TROP2 protein was measured to be 2.172 nM. The detection results are as follows: Figure 11 As shown.

[0132] Example 8 We constructed an amino acid-mutated nanobody NbA9 and prepared a nanobody ADC drug.

[0133] Primers A9-F (SEQ ID NO:20) and A9(S126C)-R (SEQ ID NO:11) were synthesized for PCR of the NbA9 nanobody gene fragment. Serine at position 126 was mutated to cysteine. The amplified NbA9(S126C) nanobody gene fragment was inserted into the pADL-23c vector via SFI-I ​​digestion and ligation, constructing the pADL-23c-NbA9(S126C)-His prokaryotic expression vector. NbA9(S126C)-His nanobody was expressed in prokaryotes by IPTG induction. Affinity chromatography was performed using Ni Sepharose 6 Fast Flow packing material, and SDS-PAGE analysis revealed high-purity nanobodies. Results are shown below. Figure 12 As shown.

[0134] Synthesize primer A9-F (SEQ ID NO:20).

[0135] 5'-CGCGGCCCAGCCGGCCATGGCCGATGTGCAGCTGCAGGAGTC-3'.

[0136] A9(S126C)-R (SEQ ID NO:11).

[0137] 5'-GTTGGCCTCCCGGGCCgTTATTAgtgatggtgatggtgatgACAGGAGACGGTGACCTGGGTC-3'.

[0138] Subsequently, the nanobody was conjugated to the CL2A-SN-38 linker-payload. UV-Vis spectroscopy analysis of the unconjugated nanobody NbA9(S126C)-His and the nanobody-conjugated drug NbA9(S126C)-CL2A-SN-38 was performed using NanodropOne. The results are as follows: Figure 13 As shown, the calculated drug-antibody ratio (DAR) value is approximately 1.

[0139] Biotinylated recombinant human TROP2 protein antigen was immobilized onto an SA probe. Multicycle kinetics of the conjugate were analyzed using Octet R96e. The affinity constant (KD value) between the NbA9(S126C)-CL2A-SN-38 nanobody conjugate and recombinant human TROP2 protein was determined to be 14.25 nM. The results are as follows: Figure 14 As shown.

[0140] Example 9 Detection of the specific cytotoxic effects of NbA9(S126C)-CL2A-SN-38 nanobody conjugate.

[0141] MDA-MB-231 cells, NCI-H596 cells, and 293T cells were collected from culture and single-cell suspensions were prepared. Eighteen aliquots (2 × 10⁶) of each cell type were taken. 4 Cells were transferred to 96-well plates, divided into 6 groups (3 wells per group), labeled with concentrations of 20 μg / mL, 10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, and 0 μg / mL, respectively. After overnight incubation at 37°C in a CO2 incubator to allow cell adhesion, the prepared NbA9(S126C)-CL2A-SN-38 nanobody conjugate was added to the corresponding wells at the final labeled concentration, and incubated for another 48 h at 37°C in a CO2 incubator. After incubation, CCK-8 (Beyotime) was added to each well at a 1:10 volume ratio, and the cells were incubated for another 2 h. OD450 was measured using a Biotek microplate reader. The viable cell percentage per well was calculated based on the readings, and the results are statistically presented as follows: Figure 15 As shown.

[0142] from Figure 15It is known that the NbA9(S126C)-CL2A-SN-38 nanobody conjugate has specific cytotoxic effects on TROP2-positive tumor cells MDA-MB-231 and NCI-H596, but has no non-specific cytotoxic effects on TROP2-negative 293T cells.

[0143] In summary, this invention provides a nanobody specifically targeting TROP2, and an ADC drug is prepared based on the nanobody. The nanobody has good TROP2 antigen binding ability and strong functional activity, and has important application prospects in the preparation of anti-tumor drugs.

[0144] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. An anti-TROP2 nanobody, characterized in that, The amino acid sequence of the heavy chain variable region CDR1 of the anti-TROP2 nanobody is shown in SEQ ID NO:1, the amino acid sequence of CDR2 is shown in SEQ ID NO:2, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

3.

2. The anti-TROP2 nanobody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-TROP2 nanobody is shown in SEQ ID NO:4 or SEQ ID NO:

6.

3. A nucleotide molecule, characterized in that, The nucleotide molecule encodes the anti-TROP2 nanobody according to claim 1 or 2, and the sequence of the nucleotide molecule is shown in SEQ ID NO:5 or SEQ ID NO:

7.

4. An expression carrier, characterized in that, The expression vector contains the nucleotide molecule as described in claim 3.

5. A method for preparing an anti-TROP2 nanobody, characterized in that, The preparation method includes the following steps: (S1) Prepare an expression vector containing the nucleotide molecule of claim 3; (S2) Transfect eukaryotic or prokaryotic host cells with the expression vector from step (S1); (S3) Culture step (S2) of transfected eukaryotic or prokaryotic host cells; (S4) Separate and purify to obtain the anti-TROP2 nanobody.

6. A nanobody-conjugated drug, characterized in that, The nanobody-conjugated drug contains the anti-TROP2 nanobody as described in claim 1 or 2.

7. The use of the nanobody-conjugated drug according to claim 6 in the preparation of a tumor therapeutic drug; wherein the tumor is breast cancer and / or lung cancer.

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