Proantibody drug conjugate capable of being activated by multiple proteases as well as preparation method and application of proantibody drug conjugate

Abstract

The invention discloses a proantibody drug conjugate capable of being activated by multiple proteases as well as a preparation method and application of the proantibody drug conjugate. The proantibody drug conjugate is named as PDC-PC1, a section of connecting peptide capable of being split by protease is added to each of amino terminals of two light chains of HcHAb18, on this basis, masking peptides used for preventing the antibody from being combined with an antigen are fused to the connecting peptides to form the proantibody PC1, a small-molecule tubulin inhibitor MMAE is cross-linked to an inter-chain disulfide bond of the PC1 in a fixed-point mode, and the proantibody drug conjugate is obtained. Therefore, the proantibody drug conjugate PDC-PC1 is formed. The new-generation PDC drug PDC-PC1 provided by the invention endows parent antibody with dual characteristics of conditional selective targeting combination and tumor microenvironment (TME) response delivery treatment, the targeting selectivity is remarkable, the specificity is stronger, the cross toxic and side effects are effectively reduced, and the accurate targeting curative effect is further improved through the introduction of MMAE fixed-point crosslinking.

Description

Technical Field

[0001] The present application relates to the field of antibody drug research and development, and particularly relates to a pro-antibody drug conjugate activatable by multiple proteases, and a preparation method and application thereof. Background Art

[0002] Antibody drugs are important varieties in the biopharmaceutical industry and have become a popular research field due to their targeting and effectiveness. With the increasing maturity of antibody drug research, development and production technologies, multiple therapeutic antibody drugs have been approved for marketing or are in different clinical trial stages. However, for antibody drugs that bind to antigens on the cell membrane, the antigens they target not only exist on the target cells of the diseased tissue, but also on the cell surface of non-diseased tissues, resulting in the interference of antibody drugs with the signal transduction of non-target cells, and thus causing cross-off-target toxic side effects. In the whole process of antibody drug research and development, many clinical trials ultimately fail, and a considerable part of the reason is the unforeseen cross-off-target toxicity of antibody drugs. Therefore, eliminating the cross-off-target toxicity of antibody drugs and at the same time improving their precision treatment efficiency is the forefront hot spot direction and key development trend in the field of antibody therapeutic drugs.

[0003] CD147 (also known as EMMPRIN or Basigin) belongs to the immunoglobulin superfamily and is a type I transmembrane glycoprotein composed of an extracellular domain, a transmembrane domain and an intracellular domain, and its extracellular domain is highly glycosylated. Studies have found that CD147 mediates a variety of signal transduction and has many biological functions in pathological processes such as tumor progression, inflammatory response, Plasmodium invasion, and viral infection. CD147 is also a tumor-associated membrane antigen and plays an extremely important role in the adhesion, invasion and metastasis of tumor cells. In recent years, CD147 has been widely concerned as a popular target for targeted therapy of various diseases. More and more studies have shown that CD147 is not only highly overexpressed in various types of tumors, including ovarian cancer (OC), lung cancer, prostate cancer and liver cancer, but is also closely related to their poor prognosis. In addition, the therapeutic efficacy of antibody drugs targeting CD147 has also been widely confirmed in preclinical models of various cancers. In a latest study, researchers evaluated an anti-CD147 antibody-drug conjugate using a cynomolgus monkey model, indicating that it is a very promising clinical treatment plan for malignant liver cancer, which once again verifies the superiority of CD147 as a drug development and treatment target. So far, such studies have shown that developing novel therapeutic antibody drugs targeting human CD147 antigen for precision treatment of various tumors including OC has important clinical translational application value.

[0004] HcHAb18 monoclonal antibody injection is a humanized antibody drug targeting CD147, which is used for patients with primary or postoperative recurrent and metastatic liver cancer that cannot be surgically removed. Although HcHAb18 has shown good efficacy in clinical trials, some patients have experienced various toxic and side effects such as rash, hair loss, diarrhea and colitis. The main reason is that the CD147 target is expressed at a low level in non-target tissues (such as skin, intestinal epithelium, etc.), which makes HcHAb18 appear "on-targetoff-tumor" phenomenon in actual application, that is, non-tumor targeted distribution or cross-off-target effect.

[0005] In order to solve the off-target toxicity of the HcHAb18 antibody, the present invention reshapes its characteristics based on the pre-antibody drug design concept and the disulfide bridged drug coupling strategy. The developed PDC-PC1, which can be activated by three proteases, is a new type of antibody drug that accurately, efficiently and selectively targets solid tumors such as OC. Specifically, the present invention uses a specific Masking peptide fused to the amino terminus of the light chain and a chemotherapy drug with a strong cytotoxic effect to cross-link and upgrade the parent antibody HcHAb18, which essentially realizes the clinical precision treatment value that takes into account its high tumor selectivity and specific targeting. After the transformation, the newly formed PDC-PC1 specifically recognizes and binds to the CD147 antigen only on the surface of tumor cells, and after internalization and lysosomal cleavage, the cross-linked drug is released into the cytoplasm of tumor cells, thereby exerting a precise and powerful anti-tumor effect and immunological effect. Summary of the invention

[0006] In order to overcome the shortcomings of the parent antibody cross-off-target toxicity in the prior art, the main purpose of the present invention is to provide a novel pre-antibody drug conjugate that can be activated by multiple proteases and its preparation method and application, so as to solve the problem of off-target toxicity of HcHAb18 antibody.

[0007] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0008] A pro-antibody drug conjugate that can be activated by multiple proteases, the pro-antibody drug conjugate is named PDC-PC1, and the specific structure is as follows:

[0009]

[0010] Among them, R is the pro - antibody of the parental antibody HcHAb18, named PC1; PC1 is obtained by fusing a masking peptide to the amino - terminus of the light chain of the HcHAb18 antibody through a protease - cleavable linker peptide; the HcHAb18 antibody comprises a light chain and a heavy chain, the amino acid sequence of the HcHAb18 light chain is as shown in SEQ ID NO:4, and the amino acid sequence of the HcHAb18 heavy chain is as shown in SEQ ID NO:1.

[0011] For the multi - protease - activatable pro - antibody - drug conjugate described above, the amino acid sequence of the masking peptide is as shown in SEQ ID NO:2, and the linker peptide is composed of the cleavage sequences corresponding to three proteases, namely matrix metalloproteinase 9, podoplaninase, and urokinase - type plasminogen activator, and its amino acid sequence is as shown in SEQ ID NO:3.

[0012] The preparation method of the multi - protease - activatable pro - antibody - drug conjugate comprises the following steps:

[0013] 3 - 1) Add a protease - cleavable linker peptide to the amino - terminus of both light chains of HcHAb18. On this basis, fuse the masking peptide used to prevent the antibody from binding to the antigen to the linker peptide to form the pro - antibody PC1;

[0014] 3 - 2) Site - specifically cross - link the small molecule microtubule inhibitor MMAE to the inter - chain disulfide bond of PC1 in step 3 - 1 to form the pro - antibody - drug conjugate PDC - PC1.

[0015] For the preparation method of the multi - protease - activatable pro - antibody - drug conjugate, the amino acid sequence of the masking peptide in step 3 - 1 is as shown in SEQ ID NO:2, and the linker peptide in step 1 is composed of the cleavage sequences corresponding to three proteases, namely matrix metalloproteinase 9, podoplaninase, and urokinase - type plasminogen activator, and its amino acid sequence is as shown in SEQ ID NO:3.

[0016] For the preparation method of the multi - protease - activatable pro - antibody - drug conjugate, the coupling process of PC1 and MMAE in step 3 - 2 includes the following chemical reactions:

[0017] 5 - 1) Add NaBH4 to the tetrahydrofuran solution of 4 - bromo - 2 - furan - carbaldehyde. After the reaction is complete, add dichloromethane for extraction, add anhydrous MgSO4 for drying and then filter. After rotary evaporation, dissolve it in pyridine, add acetic anhydride, and after the reaction is complete, rotary evaporate again. Dilute with ethyl acetate, wash successively with saturated aqueous NaHCO3 and saturated brine, discard the aqueous phase, then dry with anhydrous MgSO4, filter, and after rotary evaporation, purify by column chromatography to obtain compound 2, and the structure of compound 2 is as shown below:

[0018]

[0019] 5-2) Dissolve the compound 2 in step 5-1 in a THF / PBS solution, add N-bromosuccinimide for reaction, then add tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate thereto, continue the reaction at room temperature, spin-dry, dilute with DCM, add anhydrous MgSO4 for drying, filter, spin-dry, and after purification, obtain compound 3b. The structure of compound 3b is shown as follows:

[0020]

[0021] 5-3) Dissolve the compound 3b in step 5-2 in DCM, add trifluoroacetic acid, spin-dry after the reaction is complete, and dry under vacuum to obtain compound 3. The structure of compound 3 is shown as follows:

[0022]

[0023] 5-4) Couple the compound 3 in step 5-3 with N-hydroxysuccinimide ester-valine-citrulline-p-aminobenzyloxycarbonyl-MMAE to synthesize 3-bromo-5-methylenepyrrolidone-modified MMAE with bis-cysteine bridging reactivity, named 3Br-5MP-MMAE;

[0024] 5-5) After completely reducing PC1 with tris(2-carboxyethyl)phosphine and desalting, add 3Br-5MP-MMAE, thereby connecting the 3Br-5MP-MMAE drug molecule to the cysteine thiol group generated after the reduction of the interchain disulfide bond of PC1, and use NaBH4 to reduce to obtain PDC-PC1.

[0025] Use of the multi-protease activatable pro-antibody drug conjugate in the preparation of an anti-tumor drug.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] The new generation of PDC drug PDC-PC1 provided by the present invention endows the parental antibody with dual characteristics of conditional selective targeting binding and tumor microenvironment (TME)-responsive delivery treatment, with significant targeting selectivity and stronger specificity, effectively reducing cross-toxic side effects, and further improving its precise targeting efficacy through the site-specific cross-linking introduction of monomethyl auristatin E (MMAE). More importantly, the present invention provides a rapid transformation strategy and specific application implementation plan for improving both in vivo safety and killing efficiency in the property transformation of other therapeutic antibody drug varieties with similar cross-off-target problems. Description of the Drawings

[0028] Figure 1Schematic diagram of the process for expressing and purifying PC1 in Example 1;

[0029] Figure 2 High performance liquid chromatography (HPLC) chromatogram for detecting the purity of PC1 in Example 2;

[0030] Figure 3 Silver staining image for detecting the enzymatic cleavage responsiveness of PC1 by SDS-PAGE gel electrophoresis in Example 3;

[0031] Figure 4 High performance liquid chromatography (HPLC) chromatogram for detecting and verifying the purity of chemically synthesized 3Br-5MP-MMAE by UPLC-MS in Example 4;

[0032] Figure 5 Mass spectrometry (MS) spectrum for detecting the molecular weight of chemically synthesized 3Br-5MP-MMAE by UPLC-MS in Example 5;

[0033] Figure 6 Coomassie brilliant blue staining image for analyzing the reduction of interchain disulfide bonds of PC1 and the coupling process with 3Br-5MP-MMAE by SDS-PAGE in Example 6;

[0034] Figure 7 Representative image for observing and comparing the binding differences of PDC-PC1 and control antibody to OC cells by laser scanning confocal microscopy in Example 7;

[0035] Figure 8 Statistical result graph for detecting the killing efficiency of PDC-PC1 and control antibody to OC cells by CCK-8 method in Example 8;

[0036] Figure 9 Graph of tumor volume changes in nude mice bearing human OC during the treatment with PDC-PC1 and control antibody in Example 9;

[0037] Figure 10 HE staining image for comparing the toxicity of PDC-PC1 and control antibody to OC or normal organs in Example 10;

[0038] Figure 11 Structural diagram of the novel PDC drug PDC-PC1 with multiple protease-selective activation of the present invention. Detailed implementation manners

[0039] The following examples are exemplary descriptions of the main experimental evidence, rather than limiting the core content and application scope of the present invention disclosed by the quantity of evidence. It should be noted that in all these drawings and corresponding descriptions, only the concepts, principles and representative experimental evidence of the disclosed embodiments of the present invention are shown exemplarily. When the evidence chain is complete, it is not necessary to show all the specific detailed information and extended details of each embodiment listed in the present invention.

[0040] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. The experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified.

[0041] Term Explanation

[0042] PDC: Pro-antibody drug conjugate

[0043] Example 1: Expression and preparation process of PC1

[0044] 1) Design of PC1: Given the off-target toxicity of the anti-human CD147 HcHAb18 antibody, we modified the antibody structure of HcHAb18 according to the action principle of pro-antibody drugs, and designed a drug PC1 with higher safety. The heavy chain of PC1 is the same as the heavy chain of HcHAb18 (SEQ ID NO: 1), and its light chain is composed of a masking peptide (SEQ ID NO: 2), a linker peptide (SEQ ID NO: 3), and the light chain of HcHAb18 (SEQ ID NO: 4) connected together. Specifically, on the premise of keeping the heavy chain of the parental antibody HcHAb18 unchanged, a masking peptide containing 12 amino acids was fused to the amino terminus of the HcHAb18 light chain through a linker peptide, thus forming PC1. This masking peptide is derived from the extracellular domain of the CD147 antigen and is the antigen epitope that HcHAb18 can recognize. This linker peptide consists of 23 amino acids: GPLGVR GG AAN GG SGRSA GGGGS, carrying sequences cleavable by three proteases, matrix metalloproteinase 9 (MMP9), legumain, and urokinase-type plasminogen activator (uPA), where GPLGVR is the cleavage sequence of MMP9, AAN is the cleavage sequence of legumain, and SGRSA is the cleavage sequence of uPA.

[0045] 2) Expression of PC1: The specific process is as Figure 1As shown, the gene sequences encoding the heavy chain of PC1 and the modified light chain were amplified separately using polymerase chain reaction (PCR) technology and cloned into eukaryotic expression vectors. Subsequently, the successfully constructed vectors were sequenced and verified, and the correct plasmids were amplified. The plasmids were transiently transfected into HEK293 cells after being mixed with transfection reagents in proportion. Under stirring at 37 °C, the cells were expanded in serum-free medium.

[0046] 3) Purification of PC1: After centrifugation of the cell culture medium, PC1 was purified using a Protein A affinity chromatography column, and finally, the fully recombinant product PC1 was obtained.

[0047] 4) To better verify the key role of the masking peptide, the present invention also designed a control probody NC2, which has the same linker peptide and parental antibody structure as PC1, but its light chain amino terminus is fused with a peptide segment (SEQ ID NO: 5) that does not block the antigen-antibody binding potential through a linker peptide. Using the same technical means described in this example, NC2 was expressed and purified.

[0048] Example 2: Purity Detection of PC1

[0049] 1) Column equilibration: When using SEC-HPLC to detect the purity of PC1, a Cytiva chromatography system and a Cytiva Superdex 200 Increase 10 / 300GL 10x 300mm protein molecular sieve column were used, and PBS with pH = 7.0 was used as the mobile phase for rinsing.

[0050] 2) Detection: After the conductivity Cond increased and flattened, and UV280 decreased and flattened, the PC1 solution was filtered through a filter membrane and injected. Flow rate: 0.5 mL / min. The peak elution time and peak area were observed through UNICORN 7 software.

[0051] After the purity detection of PC1 was carried out according to the above method, the results showed that the purity of PC1 was above 96%, indicating that PC1 had a high purity level. As Figure 2 shown, this target product was suitable for subsequent verification experiments and synthesis reactions.

[0052] Example 3: In Vitro Proteolytic Activation Potential of PC1

[0053] 1) Sample preparation: The following solutions are used for enzymatic digestion reactions. TCNB buffer solution (20 mL): 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w / v) Brij 35, pH = 7.5, stored at 4°C; APMA activator (100 mM, 3.33 mL): with DMSO as the solvent, stored at 4°C; activation buffer (20 mL): 50 mM sodium acetate, 100 mM NaCl, pH = 4.0, stored at 4°C. Take 8 μL of the Legumain stock solution (20 ng / μL), add 8 μL of the activation buffer with pH = 1.5, dilute it, and place it in an EP tube (C), incubate at 37°C for 2 h. Take 8 μL of the uPA stock solution (20 ng / μL), add 8 μL of PBS, and place it in an EP tube (D). Take 8 μL of the MMP9 stock solution (20 ng / μL), dilute it with 4 μL of the TCNB buffer solution with pH = 2.3, add 4 μL of the 10 mM APMA dilution, and incubate at 37°C for 2 h in an EP tube (E). Add 4 μL of the PC1 antibody stock solution (200 ng / μL) to each of the three EP tubes, carefully mix each tube, take 4 μL of the HcHAb18 stock solution (200 ng / μL) and add it to 16 μL of PBS to obtain the sample for EP tube (A), stored at 4°C. Take 4 μL of the HcHAb18 stock solution (200 ng / μL) and add it to 16 μL of PBS to obtain the sample for EP tube (B). After incubating at 37°C for 24 h, add 4 μL of 5× loading buffer (reducing electrophoresis) to each of the five EP tubes A, B, C, D, and E, place them in a 100°C metal bath (preheated half an hour in advance) for 10 min to denature them, and store at -20°C.

[0054] 2) Electrophoresis: Prepare a 10% separating gel, load the previously prepared sample proteins (A - E) onto it, perform electrophoresis on ice, with a voltage of 80 V, and stop electrophoresis after about 3 h when the bromophenol blue just runs out of the gel.

[0055] 3) Solution preparation: The following solutions are used for silver staining, prepared and used immediately. Sensitizing solution: 0.01569 g of Na2O3S2·5H2O dissolved in 50 mL of ddH2O (protected from light); silver solution: 0.05 g of AgNO3 dissolved in 50 mL of ddH2O (protected from light); developing solution: 2.5 g of Na2CO3 dissolved in 125 mL of ddH2O, add 50 μL of 37% formaldehyde.

[0056] 4) Silver staining: Fix the gel with 50% ethanol - 5% acetic acid (50 mL) and gently shake it at room temperature for 60 minutes. Discard the liquid, and gently shake it with 50% ethanol (50 mL) at room temperature for 30 minutes. Discard the liquid, and gently shake it with ddH2O at room temperature for 30 minutes. Discard the liquid, and gently shake it with the sensitizing solution at room temperature for 1 minute. Discard the liquid, and gently shake it with ddH2O twice at room temperature, 1 minute each time. Discard the liquid, and gently shake it with the silver solution at room temperature for 30 minutes. At the end, collect it into the waste liquid bottle to prevent environmental pollution. Gently shake it with ddH2O twice at room temperature, 1 minute each time. Discard the ddH2O, develop the gel with 50 mL of the developing solution for 30 s, then discard the liquid, add the remaining developing solution for development until the bands meet the requirements, and observe in time. Stop the development by discarding the developing solution and adding the stop solution (5% acetic acid), wash it with ddH2O and then store it, and take careful photos.

[0057] The results are as Figure 3 shown: Compared with HcHAb18, the light chain band of PC1 moves to a position with a higher molecular weight, indicating that due to the introduction of the masking peptide, the molecular weight of the light chain of PC1 increases. When recombinant human Legumain, uPA or MMP9 protease is incubated with PC1, a light chain band with a slightly lower molecular weight appears, indicating that the masking peptide is cleaved. Therefore, PC1 has the potential for enzymatic cleavage and activation in vitro.

[0058] Example 4: Technical route for the chemical synthesis of 3Br - 5MP - MMAE

[0059] The technical route for the chemical synthesis of 3Br - 5MP - MMAE is shown in the following formula:

[0060]

[0061] 1) Synthesis of compound 1: Dissolve 3.92 g of 4 - bromo - 2 - furfuraldehyde (22.4 mmol) in 100 mL of freshly distilled tetrahydrofuran (THF). Slowly add 3.39 g of sodium borohydride (89.6 mmol) to the solution in portions at 0 °C, and continuously stir for 4 h. Monitor the reaction by thin - layer chromatography (TLC) (the eluent is petroleum ether:ethyl acetate = 4:1) to check if the reaction is complete. Quench the reaction by adding excess water. After no bubbles are generated, extract with dichloromethane (DCM), dry over anhydrous MgSO4, filter, and evaporate to dryness. The crude product is used directly in the next step without purification.

[0062] When synthesizing Compound 2, the unpurified Compound 1 from the previous step was dissolved in 110 mL of pyridine (py). 10.52 mL (112 mmol) of acetic anhydride (Ac2O) was slowly added dropwise under an ice bath. After stirring at room temperature for 3.5 h, TLC was used to monitor whether the reaction was complete. The reaction was quenched by adding excess water and then rotary evaporated. It was diluted with ethyl acetate and washed successively with saturated aqueous NaHCO3 solution and saturated brine solution. After discarding the aqueous phase, it was dried with anhydrous MgSO4, filtered, rotary evaporated to obtain the crude product, and purified by column chromatography (petroleum ether:ethyl acetate = 10:1) to obtain Compound 2 as a pale yellow liquid.

[0063] 2) Synthesis of Compound 3b: 94.18 mg (0.43 mmol) of Compound 2 was dissolved in 10 mL of a THF:PBS = 1:1 solution. 76.53 mg (0.43 mmol) of N-bromosuccinimide (NBS) was added under an ice bath and reacted for 1 h. Then 125.8 μL (0.516 mmol) of tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate was added, and stirring was continued at room temperature for 6 h. TLC was used to monitor whether the reaction was complete. The reaction system was directly rotary evaporated and then diluted with DCM, dried with anhydrous MgSO4, filtered, rotary evaporated, and purified by preparative thin layer chromatography (PTLC) to obtain Compound 3b as a yellow liquid.

[0064] 3) Synthesis of Compound 3: Compound 3b was dissolved in DCM, trifluoroacetic acid was added, and LC-MS was used to monitor whether the reaction was complete after reacting at room temperature for 40 minutes. The system was directly rotary evaporated and then dried under vacuum for 6 h to obtain Compound 3 as a yellow liquid.

[0065] 4) Synthesis of Compound 4: 5 mg of N-hydroxysuccinimide ester-valine-citrulline-p-aminobenzyloxycarbonyl-MMAE (Compound 3c) and 24 mg of Compound 3 were dissolved in 1 mL of anhydrous acetonitrile. Then 300 μL of an anhydrous acetonitrile solution of 0.1 mol / L triethylamine (TEA) was added, and the reaction was carried out at 37 °C for 1 h. LC-MS was used to monitor whether the reaction was complete. After purification by HPLC and freeze-drying, Compound 4 was obtained as a white solid.

[0066] 5) UPLC-MS Detection: Take a small amount of the freeze-dried product into a 1.5 mL EP tube, add 1 mL of acetonitrile to dissolve and mix evenly. Aspirate with a syringe needle, insert an organic phase filter membrane tightly into the syringe, take a conical bottom flask, filter, tighten the lid and place it in the machine position. Turn on the machine and the ultraviolet detector. First, flush the column with 0.3 mL / min of A 100% for 10 min. Select column 1 for small molecules, at 35 °C, with solvents A and B. Set the program in Xevo G2-XS QTof, in the electrospray positive ion mode ES+, set the Temperatures Source temperature to 100 °C, Open, No, Small Molecule.ipr, Open. Flush the column with 0.3 mL / min of A 95% and B 5%, set the program, select the small molecule mode Small Molecule pos res for 10 min, Load method to load, and wait for the column to equilibrate (the column pressure in the Console is 9000, and Delta is around 200). Observe the product peak and observe the peak area of the MMAE reactant at 254 nm. After the run is completed, obtain the chromatogram.

[0067] The test results are as Figure 4 shown. On the chromatogram of 3Br-5MP-MMAE, the target peak has no tailing and contains only one component, indicating that the product 3Br-5MP-MMAE with high purity is obtained, which is suitable for subsequent verification experiments and synthesis reactions.

[0068] Example 5: Purity Verification of Synthesized 3Br-5MP-MMAE

[0069] After synthesizing 3Br-5MP-MMAE, use UPLC-MS to verify its molecular weight. The specific method is as follows:

[0070] After following the steps in Example 4, according to the peak elution time and position in the chromatogram, perform mass spectrometry detection. In the software, select Chromatogram, Analog Chromatogram, and you can select the liquid chromatogram of different channels. After zooming in with the left mouse button and then looking at the specific molecular weight with the right mouse button, obtain the mass spectrum.

[0071] The experimental results are as Figure 5 shown. On the mass spectrum of 3Br-5MP-MMAE, a molecular ion peak is shown at m / z 1455 and corresponds to its molecular weight, indicating that we synthesized the product 3Br-5MP-MMAE with the correct structure through the above steps.

[0072] Example 6: Reduction of the Interchain Disulfide Bond of PC1 and the Coupling Process with 3Br-5MP-MMAE

[0073] 1) Reduction: Label a 50 mL centrifuge tube, transfer 20 mL of 0.5 M borate buffer solution (BBS, pH = 8.0) to the upper layer of a 4 °C refrigerator, add 0.5 M (0.5844 g) NaCl, and mix well by shaking. Take 5 mL from it and dilute it to 50 mL with ddH2O to form 1×buffer. Take 100 μL of 1×buffer and add 2.5 μL of 0.5 M tris(2-carboxyethyl)phosphine (TCEP) stock solution and mix well to form 12.2 mM TCEP for immediate use. Take 117.6 μL of 17 mg / mL PC1 and add 20 μL of 12.2 mM TCEP (molar ratio 1:18). Let it stand at room temperature for 1 h and then at 37 °C for 20 min. Add 100 μL of 1×buffer to make the total volume greater than 200 μL and desalt it through a 15 mL desalting column.

[0074] 2) Desalting: Add 1×buffer to the column, block the opening with a syringe and press the buffer down, rinse 6 times. After the last rinse, centrifuge at 1000 rcf for 2 min. Operate quickly to avoid leakage of the solution before centrifugation. Pour out the waste liquid, place the column in a new 15 mL centrifuge tube, label it, add the antibody, balance and centrifuge at 1000 rcf for 2 min. After that, put the column back into the original tube, cover the lid, and the first desalting is completed. Repeat the operation for another desalting (rinse 6 times, add the antibody, centrifuge in a new tube and collect).

[0075] 3) Detect protein concentration: Open the NanoDrop2000 software, select the Protein A280 mode, wash it three times with ddH2O, add 2.5 μL of ddH2O, Blank. After that, the Measure button lights up. Wipe off the ddH2O, add the antibody collected by centrifugation, Measure, observe the position and shape of the absorption peak, record the concentration to calculate the dosage of 3Br-5MP-MMAE, wash the 50 μL pipette three times, and close the software.

[0076] 4) Conjugation: Operate on ice, add 3Br-5MP-MMAE to 200 μL of antibody solution, and detect the peak area of the product by HPLC after standing at room temperature for 4 h.

[0077] 5) Characterization: When detecting by HPLC, replace the protein column, drip water into the column with the inlet pipe, then install the inlet pipe, place it upright, and install the outlet pipe after the column overflows with water. Keep the flow rate at Flow 1 mL / min and flush with 100% mobile phase A for half an hour, and pay attention to observing whether the column leaks. Mobile phase C: 2 M ammonium sulfate, mobile phase D 50 mM ammonium sulfate. After preparation, filter through a membrane and then sonicate. Inject the sample for detection and observe the elution time.

[0078] 6) Reduction: Measure the protein concentration and volume, calculate the amount of substance of the antibody, add NaBH4 at a molar ratio of 1:50, react for 0.5 h, and the NaBH4 solution needs to be prepared and used immediately.

[0079] 7) Purification: Remove small molecules through a molecular sieve column. The mobile phase flows from top to bottom. First, load the upper end of the column. After the lower end flows out, load the lower end, tighten it, wait for a while, and check for leakage. Dilute to 1× with 20× PBS, filter and sonicate, and prepare Watson water. Ensure that the solution in the bottle is sufficient. Take out the A1 pipeline from the ddH2O bottle and put it into the PBS bottle, wrap the bottle mouth with tin foil, exhaust air bubbles. In the UNICORN 7.0 software, System Control < Manual < Execute Manual Instructions < Pumps < Pump A wash < Inlet A1 < Insert < Execute. After that, flush the column Manual < Execute Manual Instructions < Pumps < System flow 0.5ml / min < Insert, and set the overpressure alarm Alarms < Alarm pre column pressure < High alarm 3.00MPa < Insert < Execute until the conductivity Cond rises and flattens, and the UV280 and UV254 are always low. After 1 hour, that is, 30 mL of equilibration. During this period, ① Take 10 mL from the PBS bottle and put it into a 50 mL tube. After filling the syringe, change the needle and inject three needles of PBS; ② Insert the lid of the 1.5 mL EP tube inward and check if it can be switched smoothly; ③ Prepare the antibody. Dilute PDC-PC1 to 550 uL with 1× PBS, filter through a yellow filter membrane to obtain 500 uL and temporarily store it in an EP tube, and prepare for syringe injection. After equilibration, End, Manual < Execute Manual Instructions < Pumps < System flow 0.5ml / min < Insert, Flow path < Injection value < Inject < Insert, Fraction collection < Fractionation < 0.50ml < Insert, Alarms < Alarm pre column pressure < High alarm 3.00MPa < Insert < Execute. Place the tube in the first position, inject the antibody to avoid air bubbles, adjust the abscissa, right-click Customize < X-Axis < Fixed 1-40ml < OK, and adjust back to Manual load after 10 minutes. After all the peaks are eluted, Manual < Execute Manual Instructions < Fraction collection < Stop fractionation < Insert < Execute, End. Take out the 1.5 mL EP tubes according to the tube numbers corresponding to the peaks, make marks, and clean the other 1.5 mL EP tubes.Wash the A1 head with a wash bottle, put it back into the water, cover it with the PBS lid, remove the air bubbles from A1. System Control<Manual<Execute Manual Instructions<Pumps<Pump A wash<Inlet A1<Insert<Execute. Flush the column in the same way as at the start, but collect into Waste and fractionate into a 15 mL centrifuge tube (set the collection volume to 10 mL and place the mobile phase in the position of the 1st centrifuge tube before Execute) to flush the entire pipeline until the conductivity Cond drops and levels off. After filling a syringe, change the needle and inject three needles of ddH2O. Disassemble the column (first disassemble the lower part, then the upper part after installing the lower part, and install the upper part after the upper part is liquid-sealed). After completion, pour out the waste liquid in the 15 mL centrifuge tube. Ultrafiltrate and centrifuge in a 4°C refrigerator, centrifuge at 15000 rcf for 5 min each time, pipette up and down to prevent the filter membrane from being blocked, resuspend thoroughly after concentration, and detect the protein concentration, mass spectrometry, and liquid chromatography to confirm the content and structure, and make a mark for storage in an -80°C refrigerator.

[0080] 8) Electrophoresis: Prepare a 10% separating gel. Add PC1, PC1 after reduction with TCEP, and PDC-PC1 to non-reducing loading buffer without dithiothreitol respectively, and place them in a 100°C metal bath for 10 min to denature. After the samples are cooled on ice, centrifuge and load the samples. Subsequently, perform non-reducing SDS-PAGE and electrophorese at a constant voltage of 80 V in an ice bath for about 3 hours until the bromophenol blue just runs out of the gel.

[0081] 9) Staining: After stopping the electrophoresis, perform Coomassie Brilliant Blue staining. Immediately stain the gel with Coomassie Brilliant Blue for 1 hour, rinse it with the decolorizing solution for 1 hour to wash off the surface floating color, then add fresh decolorizing solution and decolorize overnight at the lowest rotation speed of the shaker. Take pictures and analyze with a Bio-rad instrument to observe the connection of the antibody light chain and heavy chain to verify the reduction effect of TCEP and the cross-linking reaction of 3Br-5MP-MMAE with the antibody.

[0082] During the production process of PDC-PC1, we can easily detect the opening of disulfide bonds and the coupling of 3Br-5MP-MMAE. From Figure 6 the experimental results, it can be seen that after treatment with TCEP, PC1 changes from a complete antibody to independent light chain and heavy chain fragments ( Figure 6 , lane 2), while PC1 without TCEP treatment shows a main band at 151 kDa ( Figure 6 , lanes 1 and 3), indicating that the treatment with TCEP can open the interchain disulfide bonds of PC1. After reduction with TCEP and coupling with 3Br-5MP-MMAE, only one main band appears in the final product PDC-PC1 ( Figure 6, lane 4), indicating that the intermolecular disulfide bonds of PC1 that have been opened are re-bridged by 3Br-5MP-MMAE. Therefore, in this example, PDC-PC1 was successfully prepared, confirming that the structure of PDC-PC1 is Figure 11 as shown, and demonstrating the feasibility of the design and implementation of the above synthetic route. In addition, using the same technical means described in this example, 3Br-5MP-MMAE was respectively coupled with HcHAb18 and NC2 to synthesize ADC and PDC-NC2, and the two were used as control treatment groups for subsequent experiments.

[0083] Example 7: Binding differences of PDC-PC1 to OC cells before and after activation

[0084] This example aims to compare the binding differences of PDC-PC1 to OC cells before and after activation. PDC-PC1 (Act PDC-PC1) and PDC-NC2 (Act PDC-NC2) after cleavage by uPA enzyme were used as antibodies after in vitro activation, and IgG was used as an isotype control antibody to analyze the binding of different antibodies to SKOV3 cells. The specific method is as follows:

[0085] 1) Seeding cells on coverslips: Take a 24-well plate, place 1 round coverslip in each well (wash it once with PBS after air-drying to remove ethanol), digest the cells with trypsin and plate them. The number of cells should not be too many, about 30%. Add 0.5 mL of cell suspension with a concentration of 0.2×10 5 cells / mL diluted with complete culture medium to each well (i.e., 0.1×10 5 cells / well / 0.5 mL), and label the groups. Incubate for 24 h to allow the cells to spread.

[0086] 2) Fixation: Prepare ice in advance and operate on ice. Aspirate the culture medium and wash the coverslips 2 times with PBS. Add 200 μL of cold 4% PFA to each well, fix on ice / at room temperature for 20 min, then aspirate and wash the coverslips 3 times with PBS, aspirating each time after 3 min.

[0087] 3) Blocking: Add 200 μL of 5% BSA / PBST blocking solution to each well of the coverslip, block at room temperature for 1 h, then aspirate the blocking solution.

[0088] 4) Incubation with primary antibody: Drop 200 μL of PDC-PC1 and control antibody solutions diluted with 5% BSA / PBST onto each well of the coverslip, incubate overnight at 4 °C, then aspirate the primary antibody and wash 3 times with PBST, 3 min each time.

[0089] 5) Secondary antibody incubation: Add 200 μL of rhodamine-labeled anti-human secondary antibody solution dropwise onto each well of the cover glass. Wrap the secondary antibody with tin foil to avoid light. Dilute it with 5% BSA / PBST at a ratio of 1:500. Incubate at room temperature in the dark for 1 h, then aspirate the secondary antibody and wash the cells 3 times with PBST, 3 min each time.

[0090] 6) Nuclear staining: Take it out in advance to melt at room temperature in the dark. Stain the nuclei with Hoechst 33342 / PBS (diluted at 1:3000) for 5 min, then aspirate it and wash 3 times with PBST solution, 3 min each time.

[0091] 7) Mounting: Label the glass slides. Place a small round piece on each glass slide. Dip a white pipette tip into a small drop of mounting medium on the glass slide. Take out the round cover glass, absorb the excess liquid by sticking the edge to the paper, then slowly place it cell-side down into the drop of mounting medium. Gently remove the mounting medium on the cover glass with a tissue paper, apply nail polish to the edge for sealing, store in the dark, and take pictures after drying.

[0092] 8) Image collection: Detect and collect images with a confocal excitation scanning microscope. Select Auto HV, select SU Auto for the pinhole, select Sequential. The AF 405 channel is for the nuclei. Change the pseudo-color of the Ch2 channel to Red and keep the Gain value at 1. Use a 40× oil immersion objective and then Zoom to the appropriate magnification, find the focal plane, and collect pictures.

[0093] The binding of different antibodies to SKOV3 cells is shown in Figure 7 , in the unactivated PDC-PC1 experimental group, we detected very low fluorescence signals, indicating that PDC-PC1 hardly binds to CD147. However, when PDC-PC1 was incubated with protease, we detected strong fluorescence signals, which could effectively restore the binding to CD147. This indicates that removing the masking peptide by protease significantly enhances the binding ability of PDC-PC1 to CD147-positive cells.

[0094] Example 8: Comparison of the killing effects of PDC-PC1 and control antibodies on OC cells

[0095] 1) Seeding cells: Digest with trypsin, resuspend the cells for counting, dilute to 2×10 4 cells / mL, plate in a 96-well plate. Add 100 μL to each well for three replicates (i.e., 2000 cells per well. Note that the cell suspension must be well mixed during inoculation to avoid cell precipitation), add a circle of PBS around, label the groups, and culture for 24 h.

[0096] 2) Drug administration: Prepare ice in advance and operate on ice. Take an EP tube and label the concentration group at the tube mouth. Dilute in gradients and add 100 μL of PDC-PC1 and control antibody solutions diluted with culture medium to each well.

[0097] 3) Detection: Keep the laminar flow hood in the dark. Dilute the CCK-8 reagent with pure 1640 (each well finally contains 100 μL of pure 1640 and 10 μL of CCK-8 solution) and mix well. Then, aspirate the original culture medium and add the diluted solution. Continue culturing in the incubator for 0.5 - 4 h. Open the oven, observe the color change every 20 min, and then measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader (mix well on a plate shaker before measurement).

[0098] 4) Data processing: After discarding the outliers, calculate the survival rate. Survival rate = (absorbance of the experimental group - absorbance of the blank group) / (absorbance of the control group - absorbance of the blank group). Plot the graph using GraphPad to compare the differences between groups.

[0099] We used a CCK-8 kit to determine the killing effect of different antibodies on SKOV3 cells. The results of CCK-8 are shown in Figure 8 , and the results showed that conditionally activated PDC-PC1 could cause significant tumor cell death, subsequently leading to a sharp decline in cell viability, while HcHAb18 or the pro-antibody only slightly decreased cell viability, indicating that MMAE-mediated cytotoxicity greatly enhanced the anti-tumor effect of PC1.

[0100] Example 9: Anti-tumor efficacy of PDC-PC1 and control antibodies in a human OC xenograft nude mouse model

[0101] This example aims to illustrate the anti-tumor efficacy of PDC-PC1 and control antibodies in a human OC xenograft nude mouse model. The specific method is as follows:

[0102] 1) Tumor-bearing: Subcutaneously xenograft human OC cells (5×10 6 cells / 0.1 mL / mouse) into 6-week-old BALB / c nude mice to establish a tumor-bearing mouse model. When the tumor volume reaches 100 to 200 mm 3 , randomly divide the mice into groups according to their body weight and tumor volume, with 5 mice in each group.

[0103] 2) Administration: Intravenous injection of 15 mg / kg of PDC-PC1 or control antibody via the tail vein, once every four days. A total of five administrations are carried out until the observation ends.

[0104] 3) Efficacy monitoring: Measure the body weight and tumor volume of the mice every other day, and measure the long and short diameters of the tumor using vernier calipers. V = 1 / 2 × a × b 2 (a is the long diameter, b is the short diameter). After sacrificing the mice by cervical dislocation, dissect the heart, liver, spleen, lungs, kidneys, stomach, and tumor, weigh the organs and tumor, and take pictures for record.

[0105] Since small molecule drug MMAE was not conjugated, HcHAb18 and PC1 could not significantly reduce the tumor volume. However, in the PDC-PC1 treatment group formed after conjugating MMAE, the antitumor effect was significantly enhanced. After treatment, the tumor volume of the mice decreased significantly, and the tumors of one mouse completely disappeared at the end of the observation period. Therefore, PDC-PC1 showed good therapeutic effects and could eliminate xenografted tumors. In contrast, due to the lack of tumor targeting ability and the absence of conjugated mitotic inhibitor MMAE, the tumor inhibitory effect of HcHAb18 was not obvious( Figure 9 ).

[0106] Example 10: Differences in the toxicity profiles of PDC-PC1 and control antibody treatments on tumors or normal organs

[0107] This example aims to illustrate the differences in the toxicity profiles of PDC-PC1 and control antibody treatments on tumors or normal organs in a human OC nude mouse model. The specific method is as follows:

[0108] 1) Sample collection: The process of tumor-bearing and drug administration was the same as in Example 9. When making paraffin sections, the tissues were fixed with 4% PFA for 24 h, and the tissue blocks were placed in embedding cassettes and immersed in PBS for 2 h.

[0109] 2) Tissue dehydration: The embedding cassettes were placed in a beaker for the following dehydration operations: heart, kidney, spleen: 70% ethanol for 1 h, 80% ethanol for 1 h, 90% ethanol for 1 h, 95% ethanol for 30 min, absolute ethanol in two groups of 20 min each, xylene in two groups of 30 min each. Lung, liver, stomach tumor: 70% ethanol for 1 h, 80% ethanol for 1 h, 90% ethanol for 1 h, 95% ethanol for 40 min, absolute ethanol in two groups of 30 min each, xylene in two groups of 30 min each, and then embedding operations were carried out.

[0110] 3) Sectioning: The embedded tissues were soaked in ice water at 4 °C for 12 h, then serially sectioned at a thickness of 5 μm and baked for 1 h.

[0111] 4) Staining: The prepared paraffin sections were dewaxed and hydrated: xylene for 10 min × 3 times, absolute ethanol for 3 min × 3 times, 95% ethanol for 3 min × 2 times, 75% ethanol for 3 min × 2 times, distilled water for 1 min, and then placed in PBS buffer. The sections were immersed in hematoxylin stain for 3 min to stain the cell nuclei, washed with tap water, differentiated with 1% hydrochloric acid alcohol for 3 s, and rinsed with tap water for 5 min. The sections were immersed in eosin stain for 2 min to stain the cytoplasm. Rinsed with tap water for 20 s. The sections were successively placed in 95% alcohol I for 5 min, 95% alcohol II for 5 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min for dehydration and clearing. The sections were taken out from xylene and air-dried, and then sealed with neutral gum. Image acquisition and analysis were performed using a microscope.

[0112] Paraffin sections were prepared from the heart, liver, spleen, lung, kidney, stomach, and tumor tissues of mice and subjected to HE staining. The results are as Figure 10 shown. After administration of PDC-PC1, no abnormal changes were observed in the normal organs of the mice. At the same time, the area where tumor cells disappeared in the tumor tissues was relatively wide. The above results indicate that PDC-PC1 inhibits cell proliferation and induces apoptosis in a tumor-specific manner, and it has significant anti-tumor effects and high safety.

[0113] In summary, the present invention proposes a new design strategy for PDC antibody drugs. Taking the development process of PDC-PC1 that can be activated by three proteases and its precise treatment on the OC model as an example, four advantages of this strategy are demonstrated: namely, higher tumor selectivity, stronger antigen-binding specificity, elimination of cross-toxic side effects of parental antibodies, and better precise efficacy of site-specific drug conjugation.

[0114] Before the filing of the present invention application, there was no relevant literature report on the research or development of PDC-type antibody drugs targeting the CD147 target of solid tumors globally. Therefore, the multi-protease-activated PDC-PC1 demonstrated in the present invention is a new type of antibody drug variety with strong originality, unique ideas, and good prospects for clinical translation and application. The PDC drug design technology and the new variety of PDC-PC1 antibody drug targeting the CD147 target substantially protected by the present invention are completely different in essence from the previously disclosed Probody technology or ADC technology. It is a comprehensively updated and iteratively upgraded technology based on the past antibody remodeling technology, with significant substantial creativity. This technical solution will provide a general demonstration for other antibody drug varieties with cross-toxic side effects in terms of characteristic remodeling and precise empowerment. In short, the present invention has developed a new type of PDC targeting human CD147 for the first time and disclosed its preclinical biological safety and precise targeting efficacy. The technical solution and the new variety of antibody drug protected by the present invention will greatly promote the new trend of antibody new drug research and development based on the PDC concept at home and abroad.

[0115] The content of the above embodiments only details a part of the specific implementation manners of the present invention, but does not limit the present invention to the currently disclosed embodiments. In addition, the substantial content protected by the present invention is not limited to this. Without departing from the design scope of the present invention, any other modifications, equivalent replacements, improvements, etc. made according to the thinking principle and technical means of the present invention all belong to the protection scope of the present invention.

Claims

1. A multi-protease activatable pro-antibody drug conjugate, characterized in that, The pre-antibody drug conjugate is named PDC-PC1, and its specific structure is as follows: Wherein, R is the pre-antibody of the parental antibody HcHAb18, named PC1; PC1 is obtained by fusing a masking peptide to the amino terminus of the light chain of the HcHAb18 antibody through a protease-cleavable linker peptide; the HcHAb18 antibody comprises a light chain and a heavy chain, the amino acid sequence of the HcHAb18 light chain is shown in SEQ ID NO:4, and the amino acid sequence of the HcHAb18 heavy chain is shown in SEQ ID NO:

1.

2. The multi-protease activatable pro-antibody drug conjugate according to claim 1, wherein, The amino acid sequence of the masking peptide is shown in SEQ ID NO:2, and the linker peptide is composed of the cleavage sequences corresponding to three proteases, namely matrix metalloproteinase 9, podoplaninase and urokinase-type plasminogen activator, and its amino acid sequence is shown in SEQ ID NO:

3.

3. The preparation method of the multi-protease activatable pro-antibody drug conjugate according to claim 1, characterized in that, It includes the following steps: 3-1) Add a protease-cleavable linker peptide to the amino terminus of both light chains of HcHAb18. On this basis, fuse the masking peptide used to prevent the antibody from binding to the antigen to the linker peptide to form the pre-antibody PC1; 3-2) Site-specific crosslinking of the small molecule microtubule inhibitor MMAE to the interchain disulfide bond of PC1 in step 3-1 to form the pre-antibody drug conjugate PDC-PC1.

4. The preparation method of the pre-antibody drug conjugate that can be activated by multiple proteases according to claim 3, characterized in that, The amino acid sequence of the masking peptide in step 3-1 is shown in SEQ ID NO:2, and the linker peptide in step 3-1 is composed of the cleavage sequences corresponding to three proteases, namely matrix metalloproteinase 9, podoplaninase and urokinase-type plasminogen activator, and its amino acid sequence is shown in SEQ ID NO:

3.

5. The preparation method of the pre-antibody drug conjugate that can be activated by multiple proteases according to claim 3, characterized in that, The coupling process of PC1 and MMAE in step 3-2 includes the following chemical reactions: 5-1) Add NaBH4 to a tetrahydrofuran solution of 4-bromo-2-furaldehyde. After the reaction is complete, add dichloromethane for extraction, add anhydrous MgSO4 for drying, filter, spin-dry, dissolve in pyridine, add acetic anhydride, spin-dry after the reaction is complete, dilute with ethyl acetate, wash successively with saturated aqueous NaHCO3 solution and saturated brine, discard the aqueous phase, dry with anhydrous MgSO4, filter, spin-dry, and purify by column chromatography to obtain compound 2. The structure of compound 2 is shown as follows: 5-2) Dissolve compound 2 obtained in step 5-1 in a THF / PBS solution, add N-bromosuccinimide for reaction, then add tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate, continue the reaction at room temperature, spin-dry, dilute with DCM, add anhydrous MgSO4 for drying, filter and spin-dry, and purify to obtain compound 3b. The structure of compound 3b is shown as follows: 5-3) Dissolve compound 3b obtained in step 5-2 in DCM, add trifluoroacetic acid, spin-dry after the reaction is complete, and dry under vacuum to obtain compound 3. The structure of compound 3 is shown as follows: 5-4) Couple compound 3 described in step 5-3 with N-hydroxysuccinimide ester-valine-citrulline-p-aminobenzyloxycarbonyl-MMAE to synthesize 3-bromo-5-methylenepyrrolidone-modified MMAE with bis-cysteine bridging reactivity, named 3Br-5MP-MMAE; 5-5) After completely reducing PC1 with tris(2-carboxyethyl)phosphine and desalting, add 3Br-5MP-MMAE, thereby linking the 3Br-5MP-MMAE drug molecule to the cysteine thiol group generated after the reduction of the interchain disulfide bond of PC1, and using NaBH4 to reduce to obtain PDC-PC1.

6. Use of the multi-protease activatable pro-antibody drug conjugate according to claim 1 in the preparation of anti-tumor drugs.

Images