Anti-trop2 antibody

Abstract

Provided are monovalent and multivalent anti-TROP2 antibodies. Such antibodies specifically bind to TROP2 protein, have a strong relative affinity and high endocytosis efficiency. The specific antibody has a wide application prospect in the development of ADC drugs. The present invention also relates to a murine monoclonal antibody, a human-murine chimeric antibody, a humanized antibody, and the use of such antibodies in the diagnosis and treatment of tumors.

Description

An anti-TROP2 antibody Technical Field

[0001] This invention relates to antibodies that specifically bind to the human TROP2 protein, particularly monoclonal antibodies of mouse, chimeric, and humanized origin, and the amino acid and nucleotide sequences encoding these antibodies. The invention also includes the use of these antibodies as diagnostic reagents or pharmaceuticals in the diagnostic and / or therapeutic treatment of malignancies or any lesions associated with overexpression of the receptor. Technical Background

[0002] Trop2 protein, also known as trophoblast antigen 2 or tumor associated calcium signal transducer 2, is encoded by a single-copy gene, TACSTD2, located at chromosome 1p32. Its corresponding mRNA synthesizes a 36 kDa nascent polypeptide, which, after N-terminal glycosylation, forms a monomeric membrane protein with a single transmembrane domain (Annie RA et al., 2015).

[0003] Within the 26-amino acid intracellular region of TROP2, there is a serine residue at position 303. This site is highly conserved across species (Basu et al., 1995; Annie RA et al., 2015) and can be phosphorylated by PKC kinase, thus becoming the binding site for the signal transduction molecule PIP2, which downstream stimulates an increase in cytoplasmic calcium ion concentration (Sewedy et al., 1998; Alberti et al., 1999). However, in in vitro cell lines, overexpression of Trop2 lacking its cytoplasmic region results in the protein losing its growth-stimulating function, indicating that the Trop2 cytoplasmic region plays a stimulatory role in other signaling pathways (Guerra et al.). Trop2 has an extracellular domain that can bind EGF growth factor, which could potentially intercept EGF and downregulate the activity of the IGF-1R / Akt signaling pathway. Therefore, a decrease in Trop2 expression could inversely activate the IGF-1R / Akt signaling pathway (Lin et al., 2011; Annie RA et al., 2015).

[0004] Trop2 plays a significant role in promoting tumorigenesis. Overexpression of Trop2 significantly enhances the tumorigenicity of NIH3T3 cells (Wang et al., 2008). Trop2 also affects epithelial-mesenchymal transition (EMT) and enhances the migration and invasion of cancer cells (Trerotola et al., 2013; Li et al., 2017). This process may be achieved by affecting the PI3K / Akt signaling pathway: in gallbladder cancer cells overexpressing Trop2, the phosphorylation activation of Akt is significantly increased; conversely, knockdown of Trop2 expression inhibits the activity of this signaling pathway (Li et al., 2017). In addition, Trop2 overexpression can also stimulate the activity of the MAPK / ERK signaling pathway, thereby leading to upregulated proliferation of pancreatic cancer cells and increased tumor progression in tumor-bearing mice (Cubas et al., 2010).

[0005] Trop2 protein expression is found in a range of epidermal-derived tissues, such as the mammary gland, kidney, and pancreas during embryonic development and adulthood (Annie RA et al., 2015). However, in tumor tissues corresponding to normal tissues, Trop2 protein expression is significantly elevated without difference, and the intensity of expression is positively correlated with the degree of tumor growth in animal models (Trerotola et al., 2013). Since the Trop2 gene sequence itself has not undergone mutations or amplification, the upregulation of Trop2 expression in cancer is considered to be due to stimulation at the transcriptional regulatory level (Trerotola et al., 2013). In patients with various types of solid tumors, overexpression of this protein usually predicts a poor prognosis (Zeng et al., 2016). In cases of gallbladder cancer (Chen et al., 2014), gastrointestinal cancer (Muhlmann et al., 2009), hilar cholangiocarcinoma (Ning et al., 2013), and pancreatic cancer (Fong et al., 2008), patients with high Trop2 expression showed significantly reduced survival rates. Given the high expression of Trop2 in various cancers and its significant impact on patient survival, this protein is considered a potential target for cancer therapy.

[0006] Among known antibody drugs, one is IMMU-132, a toxin-coupled antibody (ADC) targeting Trop2 (Goldenberg et al., 2015). The loading antibody RS7 for this ADC was obtained through hybridoma preparation, by fusing mouse spleen lymphocytes immunized with crude extracts of lung cancer cell membranes with mouse myeloma cells (Stein et al., 1990). The antigen bound to RS7 in the crude extract was later identified as Trop2 (Stein et al., 1994). The RS7 antibody itself can be internalized by various cancer cells, thus giving it the potential to be formulated into an ADC (Stein et al., 1993). After the murine RS7 was sequence-humanized, the humanized version of RS7 was used to prepare antibody-drug conjugates and loaded with SN-38 drugs and a topoisomerase inhibitor via interchain thiol coupling (Moon et al., 2008; Sahota et al., 2017). In recent Phase I clinical data, IMMU-132 has shown clinical efficacy in the treatment of various cancers, especially triple-negative breast cancer (Starodub et al., 2015; Sahota and Vahdat., 2017).

[0007] The information disclosed in this patent pertains to the pharmaceutical field. Specifically, the chimeric antibody or humanized antibody described in this patent can bind to human trophoblast antigen protein II (Trop2) and has the function of a targeted antibody in antibody-drug conjugate therapy.

[0008] Summary of the Invention

[0009] This invention does not involve antibodies in their natural form. All antibodies described herein are obtained through immunization of mice and identification and isolation methods, or through genetic recombination. According to this invention, the object of protection is an antibody, functional fragment, or derivative, characterized in that it contains at least one CDR, the amino acid sequence of which is derived from SEQ ID No. 1 to SEQ ID No. 12.

[0010] Any antibody fragment or derivative containing at least one CDR, and whose sequence, after optimized alignment with sequences SEQ ID No. 1 to SEQ ID No. 12, has at least 80% identity, or preferably 85%, 90%, 95%, or 98% identity, should be considered an equivalent of the present invention and is therefore also part of the present invention.

[0011] More specifically, the antibody or one of the functional fragments or derivatives of the present invention is characterized in that it comprises a heavy chain, said heavy chain comprising at least one CDR, said CDR being selected from sequences containing amino acid sequences SEQ ID No. 1 to SEQ ID No. 6.

[0012] More specifically, the antibody or one of its functional fragments or derivatives described in this invention is characterized in that it comprises a light chain, wherein the heavy chain comprises at least one CDR, wherein the CDR is selected from sequences containing amino acid sequences SEQ ID No. 7 to SEQ ID No. 12.

[0013] According to the aforementioned aspect, in a more specific first embodiment, the antibody or one of its functional fragments or derivatives of the present invention comprises a heavy chain, said heavy chain comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises the amino acid sequence SEQ ID No. 1, CDR-H2 comprises the amino acid sequence SEQ ID No. 2, and CDR-H3 comprises the amino acid sequence SEQ ID No. 3.

[0014] More specifically, according to the first embodiment, the antibody or one of its functional fragments or derivatives comprises a heavy chain, wherein the sequence of the heavy chain comprises SEQ ID No. 13 for chimeric antibodies and SEQ ID No. 17 for humanized antibodies.

[0015] According to the aforementioned aspect, in a more specific first embodiment, the antibody or one of its functional fragments or derivatives of the present invention comprises a light chain, said light chain comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 comprises the amino acid sequence SEQ ID No. 7, CDR-L2 comprises the amino acid sequence SEQ ID No. 8, and CDR-L3 comprises the amino acid sequence SEQ ID No. 9.

[0016] More specifically, according to the first embodiment, the antibody or one of its functional fragments or derivatives comprises a light chain, wherein the sequence of the light chain comprises SEQ ID No. 14 for chimeric antibodies and SEQ ID No. 18 for humanized antibodies.

[0017] According to the aforementioned aspect, in a more specific second embodiment, the antibody or one of its functional fragments or derivatives of the present invention comprises a heavy chain, said heavy chain comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises the amino acid sequence SEQ ID No. 4, CDR-H2 comprises the amino acid sequence SEQ ID No. 5, and CDR-H3 comprises the amino acid sequence SEQ ID No. 6.

[0018] More specifically, according to the second embodiment, the antibody or one of its functional fragments or derivatives comprises a heavy chain, wherein the sequence of the heavy chain comprises SEQ ID No. 15 for chimeric antibodies and SEQ ID No. 19 for humanized antibodies.

[0019] According to the aforementioned aspect, in a more specific second embodiment, the antibody or one of its functional fragments or derivatives of the present invention comprises a light chain, said light chain comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 comprises the amino acid sequence SEQ ID No. 10, CDR-L2 comprises the amino acid sequence SEQ ID No. 11, and CDR-L3 comprises the amino acid sequence SEQ ID No. 12.

[0020] More specifically, according to the second embodiment, the antibody or one of its functional fragments or derivatives comprises a light chain, wherein the sequence of the light chain includes SEQ ID No. 16 for chimeric antibodies and SEQ ID No. 20 for humanized antibodies.

[0021] As another aspect of the invention, the present invention relates to isolated DNA, characterized by comprising nucleic acids selected from the following DNA sequences: each nucleic acid encodes an amino acid sequence that is one of the above amino acid sequences SEQ ID No. 1 to SEQ ID No. 20.

[0022] More specifically, this includes nucleic acids selected from the following DNA sequences:

[0023] The first specific embodiment includes nucleic acid sequences of SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23 and SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29.

[0024] The second specific embodiment includes nucleic acid sequences of SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26 and SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32.

[0025] Further details

[0026] The first specific embodiment contains chimeric antibody sequences SEQ ID No. 33 and SEQ ID No. 34, and humanized antibody sequences contain nucleic acid sequences SEQ ID No. 37 and SEQ ID No. 38.

[0027] The second specific embodiment includes a chimeric antibody sequence comprising sequences SEQ ID No. 35 and SEQ ID No. 36, and a humanized antibody sequence comprising nucleic acid sequences SEQ ID No. 39 and SEQ ID No. 40. Attached Figure Description

[0028] Figure 1 shows the identification of the CDR region and the two-dimensional structure diagram of the variable region sequence of the 4D3 light chain and heavy chain;

[0029] Figure 2 shows the identification of the CDR region and two-dimensional structure of the variable region sequence of the 7F11 light chain and heavy chain;

[0030] Figure 3 shows the ELISA binding curves for ch4D3 and ch7F11;

[0031] Figure 4 shows the cell surface binding activity of ch4D3 and ch7F11;

[0032] Figure 5 shows the endocytic activity of ch4D3 and ch7F11 antibodies on live BXPC-3 cells.

[0033] Figure 6 shows the changes in the humanity (Z-score) of the light and heavy chain variable regions of the 4D3 murine antibody before and after humanization.

[0034] Figure 7 shows the changes in the humanity (Z-score) of the light and heavy chain variable regions of the 7F11 murine antibody before and after humanization.

[0035] Figure 8 shows the binding curves of the 4D3 humanized antibody and the chimeric antibody at the same dilution concentration;

[0036] Figure 9 shows the SEC detection results of the 4D3 humanized antibody at Day 7 (Figure A), Day 14 (Figure B), and Day 21 (Figure C).

[0037] Figure 10 shows the binding curves of the 7F11 humanized antibody and the chimeric antibody at the same dilution concentration;

[0038] Figure 11 shows the SEC detection results of the 7F11 humanized antibody at Day 7 (Figure A), Day 14 (Figure B), and Day 21 (Figure C).

[0039] Figure 12 shows the competition results of 4D3-humanized-biotin antibody with ch4D3 and hu4D3 antibodies at concentrations of 0.02–50 μg / ml for binding epitopes;

[0040] Figure 13 shows the results of the 7F11-humanized-biotin antibody competing with ch7F11 and hu7F11 antibodies at concentrations of 0.02–50 ug / ml for binding epitopes.

[0041] Figure 14 shows the binding data of hu7F11 antibody in three cell types.

[0042] Figure 15 shows the binding data of Hu4D3 antibody in three cell types.

[0043] Figure 16 shows the endocytosis data of the hu7F11 antibody in three cell types.

[0044] Figure 17 shows the endocytosis data of Hu4D3 antibody in three cell types. Detailed Implementation

[0045] Example 1: Immunization of 6-8 week old Balb / c mice with TROP2 antigen

[0046] A TROP2 extracellular domain expression vector was constructed, and milligram-level TROP2 protein was transiently expressed in 293F suspension cells. Six- to eight-week-old mice were selected and subcutaneously immunized with TROP2 antigen according to the immunization doses and time points shown in the table below. The final immunization was performed on mice with the highest plasma titer after three immunizations. The immunization procedure is shown in Table 1.

[0047] Immunization schedule (days) Route, dosage, etc. Primary immunization: 0 100ug / 0.25ml / mouse, complete adjuvant, subcutaneous immunization. Secondary immunization: 14 100ug / 0.25ml / mouse, incomplete adjuvant, subcutaneous immunization. Tertiary immunization: 35 100ug / 0.25ml / mouse, incomplete adjuvant, subcutaneous immunization. Third immunization blood collection: 42 TROP2 antigen-coated plate ELISA detection of tail vein plasma titer. Final immunization: 56 50ug / 0.25ml / mouse, phosphate-buffered saline, intraperitoneal immunization.

[0048] Table 1

[0049] Example 2: In vitro fusion of spleen cells from immunized mice

[0050] Mouse myeloma cells SP2 / 0 were pre-cultured in DMEM+FBS 10% complete medium. Before fusion, 5 x 10⁸ cells were pipetted using a Pasteur pipette. 7 SP2 / 0 cells were centrifuged at 1000g for 5 minutes and rinsed with serum-free DMEM preheated to 37°C to remove residual serum. Simultaneously, feeder cells were collected from the peritoneal cavity of KM mice and cultured at 5 x 10⁻⁶ cells / mL. 3Feeder cells were seeded into 96-well plates at a rate of 100 μL / well. Blood was collected from the eyeballs of mice three days after final immunization, and the mice were euthanized. After disinfection with 75% alcohol, spleen tissue was harvested under sterile conditions. Spleen cells were collected by blowing preheated serum-free DMEM, and cells from half a spleen were counted. A mixture of spleen cells and SP2 / 0 cells at a ratio of 1:1 to 10:1 was centrifuged, and residual DMEM was aspirated. 1 ml of preheated PEG-1450 was added and mixed thoroughly. After 3 min, 35 ml of preheated DMEM was added to dilute and terminate the reaction. Cells were centrifuged at 1000 rpm for 5 min, then resuspended in HAT selection medium and seeded into 10 96-well plates.

[0051] Example 3: Positive detection of hybridoma cell supernatant

[0052] Seven to ten days after fusion, cell clone formation was observed. The supernatant was changed once with DMEM + 10% FBS medium the day before detection. Simultaneously, ELISA plates were coated with 2 μg / ml TROP2 antigen. On the day of detection, using a multichannel electric pipette on a sterile operating table, the supernatant from the 96-well plate was aspirated and added to the corresponding ELISA wells. The ELISA plates were incubated at 37°C for 1 hour, followed by washing the plates three times with PBST. HRP-labeled goat anti-mouse antibody diluted 1:5000 was added. After incubation at 37°C for 1 hour, the plates were washed three times with PBST. 50 μL of TMB substrate chromogenic solution was added to each well, and the reaction was carried out at room temperature for 5–10 minutes. Then, 50 μL / well of 2M sulfuric acid solution was added to stop the chromogenic reaction. Positive clones were screened based on the OD450 readings on the ELISA plates.

[0053] Example 4: Subcloning of positive cell lines

[0054] Label the wells of fusion cells with higher OD450 values ​​and culture them for no more than 2 days. Seed and feed the cells according to the method in Example 2, and homogenize positive cells with a 200µl pipette tip. Take no more than 5µl of the cell suspension for subcloning, dilute it to 100µl, and add it to the first well of a 96-well plate pre-containing 100µl / well of feeder cell suspension. Pipe 100µl evenly from A1 to H1 to the last row, then use a multichannel electric pipette to aspirate 100µl and pipette evenly from A1 to A12 to the last column. Culture the plate for 7–10 days, label the wells with single clones, and perform supernatant positive detection as described in the example.

[0055] Example 5: Preparation of Monoclonal Antibodies from Ascites Fluid

[0056] Seven days prior to ascites preparation, Balb / c mice were intraperitoneally injected with 1 ml of paraffin oil per mouse. Subsequently, single-clone cells with stable positivity rates after the first subcloning were selected and expanded for culture. Cells were harvested when they reached at least one well in a 6-well plate, centrifuged at 1000g for 3 minutes, and washed three times with phosphate-buffered saline. Each mouse was injected with 1–2 x 10⁻⁶ cells / well. 6 Preparation was carried out per mouse. Mice were fed for 7–10 days and their peritoneal cavities were observed. Ascites fluid was collected using an 18-gauge sterile needle, and the supernatant was collected at 14000g for 5 minutes. The supernatant was purified by protein A / G affinity column to obtain monoclonal antibodies for 4D3 and 7F11 cell lines.

[0057] Example 6: Obtaining the coding sequence of the variable region of the light and heavy chains of the 4D3 antibody

[0058] Single-clonal cell lines were cultured into 6-well plates, and cells were collected using trizol when the confluence reached 90-100%. Total RNA was extracted in an RNase-free environment, and a cDNA library was synthesized using oligo dT as a reverse transcription primer. This cDNA library was then subjected to 5' end-to-5' dGTP addition with terminal transferase TdT and used as a PCR template. A primer pairing was used, with oligo dC as the upstream primer and the downstream primer corresponding to the 5' CH1 constant region of the antibody light and heavy chains, to amplify the variable region gene via 5' RACE using the high-fidelity enzyme primerSTAR. The PCR products were analyzed by DNA agarose gel electrophoresis, and the DNA fragment at approximately 750 bp was recovered for downstream TA cloning. Strains identified positive by colony PCR were sequenced. The obtained sequences were identified by sequence alignment using the online IMGT database, and two-dimensional mapping of the variable region sequence was performed.

[0059] Example 7: Obtaining the coding sequence of the light and heavy chain variable regions of the 7F11 antibody

[0060] Referring to the operation process in Implementation Case 6, the light and heavy chain variable region sequence of the 7F11 clone was obtained, as shown in Figure 2.

[0061] Example 8: Antibody Expression and Purification

[0062] Antibody expression was performed using Freestyle™ 293-F (Invitrogen) suspension cells. One day prior to transfection, cells were seeded at a density of 6 × 10⁵ cells / mL in 1L shake flasks containing 300 mL of F17 complete medium (Freestyle™ F17 expression medium, Gibco) and cultured overnight at 37°C, 5% CO₂, and 120 rpm on a cell culture shaker. The following day, transfection with the antibody expression plasmid was performed using PEI at a ratio of 2:1. One day after transfection, TN1 feed medium was added at 2.5% (v / v), and the cells were cultured for another 4 days before centrifugation to collect the supernatant. The collected expression supernatant was eluted with 0.1M citric acid (pH 3.0) using a Protein A affinity chromatography column (Mabselect Sure LX, GE). The captured antibody was adjusted to pH 7.0 with 1M Tris-HCl (pH 9.0) at a 1 / 10 (v / v) ratio. Then, it was passed through a gel filtration chromatography column (SEC, Superdex 200, GE) to remove impurities such as polymers and endotoxins. At the same time, the antibody buffer was replaced with PBS (pH 7.4). The antibody obtained by this method had a target antibody monomer (POI%) greater than 99% and was used for subsequent experiments.

[0063] Example 9: ELISA method for assessing antibody affinity

[0064] The variable region gene was cloned into an expression plasmid containing the constant region of the human antibody. Following the protocol in Example 8, eukaryotic 293F cells were transiently transfected, and the secreted 4D3 and 7F11 chimeric antibodies were purified. The chimeric antibody was diluted to a concentration of 50 μg / ml and added to wells A1–H1 of a TROP2-coated ELISA plate. A three-fold horizontal dilution was then performed from A1 to A12. After incubation at 37°C for 1 hour, the plate was washed, and mouse anti-human Fc HRP-labeled antibody was added. The plate was incubated at 37°C for color development.

[0065] Figure 3 shows that both 4D3 and 7F11 have good relative affinity. EC50 (B4): 0.047 ug / ml; EC50 (B7): 0.071 ug / ml. Example 10: Evaluation of antibody biological activity using cellular immunofluorescence methods.

[0066] BXPC-3 cells were seeded and grown for 24–48 hours to achieve a cell confluence of 40–50% in the 96-well cell culture plates. On the day of the experiment, the supernatant was discarded and the cells were washed twice. PBS solution containing 3% BSA was added, and the cells were blocked at 37°C for 1 hour. Chimeric antibodies and hRS7 antibodies were diluted to 10 μg / ml and incubated at 37°C for 1 hour. The primary antibody solution was discarded, and the cells were washed four times. 100 μL / well of 4% paraformaldehyde solution was added, and the cells were incubated at room temperature for 20 minutes. This was repeated twice. Secondary antibody was diluted 100 μL / well with 1% BSA solution at a ratio of 1:800. The secondary antibody suspension was discarded, and the cells were washed four times. 100 μL / well of 2 μg / ml DAPI staining solution was added, and the cells were incubated at room temperature in the dark for 5 minutes. The DAPI staining solution was discarded, and the cells were washed four times. 100 μL / well of 1×DPBS solution was added, and the results were observed and photographed under a fluorescence microscope.

[0067] Figure 4: Cell surface binding activity of ch4D3 and ch7F11 under the same antibody concentration and treatment conditions.

[0068] Example 11: Assessing antibody biological activity using a cell endocytosis method

[0069] BXPC-3 cells were plated according to the method described in Example 10. On the day of the experiment, the cell culture medium was aspirated, PBS solution was added, and the cells were washed twice. The chimeric antibody and hRS7 antibody were diluted to 10 μg / ml in medium containing 1% FBS and incubated at 4°C for 1 h. The primary antibody solution was aspirated, and complete culture medium for the cells to be tested was added, and the cells were incubated at 37°C for 1 h. The cell culture medium was aspirated, and the cells were washed four times. 100 μL of 4% paraformaldehyde solution was added to each well and the cells were incubated for 20 min. After washing twice, 100 μL of 3% BSA blocking solution containing 0.5% Triton-X100 was added to each well and the cells were incubated at room temperature for 1 h. The punching solution was aspirated, and the cells were washed twice. The anti-human IgG fluorescent secondary antibody was diluted 1:800; 100 μL was added to each well and the cells were incubated at 37°C for 1 h. The fluorescent secondary antibody solution was aspirated, and the cells were washed four times. DAPI staining was performed, and the cells were incubated at room temperature in the dark for 15 min. Repeat the washing process 4 times, add 1×DPBS solution (100 μL / well), and observe the fluorescence staining results under a fluorescence microscope, as shown in Figures 4 and 5.

[0070] Example 12 4D3 Humanized Sequence Modification

[0071] The nucleic acid sequence of the 4D3 variable region was obtained and entered into the V-QUEST sequence lookup window provided by IMGT (http: / / www.imgt.org / IMGT_vquest / vquest). This yielded the sequence characteristics of the light and heavy chain variable regions, including three CDR regions and four FR regions, as well as the sequence of the most closely related germline gene family. In the IMGT-DomainGapAlign amino acid lookup window, the human germline gene family sequence with the highest sequence similarity was searched. The 4D3 light chain corresponds to the human IGKV1-27*01+IGKJ2*02 family sequence, and the 4D3 heavy chain corresponds to the human IGHV1-3*01+IGHJ4*01 family sequence. The CDR regions on this human germline gene sequence were replaced with the CDRs of the 4D3 light and heavy chains, and then the antibody structure information was checked using the IMGT-Structural query. Finally, the 4D3-Hum version of the humanized light and heavy chain sequences was obtained. The 4D3 mouse-derived sequence and humanized sequence were input into an online website to evaluate the similarity score of human antibodies.

[0072] Figure 6 shows the Z-score distribution range and frequency of the mouse antibody library (blue line) and the human antibody library (green line). The red line represents the Z-score scores obtained by the 4D3 light and heavy chains. After humanization, the Z-score score of 4D3 increased significantly.

[0073] Example 13: Humanization of 7F11 Sequence Modification

[0074] Following the procedure described in Implementation Case 12, the humanized light and heavy chain sequences of the 7F11 mouse antibody were obtained. The 7F11 mouse sequence and the humanized sequence were then input into an online website to assess the similarity score with human antibodies.

[0075] In Figure 7, the blue line represents the Z-score distribution range and frequency of the mouse antibody library, and the green line represents the Z-score distribution range and frequency of the human antibody library. The red line represents the Z-score scores obtained by the 4D3 light and heavy chains. After humanization, the Z-score score of 7F11 increased significantly.

[0076] Example 14: Relative Affinity Analysis of 4D3 Humanized Antibody

[0077] The humanized 4D3 antibody sequence was cloned into a eukaryotic expression vector and transiently transfected into eukaryotic 293F cells according to the protocol in Example 8. The purified antibody was uniformly diluted to 2 μg / ml and added together with the unhumanized mouse antibody to wells A1–H1 of a TROP2-coated ELISA plate. The plate was then 3-fold diluted from A1 to A12. After incubation at 37°C for 1 hour, the plate was washed, and then HRP-labeled anti-human Fc antibody was added. The plate was incubated at 37°C for color development. Finally, the relative affinity between the ch4D3 and hum4D3 antibodies was compared using EC50 and curve morphology.

[0078] Figure 8, EC50(4D3-chimeric): 0.056ug / ml; EC50(4D3-humanized): 0.0502ug / ml.

[0079] Example 15: Thermal stability analysis of 4D3 humanized antibody

[0080] The purified hum4D3 antibody was dialyzed with PBS buffer and calibrated to a final concentration of 2 mg / ml. Two batches of 70 μL each were dispensed, with three tubes per batch. The two batches were stored at 4°C and 37°C, respectively, and the tubes were removed on days 0, 7, and 14. The samples were then used for SEC analysis to assess antibody degradation and aggregation.

[0081] Figure 9: SEC detection results of 4D3 humanized antibody at Day 7 (Figure A), Day 14 (Figure B), and Day 21 (Figure C). The monomers and aggregates of 4D3-humanized antibody at each time point under 37°C conditions, as well as their percentage of the detected molecules (%), are shown in Table D.

[0082] Example 16: Analysis of relative affinity and binding epitope consistency of 7F11 humanized antibody

[0083] The relative affinity of the 7F11 humanized antibody was assessed according to the procedure in Example 14.

[0084] Figure 10: Binding curves of 7F11 humanized antibody and chimeric antibody at the same dilution concentration, EC50 (7F11-chimeric): 0.061ug / ml; EC50 (7F11-humanized): 0.0601ug / ml.

[0085] Example 17: Thermal stability analysis of 7F11 humanized antibody

[0086] The thermal stability of the 7F11 humanized antibody was analyzed according to the procedure in Example 15.

[0087] Figure 11: SEC detection results of 7F11 humanized antibody at Day 7 (Figure A), Day 14 (Figure B), and Day 21 (Figure C). The monomers and aggregates of 7F11-humanized antibody at each time point under 37°C, and their percentage of the detected molecules (%) are shown in Table D.

[0088] Example 18: Antigen affinity analysis between humanized antibodies and parental antibodies

[0089] The absolute affinity of antibody-antigen binding was evaluated using the Pall ForteBio Octet optical analysis platform. In this method, biotin-labeled antigens were immobilized on the surface of a streptavidin biosensor chip. After baseline equilibration for 180 seconds, binding occurred for 30 seconds with gradient-diluted antibodies in solution, increasing the optical thickness of the chip and causing a wavelength shift (Δλ), followed by a 30-second dissociation phase. The interaction between the Trop2 antigen and the corresponding antibody was measured in real time, allowing for precise and accurate detection of binding specificity, binding rate, dissociation rate, or sample concentration at various concentrations. The k-on and k-off values ​​at at least five concentration gradients were summarized to obtain the KD binding constant.

[0090] Ab CodeKD(M)ka(1 / Ms)kd(1 / s)R 2 ch4D36.89E-092.35E+051.62E-030.9935Hu4D33.07E-081.39E+054.28E-030.9916c h7F116.11E-112.43E+051.45E-050.9925Hu7F119.94E-111.86E+051.85E-050.9966

[0091] Example 19 Activity analysis and epitope binding consistency analysis of 4D3 humanized antibody and chimeric antibody

[0092] Biotin-labeled 4D3-humanized antibody was used, and the inflection point of the binding curve was measured by ELISA at 0.5 ng / ml. An ELISA blocking buffer containing 0.5 ng / ml of 4D3 biotin-labeled antibody was prepared. Based on this solution, 50 μg / ml of competitive antibodies 4D3-chimeric and 4D3-humanized were prepared. The solutions containing the biotin-labeled antibody and the competitive antibody were added to wells A1-A12, 150 μl per well. Then, 50 μl was added to wells B2-B12, thoroughly mixed with 100 μl of pre-added biotin-labeled antibody solution, and then diluted 3-fold to wells H1-H12. The mixture was incubated at 37°C for 1 hour, followed by washing and incubation with anti-human IgG Fc secondary antibody at 37°C for 1 hour. After washing three times, the mixture was developed and the values ​​were read after 25 minutes.

[0093] Figure 12: The 4D3-humanized-biotin antibody competed with ch4D3 and hum4D3 antibodies at concentrations ranging from 0.02 to 50 μg / ml. Both competing antibodies exhibited equal competitive activity and bound to the same epitopes. EC50 (ch4D3): 0.336 μg / ml. EC50 (hum4D3): 0.326 μg / ml.

[0094] Example 20: Activity analysis and epitope consistency analysis of 7F11 humanized antibody and humanized antibody and chimeric antibody.

[0095] Referring to the operating procedure in Example 18, the epitope competition activity and binding epitope consistency of 7F11 were analyzed.

[0096] Figure 13: The 7F11-humanized-biotin antibody competed with ch7F11 and hum7F11 antibodies at concentrations ranging from 0.02 to 50 ug / ml. Both competing antibodies exhibited equal competitive activity and bound to the same epitopes. EC50 (ch7F11): 0.732 ug / ml. EC50 (hum7F11): 0.856 ug / ml.

[0097] Example 21 Cell binding and endocytosis assay of humanized antibodies hu7F11 & hu4D3

[0098] HEK293 cells were used as negative cells, and BXPC-3 and MCF-7 cells were used as positive cells to test the binding and endocytosis of various antibodies at gradient concentrations. Cell binding assays were performed at 4°C for 1 hour, followed by the addition of standard FITC-labeled fluorescent secondary antibody, and data were collected by flow cytometry.

[0099] The endocytosis assay used Phrodo-Red goat anti-human secondary antibody, a small molecule dye labeled with an acid-sensitive dye. This secondary antibody was first co-incubated with primary antibodies of various concentrations to form a complex, and then co-incubated with each cell line for 16 hours. Samples were then taken, with at least 10,000 cells seeded in each well of a 96-well plate. Data were collected and analyzed using flow cytometry. The average fluorescence intensity of cells at each concentration in the far-red light channel was calculated, and an endocytosis curve was plotted with antibody concentration on the x-axis.

[0100] A comparison of the binding levels of hu7F11 and Hu4D3 antibodies in the three cell types showed that BxPC-3 cells exhibited the highest binding level. A comparison of the endocytosis levels of hu7F11 and Hu4D3 antibodies in the three cell types also showed that BxPC-3 cells exhibited the highest endocytosis level.

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Claims

1. An anti-TROP2 antibody or one of its Fv, scFv, Fab, F(ab’)2, Fab’, scFv-Fc fragments, characterized in that the antibody consists of a heavy chain and a light chain, and is characterized in that the antibody consists of a heavy chain with the amino acid sequence SEQ ID No.13 and a light chain with the amino acid sequence SEQ ID No.14, or consists of a heavy chain with the amino acid sequence SEQ ID No.15 and a light chain with the amino acid sequence SEQ ID No.

16.

2. The antibody or one of its Fv, scFv, Fab, F(ab’)2, Fab’, scFv-Fc fragments according to claim 1, the heavy chain derivative sequence of which contains SEQ ID No.17 and SEQ ID No.

18.

3. The antibody or one of its Fv, scFv, Fab, F(ab’)2, Fab’, scFv-Fc fragments according to claim 1, the heavy chain derivative sequence of which contains SEQ ID No.19 and SEQ ID No.

20.

4. The antibody or one of its Fv, scFv, Fab, F(ab’)2, Fab’, scFv-Fc fragments according to claim 1, any sequence with more than 95% homology with the sequence described in claim 1 should be understood as an equivalent of the present invention and is therefore also part of the present invention.

5. The antibody or one of its Fv, scFv, Fab, F(ab’)2, Fab’, scFv-Fc fragments according to claim 1, the amino acids contained in the heavy chain thereof are respectively composed of CDRH1, CDRH2 and CDRH3 of SEQ ID No.1, 2 and 3, or composed of CDRH1, CDRH2 and CDRH3 of SEQ ID No.4, 5 and 6, and the amino acids contained in the light chain are respectively composed of CDRL1, CDRL2 and CDRL3 of SEQ ID No.7, 8 and 9, or composed of CDRL1, CDRL2 and CDRL3 of SEQ ID No.10, 11 and 12.

6. Any antibody fragment or derivative that contains at least one CDR, and the sequence of the CDR has at least 80% identity, or preferably 85%, 90%, 95% or 98% identity after optimal alignment with the sequences SEQ ID No.1 to SEQ ID No.12, should be understood as an equivalent of the present invention and is therefore also part of the present invention.

5. The antibody or one of its Fv, scFv, Fab, F(ab’)2, Fab’, scFv-Fc fragments according to any one of claims 1-3, characterized in that it is a monoclonal antibody or its derivative.

6. The antibody or one of its Fv, scFv, Fab, F(ab’)2, Fab’, scFv-Fc fragments of the antibody according to claim 1, characterized in that it is murine-derived.

7. One of the antibodies or its Fv, scFv, Fab, F(ab’)2, Fab’, scFv-Fc fragments as claimed in claims 2 and 3, characterized in that it is one of the humanized sequences of the sequence as claimed in claim 1.

9. The nucleic acid encoding the antibody as claimed in claim 1, characterized in that the nucleic acid encodes the antibody as claimed in claims 1-3 or one of its Fv, scFv, Fab, F(ab’)2, Fab’, scFv-Fc fragments.

10. The nucleic acid isolated according to claim 9, characterized in that the nucleic acid contains the sequence SEQ ID No.21, 22, 23 or SEQ ID No.24, 25, 26 of the heavy chain, and the nucleic acid contains the sequence SEQ ID No.27, 28, 29 or SEQ ID No.30, 31, 32 of the light chain.

11. The nucleic acid according to claim 10, characterized in that the nucleic acid contains SEQ ID No.33 and SEQ ID No.34, or SEQ ID No.35 and SEQ ID No.

36.

12. The nucleic acid according to claim 10, characterized in that the nucleic acid contains SEQ ID No.37 and SEQ ID No.38, or SEQ ID No.39 and SEQ ID No.

34.

13. The RNA nucleic acid corresponding to the nucleic acid defined in claims 10-12, or the nucleic acid complementary to the nucleic acid defined in claims 10-12.

14. An expression vector comprising the nucleic acid according to any one of claims 10-12.

15. An expression host related to claim 14.

16. The expression vector as claimed in claim 14 and the expression host as claimed in claim 15 can produce the amino acid sequence as claimed in claims 1-3.

17. One of the antibodies or its Fv, scFv, Fab, F(ab’)2, Fab’, scFv-Fc fragments as claimed in claims 1-6 or obtained by the method as claimed in claim 16 is used for diagnosing or treating malignant tumors related to the TROP2 target.

18. One of the antibodies or its Fv, scFv, Fab, F(ab’)2, Fab’, scFv-Fc fragments for the use as claimed in claim 17 is conjugated with a related cytotoxic compound or a radioactive element.

19. The cytotoxic compound as claimed in claim 18 is selected from alkylating agents, antimetabolites, anti-tumor drugs, mitosis inhibitors, chromatin function inhibitors, anti-angiogenic agents, anti-androgens, anti-estrogens or immunomodulators, etc.

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