Anti-trop2 antibody

Abstract

To provide antibodies, in particular monoclonal antibodies of murine, chimeric, and humanized origin, that specifically bind to the human TROP2 protein, and amino acid and nucleotide sequences encoding these antibodies.SOLUTION: The present invention provides monovalent and multivalent anti-TROP2 antibodies, wherein the antibodies specifically bind to a TROP2 protein and have high relative affinity and high endocytosis efficiency, offering broad potential for application in ADC drug development. The present invention also provides murine monoclonal antibodies, human-murine chimeric antibodies, and humanized antibodies, as well as a use of these antibodies in tumor diagnosis and treatment.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to antibodies capable of specifically binding to the human TROP2 protein, in particular monoclonal antibodies of murine, 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 drugs for the diagnosis and / or therapeutic treatment of malignancies or any disease associated with receptor overexpression. [Background technology]

[0002] The TROP2 protein, also known as trophoblast antigen 2 or tumor associated calcium signal transducer 2, is encoded by the single-copy gene TACSTD2 at chromosome 1p32. The corresponding mRNA synthesizes a 36 kDa nascent polypeptide that is N-terminally glycosylated to form a monomeric membrane protein with a single transmembrane domain (Annie RA et al., 2015).

[0003] The intracellular domain sequence of TROP2 (length: 26 amino acids) contains a serine site at position 303. This site is highly conserved among species (Basu et al., 1995; Annie RA et al., 2015) and can be phosphorylated by PKC kinase to become a binding site for the signaling molecule PIP2 and stimulate an increase in downstream cytoplasmic calcium concentration (Sewedy et al., 1998; Alberti.et al., 1999). When Trop2 lacking the cytoplasmic domain is overexpressed in in vitro cell lines, the protein is deprived of its growth stimulatory function, indicating the stimulatory effect of the Trop2 cytoplasmic domain on other signaling pathways (Guerra.et al.). The extracellular domain of Trop2 contains a structural domain capable of binding to EGF growth factor and can intercept EGF to downregulate the activity of the IGF-1R / Akt signaling pathway. Therefore, decreased Trop2 expression may inversely activate the IGF-1R / Akt signaling pathway (Lin.et al.,2011; Annie RAet al.,2015).

[0004] Trop2 plays a key role in promoting tumorigenesis. Overexpression of Trop2 can significantly enhance the tumorigenicity of NIH3T3 cells (Wang.et al.,2008). Trop2 can also affect epithelial-mesenchymal transition (EMT) and enhance the migration and invasion ability of cancer cells (Trerotola.et al.,2013; Li.et al.,2017). This process can be achieved by affecting the PI3K / Akt signaling pathway. In gallbladder cancer cells overexpressing Trop2, the activation of Akt phosphorylation is significantly increased, while knocking down the expression of Trop2 can inhibit the activity of this signaling pathway (Li.et al.,2017). Overexpression of Trop2 also stimulates the activity of the MAPK / ERK signaling pathway, upregulating the proliferation of pancreatic cancer cells and promoting tumor progression in tumor-bearing mice (Cubas.et al.,2010).

[0005] Trop2 protein expression is found in a series of epidermis-derived tissues, including breast, kidney, and pancreas during embryonic development and adulthood (Annie RA et al., 2015). However, in tumors of corresponding normal tissues, Trop2 protein expression is significantly increased and positively correlated with tumor growth progression in animal models (Trerotola.et al., 2013). As the Trop2 gene sequence itself is neither mutated nor amplified, the upregulation of Trop2 expression in cancer is thought to be due to stimulation at the transcriptional regulatory level (Trerotola.et al., 2013). In patients with various types of parenchymal tumors, overexpression of this protein usually indicates poor tumor prognosis (Zeng.et al., 2016). The survival rate of patients with Trop2-high expression disease was significantly decreased in 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). Considering the high expression of Trop2 in various cancers and its significant impact on patient survival, this protein is considered to be a potential target for cancer therapy.

[0006] Known antibody drugs include IMMU-132, a toxin-conjugated antibody (ADC) against the Trop2 target (Goldenberg et al., 2015). The antibody RS7 of this ADC was obtained by hybridoma preparation and fusion screening of lung cancer cell membrane crude extracts and mouse splenic lymphocytes immunized with mouse myeloma cells (Stein et al., 1990). In the crude extracts, the antigen bound by RS7 was found to be Trop2 (Stein et al., 1994). The RS7 antibody itself can be endocytosed by various cancer cells, so the RS7 antibody may be applied to the preparation of ADCs (Stein et al., 1993). After sequential humanization of mouse-derived RS7, the humanized version of RS7 was used to prepare antibody-conjugated drugs and loaded with the topoisomerase inhibitor SN-38 drug by interchain sulfhydryl coupling (Moon, et al., 2008; Sahota, et al., 2017). Recent clinical phase I data showed that IMMU-132 is clinically effective in treating multiple cancers, especially triple-negative breast cancer (Starodub, et al., 2015; Sahota and Vahdat, 2017).

[0007] The information disclosed in this application relates to the field of therapy, specifically, the chimeric or humanized antibodies described in this application are capable of binding to human type II trophoblast antigen protein (Trop2) and have antibody targeting function in targeting antibody parasite antibody conjugate drug therapy. Summary of the Invention

[0008] The present invention does not relate to natural antibodies. The antibodies according to the present invention are obtained by mouse immunization, identification and isolation procedures or by genetic recombination. According to the present invention, the protected subject is an antibody, a functional fragment or a derivative. The antibody comprises at least one CDR and the amino acid sequence is derived from SEQ ID NO: 1 to SEQ ID NO: 12.

[0009] Any antibody fragment or derivative should be understood to be part of the present invention, since it is an equivalent of the present invention, if it comprises at least one CDR and the sequence of this CDR has at least 80% identity, preferably 85%, 90%, 95% or 98% identity with SEQ ID NO:1 to SEQ ID NO:12.

[0010] More specifically, the antibody, one of its functional fragments or derivatives according to the invention comprises a heavy chain, said heavy chain comprising at least one CDR, said CDR comprising an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:6.

[0011] More specifically, the antibody, one of its functional fragments or derivatives according to the invention comprises a light chain, said light chain comprising at least one CDR, said CDR comprising an amino acid sequence selected from SEQ ID NO: 7 to SEQ ID NO: 12.

[0012] According to said aspect, in a more specific first embodiment, the antibody, one of its functional fragments or derivatives according to 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 shown in SEQ ID NO: 1, CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 2 and CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 3.

[0013] More specifically, according to the first embodiment, the antibody, one of its functional fragments or derivatives comprises a heavy chain, in the sequence of which the chimeric antibody comprises the sequence shown in SEQ ID NO: 13 and the humanized antibody comprises the sequence shown in SEQ ID NO: 17.

[0014] According to said aspect, in a more specific first embodiment, the antibody, one of its functional fragments or derivatives described in 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 shown in SEQ ID NO: 7, CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 8 and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 9.

[0015] More specifically, according to the first embodiment, the antibody, one of its functional fragments or derivatives comprises a light chain, in the sequence of which the chimeric antibody comprises the sequence shown in SEQ ID NO: 14 and the humanized antibody comprises the sequence shown in SEQ ID NO: 18.

[0016] According to said aspect, in a more specific second embodiment, the antibody, one of its functional fragments or derivatives described in 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 shown in SEQ ID NO: 4, CDR-H2 comprises the amino acid sequence shown in SEQ ID NO: 5 and CDR-H3 comprises the amino acid sequence shown in SEQ ID NO: 6.

[0017] More specifically, according to the second embodiment, the antibody, one of its functional fragments or derivatives comprises a heavy chain, in the sequence of which the chimeric antibody comprises the sequence shown in SEQ ID NO: 15 and the humanized antibody comprises the sequence shown in SEQ ID NO: 19.

[0018] According to said aspect, in a more specific second embodiment, the antibody, one of its functional fragments or derivatives described in 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 shown in SEQ ID NO: 10, CDR-L2 comprises the amino acid sequence shown in SEQ ID NO: 11 and CDR-L3 comprises the amino acid sequence shown in SEQ ID NO: 12.

[0019] More specifically, according to the second embodiment, the antibody, one of its functional fragments or derivatives comprises a light chain, in the sequence of which the chimeric antibody comprises the sequence shown in SEQ ID NO: 16 and the humanized antibody comprises the sequence shown in SEQ ID NO: 20.

[0020] In another embodiment of the present invention, the present invention relates to an isolated DNA selected from the nucleic acids of the following DNA sequences, each of which encodes an amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 20 above.

[0021] More specifically, it includes a nucleic acid selected from the DNA sequences listed below. A first embodiment includes the nucleic acid sequences shown in 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. A second embodiment includes the nucleic acid sequences shown in 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.

[0022] More specifically, The chimeric antibody sequence of a first specific embodiment comprises an amino acid sequence encoded by the nucleic acid sequences shown in SEQ ID NO:33 and SEQ ID NO:34, and the humanized antibody sequence comprises an amino acid sequence encoded by the nucleic acid sequences shown in SEQ ID NO:37 and SEQ ID NO:38. The chimeric antibody sequence of the second embodiment comprises the amino acid sequence encoded by the nucleic acid sequences shown in SEQ ID NO:35 and SEQ ID NO:36, and the humanized antibody sequence comprises the amino acid sequence encoded by the nucleic acid sequences shown in SEQ ID NO:39 and SEQ ID NO:40. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 shows the CDR region recognition and two-dimensional structure of the 4D3 light chain and heavy chain variable region sequences. [Diagram 2] FIG. 1 shows CDR region recognition and two-dimensional structure of the 7F11 light chain and heavy chain variable region sequences. [Diagram 3] ELISA binding curves for ch4D3 and ch7F11. [Figure 4] FIG. 13 is a graph showing the cell surface binding activity of ch4D3 and ch7F11. [Diagram 5] FIG. 13 shows the endocytosis activity of ch4D3 and ch7F11 antibodies on live BXPC-3 cells. [Figure 6] FIG. 1 shows changes in humanness score (Z-score) before and after humanization of the light and heavy chain variable region sequences of the 4D3 mouse antibody. [Figure 7]FIG. 1 shows changes in humanness score (Z-score) before and after humanization of the light and heavy chain variable region sequences of 7F11 mouse antibody. [Figure 8] FIG. 1 shows the binding curves of 4D3 humanized antibody and chimeric antibody at equivalent dilution concentrations. [Figure 9] FIG. 1 shows the results of SEC detection of 4D3 humanized antibody on days 7 (A), 14 (B), and 21 (C). [Figure 10] FIG. 13 is a graph showing the binding curves of 7F11 humanized antibody and chimeric antibody at equivalent dilution concentrations. [Figure 11] FIG. 1 shows the results of SEC detection of 7F11 humanized antibody on days 7 (A), 14 (B), and 21 (C). [Figure 12] FIG. 1 shows the results of competition between 4D3 humanized biotin antibody and the binding epitopes of 0.02 to 50 μg / ml of ch4D3 and hu4D3 antibodies. [Figure 13] FIG. 1 shows the results of competition between 7F11 humanized biotin antibody and the binding epitopes of 0.02 to 50 μg / ml of ch7F11 and hu7F11 antibodies. [Figure 14] Binding data of the hu7F11 antibody in three types of cells. [Figure 15] Binding data of Hu4D3 antibody in three types of cells. [Figure 16] Endocytosis data of the hu7F11 antibody in three types of cells. [Figure 17] Endocytosis data of Hu4D3 antibody in three types of cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Example 1: Immunization of 6-8 week old Balb / c mice with TROP2 antigen A TROP2 extracellular domain expression vector was constructed, and milligram levels of TROP2 protein were transiently expressed using suspension cells 293F. Mice aged 6-8 weeks were selected and subcutaneously immunized with TROP2 antigen at the immunization dose and time points shown in the table below. After three immunizations, the mouse with the highest plasma titer was selected for the final immunization. The immunization process is shown in Table 1 below. [Table 1]

[0025] Example 2: In vitro fusion of spleen cells from immunized mice Mouse myeloma cells SP2 / 0 were precultured in DMEM + 10% FBS complete medium and 5x10 7 SP2 / 0 cells were taken and centrifuged at 1000g for 5 min, and residual serum was washed off with serum-free DMEM pre-warmed at 37℃. Feeder cells were collected from the peritoneal cavity of KM mice and 5x10 3 Feeder cells were plated in a 96-well plate at a cell volume of 100ul / well. On the third day after the final immunization, blood was collected from the eyes, and the final immunized mice were euthanized, immersed in 75% alcohol, and then placed on a sterile operating table to remove the spleen tissue. The spleen cells were pipetted in pre-warmed serum-free DMEM, 1 / 2 of the spleen cells were taken and counted, and mixed at a quantitative ratio of spleen cells:SP2 / 0 cells of 1:1 to 10:1. After centrifugation, the residual DMEM was removed by aspirating. 1ml of pre-warmed PEG-1450 was added and mixed uniformly, and after 3min, 35ml of pre-warmed DMEM medium was added to dilute and stop the reaction. The cells were centrifuged at 1000rpm for 5min, after which the cells were resuspended in HAT screening medium and inoculated into ten 96-well plates.

[0026] Example 3: Positive detection of hybridoma cell supernatants 7-10 days after fusion, the formation of cell clones was observed. The medium was replaced with DMEM+10%FBS medium the day before the supernatant detection. The ELISA plate was coated with TROP2 antigen at a concentration of 2ug / ml. On the day of detection, the medium supernatant was aspirated from the 96-well plate using a multichannel electric pipette on a sterile operating table and added to the corresponding wells of the ELISA plate. After incubating the ELISA plate at 37℃ for 1hr, the well plate was washed three times with PBST and HRP-labeled goat anti-mouse antibody diluted 1:5000 was added. After incubating at 37℃ for 1hr, it was diluted three times with PBST. A TMB substrate color development solution was prepared, 50ul was added to each well, and the reaction was allowed to proceed at room temperature for 5-10min. Then, 2M sulfuric acid solution was added at 50ul / well to stop the color development. Positive clones were screened according to the OD450 value of the microtiter plate.

[0027] Example 4: Subclone screening of positive cell lines The wells of the fusion cell plate with relatively high OD450 values ​​were labeled, and the culture was continued within 2 days. The feeder cells were plated according to the method of Example 2, and the positive cells were evenly pipetted using a 200ul pipette tip, and 5ul or less of the cell suspension was taken for subcloning. The cells were diluted to 100ul and added to the first well of a 96-well plate containing 100ul / well of the feeder cell suspension. A volume of 100ul was evenly pipetted from A1 to H1 to the last row, and then 100ul was sucked up using a multichannel electric pipette and evenly pipetted from A1 to A12 to the last row. The well plate was cultured for 7 to 10 days, and the plate wells in which single clones were formed were labeled, and the supernatant positive detection was performed with reference to the method of the Example.

[0028] Example 5: Preparation of monoclonal antibodies in ascites Balb / c mice were intraperitoneally injected with 1 ml of paraffin oil per mouse 7 days before ascites preparation. Monoclonal cells with stable positive rates after the first subcloning were then harvested and expanded. Cells were collected when they had grown to the size of at least one well of a 6-well plate. After centrifugation at 1000 g for 3 min, the cells were washed three times with phosphate buffer. Each mouse was injected with 1–2x10 6 The amount of the solution was prepared for injection into each mouse. The mice were kept for 7 to 10 days, and the mouse abdominal cavity was observed. Ascites was collected using an 18-gauge sterile needle and centrifuged at 14,000 g for 5 minutes to collect the ascites supernatant. The obtained supernatant was purified using a protein A / G affinity column to obtain monoclonal antibodies against the 4D3 and 7F11 cell lines.

[0029] Example 6: Obtaining the coding sequences for the light and heavy chain variable regions of the 4D3 antibody Monoclonal cell lines were cultured in 6-well plates, and cells were harvested with 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 used as a PCR template after adding dGTP to the 5' end with terminal transferase TdT. 5'-RACE amplification of the variable region genes was performed through the high-fidelity enzyme primerSTAR using a primer pair in which the upstream primer was oligo dC and the downstream primer corresponded to the constant regions of the 5'-end CH1 of the antibody light and heavy chains. The PCR products were analyzed by DNA agarose gel electrophoresis, and DNA fragments with a length of approximately 750 bp were recovered for downstream TA cloning. Strains identified as positive by colony PCR were sequenced. Sequence alignment and two-dimensional mapping of the variable region sequences were performed on the obtained sequences using the online IMGT database.

[0030] Example 7: Obtaining the coding sequences for the light and heavy chain variable regions of the 7F11 antibody Referring to the procedure in Example 6, the light and heavy chain variable region sequences of the 7F11 clone were obtained (FIG. 2).

[0031] Example 8: Antibody Expression and Purification Antibodies were expressed using Freestyle™ 293-F (Invitrogen) suspension cells. The day before transfection, 6 × 10 5 The cells were inoculated at a density of 100 cells / mL into 300 mL of F17 complete medium (Freestyle™ F17 expression medium, Gibco) in a 1 L shake flask and cultured overnight in a cell culture shaker at 37°C, 5% CO2, and 120 rpm. The following day, the antibody expression plasmid was transfected using PEI (plasmid:PEI=2:1). One day after transfection, TN1 feed medium was added at 2.5% (v / v), and the cells were further cultured for 4 days, after which the supernatant was collected by centrifugation. The expression supernatant obtained was eluted with 0.1 M citric acid (pH 3.0) through a Protein A affinity chromatography column (Mabselect Sure LX, GE), and the captured antibody was adjusted to pH 7.0 with 1 M Tris-HCl (pH 9.0) (1 / 10 (v / v)), and impurities such as polymers and endotoxins were removed using a gel filtration chromatography column SEC (Superdex 200, GE), and the antibody buffer was changed to PBS (pH 7.4). The antibody thus obtained had a target antibody monomer (POI%) of over 99%, and was used in subsequent experiments.

[0032] Example 9: Evaluation of antibody affinity by ELISA method The variable region genes were cloned into an expression plasmid containing the human antibody constant region, and the secreted 4D3 and 7F11 chimeric antibodies were purified by transiently transfecting eukaryotic cells 293F with reference to Example 8. The chimeric antibodies were diluted to a concentration of 50ug / ml and added to the A1 to H1 wells of a TROP2-coated ELISA plate, and then diluted horizontally in 3-fold volume from A1 to A12. After incubation at 37℃ for 1hr, the plate was washed, and a mouse anti-human Fc HRP-labeled antibody was added, incubated at 37℃, and then colored. As can be seen from Figure 3, both 4D3 and 7F11 have good relative affinity: EC50(B4): 0.047ug / ml; EC50(B7): 0.071ug / ml.

[0033] Example 10: Evaluation of antibody bioactivity by cellular immunofluorescence BXPC-3 cells were plated and grown for 24-48hr so ​​that the cell confluence in the 96-well cell culture plate reached 40-50%. On the day of the experiment, the supernatant was aspirated and removed, and the plate was washed twice. PBS solution containing 3% BSA was added and blocked at 37℃ for 1hr. Chimeric and hRS7 antibodies were diluted to 10ug / ml, incubated at 37℃ for 1h, the primary antibody solution was aspirated and removed, washed 4 times, 4% paraformaldehyde solution (100μL / well) was added and left at room temperature for 20min, washed twice, and the secondary antibody was diluted with 1% BSA solution at a ratio of 1:800 (100ul / well). The secondary antibody suspension was aspirated and removed, washed 4 times, and DAPI staining solution at a concentration of 2μg / ml was added at 100μL / well and incubated at room temperature in the dark for 5min. The DAPI staining solution was removed by aspiration, and after washing four times, 100 μL / well of 1×DPBS solution was added, and the cells were observed under a fluorescent microscope and photographed to record the experimental results. FIG. 4: Cell surface binding activity of ch4D3 and ch7F11 under the same antibody concentrations and treatment conditions.

[0034] Example 11: Evaluation of the biological activity of antibodies by cell endocytosis method BXPC-3 cells were plated with reference to the method of Example 10. On the day of the experiment, the cell culture medium was aspirated and removed, PBS solution was added, and washed twice. The chimeric antibody and hRS7 antibody were diluted to 10ug / ml using a medium containing 1% FBS and incubated at 4°C for 1h, the primary antibody solution was aspirated and removed, and a complete medium for culturing the cells to be detected was added and incubated at 37°C for 1hr. The cell medium was aspirated and removed, and after washing four times, 4% paraformaldehyde solution was added and left to stand at 100μL / well for 20min. After washing twice, 3% BSA blocking solution containing 0.5% Triton-X100 (100μL / well) was added and left to stand at room temperature for 1h. The punching solution was aspirated and removed, and washed twice. Anti-human IgG fluorescent secondary antibody was diluted at a ratio of 1:800 (100μL / well) and incubated at 37°C for 1h. The fluorescent secondary antibody solution was removed by aspiration, washed four times, and then stained with DAPI. The sections were left to stand at room temperature in the dark for 15 min. After washing four times, 100 μL of 1×DPBS solution was added per well, and the results of the fluorescent staining were observed under a fluorescent microscope (Figures 4 and 5).

[0035] Example 12: Modification of the 4D3 humanized sequence The sequencing results of the nucleic acid sequence of the 4D3 variable region were obtained, and the sequence was entered into the V-QUEST sequence check window (http: / / www.imgt.org / IMGT_vquest / vquest) provided by the website IMGT to obtain the sequence features of the light and heavy chain variable regions (three CDR region sequences and four FR region sequences, and the most closely related germline gene family sequence). The human germline gene family sequence with the highest sequence similarity was searched for in the IMGT-DomainGapAlign amino acid check window. The 4D3 light chain corresponded to the human IGKV1-27*01+IGKJ2*02 family sequence, and the 4D3 heavy chain corresponded to the human IGHV1-3*01+IGHJ4*01 family sequence. The CDR regions of the human germline gene sequence were replaced with the CDRs of the 4D3 light and heavy chains, and the antibody structure information was checked using IMGT-Structural query. Finally, the light and heavy chain humanized sequences of the 4D3-Hum version were obtained. The 4D3 mouse sequence and the humanized sequence were entered into an online website to assess the similarity score of the human antibody. In Figure 6, the blue line indicates the Z-score distribution range and frequency of the mouse antibody library, the green line indicates the Z-score distribution range and frequency of the human antibody library, and the red line indicates the Z-score obtained by the 4D3 light chain and heavy chain. After humanization, the Z-score value of 4D3 was significantly higher.

[0036] Example 13: Modification of 7F11 humanized sequence The light and heavy chain humanized sequences of the 7F11 mouse antibody were obtained by referring to Example 12. The 7F11 mouse sequence and the humanized sequence were input into an online website to evaluate the similarity score of the human antibody. In Figure 7, the blue line indicates the Z-score distribution range and frequency of the mouse antibody library, the green line indicates the Z-score distribution range and frequency of the human antibody library, and the red line indicates the Z-score values ​​obtained by the 4D3 light chain and heavy chain. After humanization, the Z-score value of 7F11 was significantly higher.

[0037] Example 14: Relative affinity analysis of 4D3 humanized antibody The 4D3 humanized antibody sequence was cloned into a eukaryotic expression vector and transiently transfected into eukaryotic cells 293F with reference to Example 8. The purified antibody was diluted to 2ug / ml and added to the A1-H1 wells of a TROP2-coated ELISA plate together with the mouse antibody before humanization, and further diluted 3-fold in the A1-A12 direction, incubated at 37°C for 1hr, washed, and added with an anti-human Fc HRP-labeled antibody, incubated at 37°C, and then allowed to develop color. Finally, the relative affinities of the ch4D3 and hum4D3 antibodies were compared according to the EC50 and curve shape. Figure 8: EC50 (4D3-chimera): 0.056ug / ml; EC50 (4D3-humanized): 0.0502ug / ml.

[0038] Example 15: Thermostability analysis of 4D3 humanized antibody The purified hum4D3 antibody was dialyzed against PBS buffer to a final concentration of 2mg / ml and divided into two batches (3 tubes / batch) at 70ul / tube. Samples from the two batches were incubated at 4℃ and 37℃, respectively, and sample tubes were taken out on days 0, 7, and 14, respectively. The samples were used for SEC analysis to evaluate the degradation and aggregation status of the antibody. Figure 9: SEC detection results of 4D3 humanized antibody on days 7 (Figure 9A), 14 (Figure 9B), and 21 (Figure 9C). Figure 9D shows the ratio (%) of 4D3 humanized antibody to monomers, aggregates, and detection molecules at each time point under 37°C conditions.

[0039] Example 16: Analysis of the relative affinity and binding epitope consistency of the 7F11 humanized antibody The relative affinity of the 7F11 humanized antibody was evaluated with reference to Example 14. Figure 10: Binding curves of 7F11 humanized and chimeric antibodies at equivalent dilution concentrations. EC50(7F11-chimera): 0.061 ug / ml; EC50(7F11-humanized): 0.0601 ug / ml.

[0040] Example 17: Thermostability analysis of 7F11 humanized antibody See Example 15, where the thermal stability of the 7F11 humanized antibody was analyzed. Figure 11: SEC detection results of 7F11 humanized antibody on days 7 (Figure 11A), 14 (Figure 11B), and 21 (Figure 11C). Figure 11D shows the ratio (%) of 7F11 humanized antibody to monomers, aggregates, and detection molecules at each time point under 37°C conditions.

[0041] Example 18: Analysis of antigen affinity of humanized and parent antibodies The absolute affinity of antibody-antigen binding was evaluated using the Pall ForteBio Octet optical analysis technology platform. In this method, biotin-labeled antigen was immobilized on the surface of a streptavidin biosensor chip, the baseline was equilibrated for 180 seconds, and then the optical thickness of the chip was increased by binding with the antibody diluted in a solution concentration gradient for 30 seconds. This resulted in a wavelength shift (Δλ) and a 30-second dissociation phase. The interaction between Trop2 antigen and the corresponding antibody was measured in real time, and the binding specificity, binding rate, and dissociation rate at each concentration or sample concentration were precisely and accurately measured. The k-on and k-off values ​​at at least five concentration gradients were compiled to obtain the KD binding constant. [Table 2]

[0042] Example 19: Activity analysis of 4D3 humanized antibody and mated antibody and analysis of binding epitope consistency The 4D3 humanized antibody was labeled with biotin, and the inflection point value of the binding curve was measured by ELISA, and was 0.5ng / ml. An ELISA blocking solution containing 0.5ng / ml of 4D3 biotin-labeled antibody was prepared, and 50ug / ml of competitive antibodies 4D3 chimeric and 4D3 humanized were prepared using this solution. The solution containing the biotin-labeled antibody and the competitive antibody was added to A1 to A12 at 150ul / well, 50ul was sucked out and added to B2 to B12, and mixed thoroughly with the 100ul of biotin antibody solution added in advance, and further diluted 3 times in order and added to H1 to H12, incubated at 37℃ for 1hr, washed, incubated with anti-human IgG Fc secondary antibody, incubated at 37℃ for 1hr, washed 3 times, developed for 25min, and the value was read. Figure 12: 4D3 humanized biotin antibody competes with ch4D3 and hum4D3 antibodies at 0.02-50ug / ml, respectively. The two competing antibodies showed similar competitive activity and shared epitopes. EC50(ch4D3): 0.336ug / ml. EC50(hum4D3): 0.326ug / ml

[0043] Example 20: Activity analysis and binding epitope consistency analysis of 7F11 humanized antibody, humanized antibody and chimeric antibody See Example 18 for analysis of the epitope competitive activity and binding epitope consistency of 7F11. Figure 13: 7F11 humanized biotin antibody competed with ch7F11 and hum7F11 antibodies at 0.02-50ug / ml, respectively. The two competing antibodies showed similar competitive activity and shared epitopes. EC50(ch7F11): 0.732ug / ml. EC50(hum7F11): 0.856ug / ml.

[0044] Example 21: Cell binding and endocytosis experiments of humanized antibodies hu7F11 & hu4D3 The binding and endocytosis of each antibody at gradient concentrations was tested using HEK293 cells as negative cells and BXPC-3 and MCF-7 cells as positive cells. For cell binding tests, after 1 hr binding at 4°C, a regular FITC-labeled fluorescent secondary antibody was added and data was collected by flow cytometry.

[0045] In the endocytosis test, the acid-sensitive small molecule dye Phrodo-Red goat anti-human secondary antibody was used, which was incubated with various concentrations of primary antibodies to form complexes, and then incubated with each cell line for 16 hours. After that, samples were taken, and at least 10,000 of these cells were inoculated into each well of a 96-well plate, and data were collected and analyzed by a flow cytometer. The average fluorescence intensity values ​​in the far-red light channel of the cells at each concentration were statistically calculated, and an endocytosis degree curve was created with the antibody concentration as the abscissa.

[0046] Comparing the degree of binding between hu7F11 and Hu4D3 antibodies in three types of cells, the degree of binding was highest in BxPC-3 cells. Comparing the degree of endocytosis between hu7F11 and Hu4D3 antibodies in three types of cells, the degree of endocytosis was highest in BxPC-3 cells.

Claims

1. an anti-TROP2 antibody or one of its Fv, scFv, Fab, F(ab')2, Fab', and scFv-Fc fragments, The antibody comprises a heavy chain and a light chain, The antibody is an anti-TROP2 antibody or one of its Fv, scFv, Fab, F(ab')2, Fab', and scFv-Fc fragments, characterized in that the antibody comprises a heavy chain having the sequence shown in amino acid sequence 13 and a light chain having the sequence shown in amino acid sequence 14, or a heavy chain having the sequence shown in amino acid sequence 15 and a light chain having the sequence shown in amino acid sequence 16.

2. The antibody or one of its Fv, scFv, Fab, F(ab')2, Fab', and scFv-Fc fragments described in claim 1, characterized in that the sequence of the heavy chain derivative comprises the sequences shown in SEQ ID NO: 17 and SEQ ID NO:

18.

3. The antibody or one of its Fv, scFv, Fab, F(ab')2, Fab', and scFv-Fc fragments described in claim 1, characterized in that the heavy chain derivative sequence comprises the sequences shown in SEQ ID NO:19 and SEQ ID NO:

20.

4. The antibody according to claim 1 or one of its Fv, scFv, Fab, F(ab')2, Fab', scFv-Fc fragments, characterized in that an amino acid sequence having 95% or more identity with the sequence according to claim 1 is an equivalent of the present invention and should therefore be understood to be part of the present invention.

5. The heavy chain comprises CDRH1, CDRH2 and CDRH3 of SEQ ID NOs: 1, 2 and 3, respectively, or CDRH1, CDRH2 and CDRH3 of SEQ ID NOs: 4, 5 and 6, The antibody or one of its Fv, scFv, Fab, F(ab')2, Fab', and scFv-Fc fragments described in claim 1, characterized in that the amino acids contained in the light chain consist of CDRL1, CDRL2, and CDRL3 of SEQ ID NOs: 7, 8, and 9, respectively, or CDRL1, CDRL2, and CDRL3 of SEQ ID NOs: 10, 11, and 12.

6. Any antibody fragment or derivative comprising at least one CDR, characterized in that if the sequence of the CDR has at least 80% identity, preferably 85%, 90%, 95% or 98% identity with SEQ ID NO: 1 to SEQ ID NO: 12, it should be understood to be part of the present invention since it is an equivalent of the present invention.

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

6. The antibody or one of its Fv, scFv, Fab, F(ab')2, Fab' and scFv-Fc fragments according to claim 1, characterized in that it is of mouse origin.

7. An antibody or one of its Fv, scFv, Fab, F(ab')2, Fab', scFv-Fc fragments according to claim 2 or 3, characterized in that it is one of the humanized sequences of the sequences according to claim 1.

9. A nucleic acid encoding the antibody of claim 1, The nucleic acid is characterized in that it is a nucleic acid encoding an antibody or one of its Fv, scFv, Fab, F(ab')2, Fab', and scFv-Fc fragments according to any one of claims 1 to 3.

10. 10. The isolated nucleic acid of claim 9, characterized in that the nucleic acid comprises a sequence as set forth in SEQ ID NO: 21, 22, 23 or SEQ ID NO: 24, 25, 26 encoding a heavy chain, and the nucleic acid comprises a sequence as set forth in SEQ ID NO: 27, 28, 29 or SEQ ID NO: 30, 31, 32 encoding a light chain.

11. 11. The isolated nucleic acid of claim 10, characterized in that the nucleic acid comprises SEQ ID NO:33 and SEQ ID NO:34, or SEQ ID NO:35 and SEQ ID NO:

36.

12. 11. The isolated nucleic acid of claim 10, characterized in that the nucleic acid comprises SEQ ID NO:37 and SEQ ID NO:38, or SEQ ID NO:39 and SEQ ID NO:

34.

13. 13. An RNA nucleic acid corresponding to or complementary to a nucleic acid according to claims 10 to 12.

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

15. An expression host related to the expression vector of claim 14.

16. 16. An expression vector according to claim 14 and an expression host according to claim 15 capable of producing an amino acid sequence according to any one of claims 1 to 3.

17. Use of an antibody described in any one of claims 1 to 6 or one of its Fv, scFv, Fab, F(ab')2, Fab', scFv-Fc fragments or an amino acid sequence described in claim 16 for diagnosing or treating malignant tumors associated with the TROP2 target.

18. 18. Use according to claim 17, comprising a cytotoxic compound or a radioactive element bound to an antibody or one of its Fv, scFv, Fab, F(ab')2, Fab', scFv-Fc fragments.

19. 19. The cytotoxic compound of claim 18, selected from alkylating agents, antimetabolites, antineoplastic agents, mitotic inhibitors, chromatin function inhibitors, antiangiogenic agents, antiandrogens, antiestrogens or immunomodulators.

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