An antibody against DXD and its derivatives and its preparation and application

Abstract

The present invention provides an antibody against DXD and its derivatives, and its preparation and use. The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the antibody are shown in SEQ ID NOs. 1-3, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR13 of the light chain variable region are shown in SEQ ID NOs. 4-6, respectively. The antibody provided by the present invention has the advantages of high affinity, good specificity, and high purity, and can be used as a tool for the development of DXD-related ADC drugs, PK testing, etc.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an antibody against DXD and its derivatives, and a preparation method and application thereof. The antibody can specifically recognize T-DXD drugs and related derivatives. Background Art

[0002] An antibody-drug conjugate (ADC) is a monoclonal antibody (antibody) targeting a tumor-specific or tumor-associated antigen, conjugated to a highly active cytotoxic drug (payload) via a linker. ADCs resemble a dumbbell, with one end connected to a tumor-recognizing antibody responsible for selectively recognizing cancer cell surface antigens and subsequently binding to them, and the other end to various tumor-killing drugs. The linker serves as the gripping point in the middle, connecting the two ends. After entering the bloodstream, ADCs recognize antigens, allowing the anticancer drugs in the ADC to enter cells via endocytosis, leading to cancer cell death. ADCs combine the dual advantages of the high targeting of monoclonal antibodies with the high activity of cytotoxic drugs in tumor tissue. They are highly effective in killing tumor cells, have fewer side effects than chemotherapy drugs, and offer superior efficacy compared to traditional antibody-based anticancer drugs. They have therefore been dubbed "biological missiles" in the field of cancer treatment.

[0003] In recent years, ADC drugs have rapidly developed in the field of oncology treatment. Since the first ADC drug was approved in 2000, 14 ADC drugs have been approved for marketing worldwide, nine of which have been launched since 2019. Trastuzumab deruxtecan (T-DXD, DS-8201) is a prominent representative of the new generation of ADC drugs. Its drug carrier, DXD, is a water-soluble non-prodrug analog of camptothecin (CPT). DXD has strong in vitro inhibitory activity against 32 malignant tumor cell lines, demonstrating broad-spectrum anticancer effects. DXD's antitumor activity is approximately 10 times that of SN-38 and 100-1000 times that of common chemotherapy drugs, effectively reducing the dosage. Unlike commonly used breast cancer chemotherapy drugs like taxanes and anthracyclines, DXD can reduce cross-resistance. DXD is highly water-soluble and can form stable ADCs with antibodies with high DAR values. DXD's half-life in the blood is significantly shortened, helping to reduce toxic side effects. DXD is specifically cleaved by lysosomal proteases highly expressed in tumors. More importantly, DXD has enhanced membrane permeability, allowing it to penetrate into neighboring cells, exerting an anti-tumor "bystander effect." In addition to T-DXD, DXD has also been successfully used in the development of other ADCs, such as Datopotamab deruxtecan (Dato-DXD, DS-1062) and HER3-DXD (U3-1402). T-DXD consists of a humanized anti-human epidermal growth factor receptor-2 (HER2) antibody, an enzymatically cleavable peptide linker, and a topoisomerase I inhibitor (i.e., DXD), and can be used in the research of HER2-positive breast cancer and gastric cancer; Dato-DXD is a trophoblast cell surface antigen-2 (TROP2)-directed ADC drug with potent anti-tumor activity; HER3-DXD is used to treat multi-line resistant breast cancer.

[0004] As a prominent representative of the new generation of ADC drugs, DXD testing is particularly important during new drug development and finished product manufacturing. Consequently, there is a significant market demand for anti-DXD antibodies that meet specific requirements, such as specificity, sensitivity, and a wide range of recognition. DXD is a small molecule with inherently weak immunogenicity and a single antigenic epitope, making antibody preparation challenging. Summary of the Invention

[0005] In view of this, the present invention aims to develop a monoclonal antibody that can be used for the detection of DXD and its derivatives.

[0006] The technical solutions of the present invention are as follows:

[0007] In a first aspect, the present invention provides an antibody against DXD and its derivatives, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3;

[0008] Specifically, the amino acid sequence of HCDR1 is as shown in SEQ ID NO.1, or has more than 80% homology to the sequence shown in SEQ ID NO.1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO.2, or has more than 70% homology to the sequence shown in SEQ ID NO.2, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO.3, or has more than 75% homology to the sequence shown in SEQ ID NO.3; the amino acid sequence of LCDR1 is as shown in SEQ ID NO.4, or has more than 80% homology to the sequence shown in SEQ ID NO.4, the amino acid sequence of LCDR2 is as shown in SEQ ID NO.5, or has more than 70% homology to the sequence shown in SEQ ID NO.5, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO.6, or has more than 75% homology to the sequence shown in SEQ ID NO.6.

[0009] Preferably, in the above antibody, the amino acid sequence of HCDR1 is shown as SEQ ID NO.1, the amino acid sequence of HCDR2 is shown as SEQ ID NO.2, and the amino acid sequence of HCDR3 is shown as SEQ ID NO.3; the amino acid sequence of LCDR1 is shown as SEQ ID NO.4, the amino acid sequence of LCDR2 is shown as SEQ ID NO.5, and the amino acid sequence of LCDR3 is shown as SEQ ID NO.6.

[0010] More preferably, in the above-mentioned antibody, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8. In some embodiments of the present invention, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO. 12, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO. 11.

[0011] The second aspect of the present invention provides biomaterials related to the above-mentioned antibodies against DXD and its derivatives, including the following:

[0012] a) An isolated nucleic acid encoding the antibody provided by the present invention; preferably, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.9, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.10;

[0013] b). An expression vector comprising the nucleic acid shown in a);

[0014] c) A host cell comprising the nucleic acid shown in a) or the expression vector shown in b).

[0015] The third aspect of the present invention provides the use of the above nucleic acid, expression vector and / or host cell in preparing antibodies against DXD and its derivatives.

[0016] A fourth aspect of the present invention provides the use of antibodies against DXD and its derivatives in the detection of DXD or the preparation of a DXD detection kit.

[0017] The beneficial effects of the present invention are as follows: the present invention adopts hybridoma fusion technology to screen and obtain monoclonal cell lines, then obtains antibody sequences through molecular biological means, and through SEC-HPLC detection and screening and verification of DS-8201 drugs, T-DXD (DS-1062) drugs, T-DXD (U3-1402) drugs, DXD-related derivatives, etc., obtains anti-DXD antibodies with the advantages of high affinity, good specificity, high purity, etc., providing a new detection tool for the research and development and detection of DXD-related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the SDS chart of the 8D12C11 ascites antibody prepared in Example 1.

[0019] Figure 2 This is the SEC-HPLC detection chart of the 8D12C11 ascites antibody prepared in Example 1.

[0020] Figure 3 This is an ELISA test chart of 8D12C11 ascites antibody under T-DXD (DS-1062) coating conditions in Example 1.

[0021] Figure 4 This is the ELISA detection chart of 8D12C11 ascites antibody under T-DXD (U3-1402) coating conditions in Example 1.

[0022] Figure 5 This is an ELISA test chart of 8D12C11 ascites antibody under T-DXD (DS-8201) coating conditions in Example 1.

[0023] Figure 6 This is an ELISA test chart of 8D12C11 ascites antibody under the DXD derivative coating condition in Example 1.

[0024] Figure 7 This is the SDS chart of the 8D12C11 recombinant antibody prepared in Example 2.

[0025] Figure 8 This is the ELISA detection diagram of the 8D12C11 recombinant antibody under T-DXD (DS-8201) coating conditions in Example 2. DETAILED DESCRIPTION

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The term "comprise" and any variations thereof in the description and claims of the present invention are intended to cover non-exclusive inclusions.

[0027] The technical solution of the present invention will be described clearly and completely below in conjunction with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0028] If no specific techniques or conditions are specified in the following examples, the procedures were carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product instructions; if no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0029] Example 1

[0030] In this example, the following steps were performed to obtain antibodies against DXD and its derivatives, as well as their heavy and light chain variable region sequences:

[0031] (1) Preparation of antigens.

[0032] The hydroxyl group in the small molecule DXD was coupled with the amino group of the carrier protein BSA using the succinic anhydride method, specifically as follows:

[0033] 15g 2,2,2-trichloroethanol, 12g succinic anhydride and 8.7ml triethylamine were dissolved in 100ml ethyl acetate; heated to reflux for 1 hour; the solvent was distilled off under reduced pressure and the residue was dissolved in 5% aqueous NaHCO3 solution; washed twice with ether and then acidified with H2SO4 (pH 2.0); the solid was washed twice with water and crystallized from chloroform-hexane (yield about 75%, melting point 88-89°C); 2.5g hemisuccinate was dissolved in 6.5ml thionyl chloride and heated at 65°C for 30 minutes; evaporated under reduced pressure and dried for 1 hour (under high vacuum conditions); the above product (2,2,2-trichloroethylsuccinyl chloride) was dissolved in 15ml N,N-dimethyl-acetamide and then mixed with 2.79g The hydroxyl groups of DXD were stirred at room temperature for 2 hours; after evaporation in vacuo at 65°C, crystals were precipitated with isopropanol; trichloroethyl was dissociated with zinc dissolved in dimethylformamide and acetic acid to obtain the hapten-hemisuccinate.

[0034] The hapten-hemisuccinate was oxidized with NaIO4 to remove the glycosidic alcohol and then coupled to BSA. 20 mg of the glycoside was dissolved in 1 ml of 100 mmol / L NaIO4 solution and reacted at 4°C in the dark for 30 minutes. One drop of ethylene glycol was added (to obtain solution A). Solution A was added to a β-galactosidase solution (20 mg / ml, dissolved in 150 mmol / L NaCl and 10 mmol / L MgCl2 aqueous solution, and the pH was adjusted to 9.0 with 3% K2CO3). The reaction was continued at 4°C for 2 hours, during which the pH was continuously adjusted to 9.0. A 50 mg / ml NaBO4 solution prepared on the spot was added at 1 / 10 of the reaction volume. The reaction was continued at 4°C overnight. The solution was dialyzed against 50 mmol / L phosphate buffer (containing 10 mmol / L MgCl2, 10 mmol / L 2-mercaptoethanol, and 100 mmol / L NaCl, pH 7.4) (the dialysate was changed several times).

[0035] (2) Animal immunization.

[0036] Mice were immunized with the above antigens according to the conventional procedure, 50 μg each time, and multiple immunizations were performed subcutaneously after emulsification with Freund's adjuvant. Blood was collected from the tail vein when the immunization was boosted to three injections, and the serum titer was detected by indirect ELISA. Mice were selected for cell fusion experiments based on the test results.

[0037] Among them, the operation of antiserum titer detection is as follows: the antigen is diluted to 2 μg / ml with CB buffer, and 100 μl per well is coated on a 96-well plate and incubated at 4°C overnight; 200 μl of 4% skim milk powder is added to each well for blocking, and incubated at 37°C for 2 hours; TBS is used to wash the plate; the antiserum is diluted with PBS, and after the dilution ratio is 1:1000, 1:2000, 1:4000, 1:8000...1:64000, 100 μl is added to each well, and incubated at 37°C for 1 hour; TBS is used to wash the plate, and goat anti-mouse secondary antibody (1:5000) is added to each well at 100 μl; after 40 minutes, TBS is used to wash the plate, and 50 μl of TMB is added to each well, and the plate is incubated at 37°C for 15 minutes, and the reading is read on a microplate reader.

[0038] (3) Cell fusion.

[0039] The cells were cultured in DMEM supplemented with 20% fetal bovine serum. The spleens of the mice were processed into splenocytes and then counted.

[0040] Preheat 1 ml of PEG and 40 ml of DMEM fusion medium in a 37°C 5% CO2 incubator. Mix myeloma cells and spleen cells in a ratio of 1:5, centrifuge at 1500 rpm for 5 minutes, and retain the cells. Add preheated PEG, add it within 60 seconds, and let it stand for another 60 seconds. Add preheated DMEM fusion medium to the fusion system to terminate the effect of PEG. Centrifuge and discard the supernatant. Suspend the fused hybridoma cells with fetal bovine serum and add culture medium containing 25% fetal bovine serum containing trophoblasts, glutamine, double antibody (i.e., penicillin-streptomycin mixture) and HAT (i.e., hypoxanthine, methotrexate and thymidine). Plate the mixed cells into a cell culture plate and culture them in a 37°C 5% CO2 incubator.

[0041] Day 0 is counted from the day of fusion. For the first three days, try not to move the culture plate. Simply monitor the cells for contamination and maintain a stable incubator environment. If the culture medium turns yellowish on days 5-7, replace it with fresh HT complete medium. After 1-2 days, collect the cell culture supernatant and perform preliminary screening for positive clones using an indirect ELISA.

[0042] Among them, the specific operation of cell supernatant ELISA verification is as follows: the antigen is diluted to 2μg / ml with CB buffer, 100μl per well is coated into a 96-well plate, and incubated at 4°C overnight; 200μl per well of 4% skim milk powder is used for blocking, and incubated at 37°C for 2 hours; TBS is used to wash the plate; 100μl of cell supernatant stock solution is added to each well, and incubated at 37°C for 1 hour; TBS is used to wash the plate, and goat anti-mouse secondary antibody (1:5000) is added to 100μl per well; after 40 minutes, TBS is used to wash the plate, 50μl of TMB is added to each well, and the plate is incubated at 37°C for 15 minutes, and the plate is read with an enzyme reader.

[0043] (4) Subcloning and identification of strains.

[0044] Take the cells that need to be subcloned and gently pipette to make a single cell suspension, add it to a counting plate for counting and calculating the cell density.

[0045] Take 100 μl of cells to be subcloned and dilute to 1×10 3 5 cells / well, the subclone concentration is 1×10 3 Take 200 μl of the cell suspension of 5 cells / ml and add it to 3.8 ml of culture medium and mix well. 100 μl / well is plated 4 strips on a 96-well plate containing trophoblast cells. 0.5 cells / well subclone is mixed in 4 ml (5 cells / well) of cell suspension. Take 700 μl and add it to 6.3 ml of culture medium and mix well (add 100 μl to a 96-well plate). 100 μl / well is plated 8 strips on a 96-well plate containing trophoblast cells. Subclones are detected by ELISA method (same as step (3)). The results of the positive clone identification obtained by the present invention are shown in Table 1.

[0046] Table 1 Titer of DXD positive cell supernatant

[0047]

[0048]

[0049] In Table 1, DS-1062, U3-1402, and DS-8201 are ADC drugs in which DXD is conjugated to different tumor target antibodies; the DXD-derivative is specifically Deruxtecan-d5.

[0050] (5) Preparation of monoclonal antibodies.

[0051] Mice were injected with adjuvant 1 week in advance, and then each mouse was injected with 0.5×10 7 cells into the mouse ascites, and the ascites was extracted after 7-10 days.

[0052] Wash with water for 5 column volumes, then with 20mM PB + 150mL NaCl (pH 7.0) for 5 column volumes. Filter the sample through a 0.45μm filter and dilute the ascites 4-fold. Load the sample, allowing the ascites to drip dropwise at least twice. Wash with 20mM PB + 150mL NaCl (pH 7.0). One drop should be colorless using a G250. Elute with 0.1M glycine (pH 3.5), controlling the flow rate while neutralizing with 2M Tris-HCl (pH 8.0). A light green color should be observed using pH paper. Stop elution when one drop is colorless using a G250. After the antibody is eluted, continue eluting the column with 3.5% acetic acid for 20 column volumes. You can slow wash for 5 column volumes and fast wash for 15 column volumes; put the eluted antibody into a dialysis bag and dialyze it against 5L 1×PBS at 4°C for 2 hours, then change to 5L 1×PBS and continue dialysis; perform quality control after ultrafiltration on the next day, and verify affinity using ELISA. The ELISA method is as follows: the coating protein is diluted to 2 μg / ml in CB buffer, and 100 μl per well is coated in a 96-well plate and incubated overnight at 4°C. Blocking is performed with 200 μl per well of 5% skim milk powder and incubated at 37°C for 2 hours. The plate is washed with TBS. The antibody is diluted in PBS to a dilution ratio of 2000 ng / ml, 1000 ng / ml, 500 ng / ml, 250 ng / ml, and 0.12207 ng / ml, and 100 μl is added to each well. The plate is incubated at 37°C for 1 hour. The plate is washed with TBS and 100 μl of goat anti-mouse secondary antibody (1:10,000) is added to each well. After 40 minutes, the plate is washed with TBS and 50 μl of TMB is added to each well. The plate is incubated at 37°C for 15 minutes and read on a microplate reader. Antibody purity is then verified by SDS-stained solids chromatography and SEC-HPLC.

[0053] After screening and verification, the monoclonal antibody produced by the cell clone number 8D12C11 has a relatively excellent effect. Among them, the SDS chart of the ascites antibody obtained is as follows Figure 1 As shown, and SEC-HPLC detection showed that its purity was 93% ( Figure 2 ); The ELISA validation results of the antibody are shown in Table 2 and Figure 3-6 shown.

[0054] Table 2 ELISA detection of EC 50 result

[0055] Plate protein (2 μg / mL) Detection of antibodies <![CDATA[EC 50 (ng / mL)]]> T-DXD(DS-1062) 8D12C11 ascites antibody 1.788-2.146 T-DXD(U3-1402) 8D12C11 ascites antibody 2.382-2.704 T-DXD(DS-8201) 8D12C11 ascites antibody 2.225-2.851 DXD derivatives 8D12C11 ascites antibody 13.20-16.57

[0056] (6) Cell sequencing.

[0057] RNA was extracted from 8D12C11 cells and reverse-transcribed into cDNA. After PCR amplification, the light and heavy chains were amplified using the cDNA as a template. Separate gels were run to recover the light and heavy chain DNA, which was then excised and ligated with the vector and PCR products. The heavy chain and vector were incubated at 50°C overnight, while the light chain and vector were incubated at 37°C overnight. The ligation reaction was incubated at 16°C overnight (approximately 16 hours). Competent E. coli cells were prepared, transformed, plate-selected, and submitted for sequencing. The monoclonal antibody sequence is shown in Tables 3-4.

[0058] Table 3 Monoclonal Antibody CDR Region Sequences

[0059]

[0060] Table 4 shows the sequence information of the heavy chain variable region and light chain variable region of the monoclonal antibody

[0061]

[0062]

[0063] Example 2

[0064] In this example, based on the antibody sequence obtained in Example 1, recombinant antibodies were prepared using CHO cells as host cells, including the following steps:

[0065] (1) Vector construction.

[0066] According to the sequencing results of Example 1, full gene synthesis, subcloning, expansion and recovery were performed; the recovered product was subjected to a ligation reaction at a ratio of product: vector (pSecTag2A) = 4:1; the recovered ligation product was transformed into a competent culture, plated, picked, and sequenced to verify the constructed vector sequence; after correct sequencing, a large amount of the ligated vector was used to amplify the 200 ml system for culture, and the plasmid was extracted using a large extraction kit, the plasmid was quality controlled, and stored for the next step.

[0067] (2) CHO expression.

[0068] Take CHO cells, centrifuge and remove the supernatant. Add about 2.5ml of electroporation solution to the cells, mix well and add an appropriate amount of plasmid. After thoroughly mixing the above cell plasmid suspension, take 5ml and add it to a 5ml electroporation tube. Place the electroporation tube into the electroporation instrument for electroporation. After the electroporation is completed, the cells in the electroporation tube are divided into a shake flask containing 100ml of culture medium prepared in advance and incubated statically for 40 minutes. After the incubation is completed, place the shake flask in 37°C, 270rpm, 8% CO2 for incubation. After 24 hours, add feed / sodium butyrate / double antibody and continue to culture for 7 days.

[0069] (3) Purification of recombinant antibodies.

[0070] Equilibrate the column with 1× PBS at a flow rate of 1 ml / min for 20 ml. Load the sample at a flow rate of 1 ml / min. Wash the column with 1× PBS at a flow rate of 1 ml / min for 20 ml. Elute the column with sodium acetate buffer (pH 3.4) at a flow rate of 1 ml / min. Collect the sample into separate tubes, approximately 500 μl per tube. Collect 10 tubes in total and read the absorbance at 280 nm using a NanoDrop instrument. Dialysis: Pipette the high-concentration protein into a dialysis bag and dialyze it into a beaker containing 1× PBS.

[0071] Figure 7 The SDS image of the recombinant antibody prepared in this example is shown in Figure 2. In addition, referring to the ELISA detection method of Example 1, the recombinant antibody obtained in this example was verified using T-DXD (DS-8201) as the coating protein. The results are shown in Figure 2. Figure 8 As shown, its EC 50 (ng / mL) was 0.9828-1.446.

[0072] In summary, the antibodies provided by the present invention (including ascites antibodies and recombinant antibodies) can well recognize T-DXD (ADC drug) and its derivatives, have the advantages of high affinity, good specificity and high purity, and can be used for DXD-related drug detection.

[0073] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solutions of the present invention rather than to limit them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. An anti-DXD antibody, characterized in that comprising a heavy chain variable region and a light chain variable region; The heavy chain variable region includes HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO.1, the amino acid sequence of HCDR2 is shown in SEQ ID NO.2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO.3; The light chain variable region includes LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is shown as SEQ ID NO.4, the amino acid sequence of LCDR2 is shown as SEQ ID NO.5, and the amino acid sequence of LCDR3 is shown as SEQ ID NO.

6.

2. The anti-DXD antibody according to claim 1, wherein The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. An isolated nucleic acid, characterized in that Encodes the anti-DXD antibody according to claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.9, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.

10.

5. An expression vector, characterized in that The expression vector comprises the nucleic acid of claim 3.

6. A host cell, characterized in that The host cell comprises the nucleic acid of claim 3 or the expression vector of claim 5.

7. A method for preparing an anti-DXD antibody, characterized in that: The host cell of claim 6 is cultured to produce the anti-DXD antibody.

8. Use of the anti-DXD antibody according to claim 1 or 2 in DXD detection.

9. A detection kit, characterized in that Comprising the anti-DXD antibody according to claim 1 or 2.

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