Antibody-drug conjugate targeting to TROP2 as well as preparation method and application of antibody-drug conjugate

Abstract

The invention discloses an antibody-drug conjugate targeting to TROP2 as well as a preparation method and application of the antibody-drug conjugate. The invention provides an antibody-drug conjugate which is named as hIMB1636-MMAE, and the antibody-drug conjugate is a compound obtained by coupling hIMB1636 and MMAE. The hIMB1636 is an IgG (immunoglobulin G) antibody specifically combined with human TROP2, and has good targeting property, affinity and endocytosis capacity. The MMAE has strong cytotoxicity. The hIMB1636-MMAE has the effects of targeting TROP2 positive cells and strongly killing cells at the same time, so that tumor cells highly expressed by TROP2 are selectively killed, the IC50 value is in the nanomole or subnanomole level, and the hIMB1636-MMAE has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biochemical medicine, and relates to an antibody-drug conjugate targeting TROP2, a preparation method thereof, and uses thereof. Background Art

[0002] Human trophoblast cell surface antigen 2 (TROP2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1), gastrointestinal tumor-associated antigen (GA733-1) or surface marker 1 (M1S1), is a 36kDa transmembrane glycoprotein expressed by the TACSTD2 gene located in the chromosome 1p32 region. TROP2 was originally considered to be a surface marker of trophoblast cells, but scientists subsequently found that its expression was elevated in a variety of solid tumors, but low in normal tissues, and it is located on the cell membrane, making TROP2 an ideal target for tumor treatment.

[0003] Antibody-drug conjugates (ADCs) are made by coupling monoclonal antibodies against tumor surface antigens with small molecule cytotoxic drugs through linkers. It is a form of targeted therapy and a perfect combination of chemotherapy and immunotherapy. ADC can selectively deliver cytotoxic drugs to tumor cells while sparing healthy cells, eliminating the main clinical obstacles of traditional chemotherapy. Compared with chemotherapy drugs, it has fewer side effects and better efficacy than traditional therapeutic antibodies. The selection of appropriate targets is crucial for the preparation of ADCs. The targets should meet the following requirements: differential expression, no expression or low expression in normal cells, high expression on the surface of tumor cells; and internalization properties. ADC has extremely high requirements for the cytotoxicity of cytotoxins. The accumulation of warhead molecules in tumors is limited, so cytotoxins usually need to be expressed at nanomolar concentrations (IC 50 <10 -9 mol / L).

[0004] At present, antibody-drug conjugates targeting CD30, CD33, CD22 and Her-2 have been approved for marketing by the FDA. At the same time, ADC drugs targeting TROP2 have achieved gratifying results in clinical cancer research, including IMMU-132, which has been approved by the US FDA for the treatment of triple-negative breast cancer. Since TROP2 can be highly expressed in a variety of tumors, ADCs targeting TROP2 can be used to treat a variety of solid tumors, expanding the types of tumor treatments for ADCs. The clinical and market prospects are broad, and can bring hope to the treatment of many tumors, especially solid tumors. Summary of the invention

[0005] The purpose of the present invention is to provide an antibody-drug conjugate targeting TROP2, a preparation method thereof and a use thereof.

[0006] First, the present invention provides an antibody-drug conjugate, named hIMB1636-MMAE.

[0007] hIMB1636-MMAE is a compound obtained by coupling hIMB1636 and MMAE.

[0008] hIMB1636 is an IgG antibody that specifically binds to human TROP2.

[0009] hIMB1636 was obtained by hybridoma screening and CDR transplantation humanization.

[0010] hIMB1636 not only has a high binding affinity to TROP2 antigen (KD value is 0.6nM), but also can selectively bind to and be internalized by TROP2-positive cells.

[0011] The three CDRs of the heavy chain variable region of hIMB1636 are shown in SEQ ID NO:1 at positions 31-35 (NYWLG), 50-66 (NIFPGGGYTNYNEKFKG), and 99-104 (EGVMDY), and the three CDRs of the light chain variable region are shown in SEQ ID NO:3 at positions 24-34 (RASQNIGTSIH), 50-56 (YASESIS), and 89-97 (QQSYSWPFT).

[0012] The heavy chain variable region of hIMB1636 is shown at positions 1-115 in SEQ ID NO:1, and the light chain variable region is shown at positions 1-107 in SEQ ID NO:3.

[0013] The heavy chain of hIMB1636 is shown in SEQ ID NO:1, and the light chain is shown in SEQ ID NO:3.

[0014] MMAE is a linear polypeptide cytotoxic molecule that acts on the β-subunit of α-β tubulin dimers, causing unregulated microtubule growth and thus inhibiting tumor growth.

[0015] The structural formula of MMAE is shown in formula (I);

[0016]

[0017] hIMB1636 and MMAE are specifically coupled via a linker.

[0018] hIMB1636-MMAE is a compound obtained by coupling hIMB1636 and MMAE via a linker.

[0019] The linker is Val-Cit dipeptide (abbreviated as VC). Val-Cit dipeptide is a cleavable linker and can be efficiently cleaved by cathepsin B. Val-Cit dipeptide is used as a linker in antibody-drug conjugates, which can be efficiently cleaved by cathepsin B to release toxins. Since cathepsin B is highly expressed in lysosomes and almost not distributed in the blood, this type of antibody-drug conjugate has high stability in the blood and can selectively release toxins in tumor tissues, thereby improving the safety of antibody-drug conjugates.

[0020] The specific implementation method of coupling hIMB1636 and MMAE through a linker can be: firstly coupling the linker with MMAE to obtain a linker-drug conjugate; then coupling hIMB1636 with the linker-drug conjugate to obtain hIMB1636-MMAE.

[0021] The linker-drug conjugate can specifically be MC-VC-PAB-MMAE.

[0022] In hIMB1636-MMAE, MC-VC-PAB-MMAE is coupled to the hIMB1636 via a carbon-sulfur bond.

[0023] MC-VC-PAB-MMAE is a compound represented by formula (II);

[0024]

[0025] MC-VC-PAB-MMAE is obtained by the reaction of MC-VC-PAB-PNP and MMAE.

[0026] MC-VC-PAB-PNP is MC-VC-PABOH and (PNP) 2 CO obtained by reaction.

[0027] MC-VC-PABOH is obtained by the reaction of VC-PABOH and MC-OSu.

[0028] VC-PABOH is Fmoc-VC-PABOH and Et 2 NH obtained by reaction.

[0029] Fmoc-VC-PABOH is obtained by the reaction of Fmoc-VC and PABOH.

[0030] Fmoc-VC is obtained by the reaction of Fmoc-Val-OSu and L-Cit.

[0031] Fmoc-Val-OSu is obtained by the reaction of Fmoc-L-Val-OH and HOSu.

[0032] The ethylene bond of the maleimide group in MC-VC-PAB-MMAE breaks to produce C, and the interchain disulfide bond of hIMB1636 breaks to produce S, and the two form a carbon-sulfur bond. One IgG antibody molecule consists of two heavy chains and two light chains, with four interchain disulfide bonds. The breakage of four interchain disulfide bonds can produce eight Ss. Therefore, one hIMB1636 molecule can be coupled to a maximum of eight MC-VC-PAB-MMAEs.

[0033] As a specific form, the structural formula of hIMB1636-MMAE3 is shown in formula (III);

[0034]

[0035] n represents DAR; 2≤n≤8.

[0036] Specifically, n=3.5-4.5.

[0037] Specifically, n=3.92.

[0038] Specifically, n=4.

[0039] The number average molecular weight of hIMB1636-MMAE is 150,000-160,000.

[0040] The number average molecular weight of hIMB1636-MMAE is 155,000-156,000.

[0041] The number average molecular weight of hIMB1636-MMAE is 155100-155200.

[0042] The number average molecular weight of hIMB1636-MMAE is 155150-155160.

[0043] The number average molecular weight of hIMB1636-MMAE is 155153-155154.

[0044] The number average molecular weight of hIMB1636-MMAE is 155153.94.

[0045] Second, the present invention provides a method for preparing an antibody-drug conjugate. MMAE is first connected to a VC linker, and the maleimide group of the connection product then undergoes an addition reaction with the interchain disulfide bond of hIMB1626 to obtain hIMB1636-MMAE. TCEP is preferably used as a reducing agent. The preparation method provided by the present invention is obtained after a large number of parameter optimizations, has good repeatability, considerable yield, and the quality of the prepared target product is stable and controllable.

[0046] The method for preparing the antibody-drug conjugate provided by the present invention comprises the following steps: breaking the interchain disulfide bond of hIMB1636, and then connecting it with MC-VC-PAB-MMAE through a carbon-sulfur bond to obtain hIMB1636-MMAE.

[0047] hIMB1636 is any of the above.

[0048] In the method, the molar ratio of hIMB1636 to MC-VC-PAB-MMAE is 1:4-8.

[0049] In the method, the molar ratio of hIMB1636 to MC-VC-PAB-MMAE is 1:6.

[0050] The interchain disulfide bonds of hIMB1636 were cleaved by reduction reaction.

[0051] The reducing agent of the reduction reaction may specifically be TCEP.

[0052] In the method, the molar ratio of hIMB1636 to the reducing agent is 1:2-4.

[0053] In the method, the molar ratio of hIMB1636 to the reducing agent is 1:3.5.

[0054] The method comprises the following steps:

[0055] (1) adding hIMB1636 and a reducing agent into a liquid phase system to carry out a reaction;

[0056] (2) MC-VC-PAB-MMAE was added to the system obtained in step (1) to carry out a reaction.

[0057] The reaction time of step (1) can be 1-2 hours.

[0058] The reaction conditions of step (1) may specifically be: 37° C., 100 rpm (nitrogen protection during the reaction).

[0059] The reaction time of step (2) can be 1-2 hours.

[0060] The reaction conditions of step (2) may specifically be: ice bath, 100 rpm (nitrogen protection during the reaction).

[0061] The method further comprises the steps of:

[0062] (3) After completing step (2), desalting is performed using a desalting column and the buffer system is replaced with PBS buffer.

[0063] The method further comprises the steps of:

[0064] (4) After completing step (3), ultrafiltration centrifugation was performed using a 30 kDa ultrafiltration tube to collect the concentrate.

[0065] The present invention also protects the antibody-drug conjugate prepared by any of the above methods.

[0066] Thirdly, the present invention provides the use of the antibody-drug conjugate in the preparation of anti-tumor drugs.

[0067] The present invention protects the use of any of the above antibody-drug conjugates in the preparation of drugs for tumor targeted therapy.

[0068] The present invention also protects a drug for tumor targeted therapy, which contains any of the above antibody-drug conjugates.

[0069] The tumor is a tumor of the reproductive system, a tumor of the digestive system, a tumor of the head and neck, a tumor of the nervous system, or a tumor of the respiratory system. Exemplarily, the tumor of the reproductive system is endometrial cancer, uterine cancer, cervical cancer, ovarian cancer, or prostate cancer. Exemplarily, the tumor of the digestive system is pancreatic cancer, colon cancer, gastric cancer, esophageal cancer, esophageal squamous cell carcinoma, bile duct cancer, or intestinal cancer. Exemplarily, the tumor of the head and neck is oral squamous cell carcinoma or pharyngeal cancer. Exemplarily, the tumor of the nervous system is a glioma. Exemplarily, the tumor of the respiratory system is lung cancer, small cell lung cancer, or non-small cell lung cancer.

[0070] Exemplarily, the tumor is endometrial cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, pancreatic cancer, colon cancer, gastric cancer, esophageal cancer, esophageal squamous cell carcinoma, bile duct cancer, intestinal cancer, oral squamous cell carcinoma, pharyngeal cancer, brain glioma, lung cancer, small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma, breast cancer, epidermoid carcinoma or pharyngeal squamous cell carcinoma.

[0071] The tumor includes, but is not limited to, pancreatic cancer, breast cancer, epidermoid carcinoma, colorectal cancer, esophageal squamous cell carcinoma, esophageal cancer, oropharyngeal squamous cell carcinoma, or non-small cell lung cancer.

[0072] The tumor is a TROP2-positive solid tumor. A TROP2-positive solid tumor refers to a solid tumor in which the numerical average value of the number of TROP2 molecules on the surface of sampled and separated tumor cells is greater than 50,000.

[0073] The tumor is caused by TROP2-positive tumor cells. TROP2-positive tumor cells refer to tumor cells with a number of TROP2 molecules on their surface greater than 50,000.

[0074] The tumor cells include, but are not limited to, pancreatic cancer cells, breast cancer cells, epidermoid cancer cells, colorectal cancer cells, esophageal squamous cell cancer cells, esophageal cancer cells, oropharyngeal squamous cell cancer cells, or non-small cell lung cancer cells.

[0075] The drug contains a pharmaceutically effective amount of an antibody-drug conjugate.

[0076] The drug further contains a pharmaceutically acceptable adjuvant.

[0077] hIMB1636 has good targeting, affinity and endocytosis ability. MMAE has strong cytotoxicity. hIMB1636-MMAE has the efficacy of targeting TROP2 positive cells and strongly killing cells, thereby selectively killing tumor cells with high expression of TROP2. 50 The value is at the nanomolar or subnanomolar level. Compared with hIMB1636, hIMB1636-MMAE better retains the affinity, endocytic activity and targeting of the antibody, and better retains its biological function. Compared with MMAE, the tumor inhibition effect of hIMB1636-MMAE is significantly improved, which can shrink or eliminate tumors, and no other toxic side effects are observed. In summary, hIMB1636-MMAE has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 This is the synthetic route of Example 1.

[0079] Figure 2 Schematic diagram of the elements of the vector pIZDHL.

[0080] Figure 3 This is the synthetic route of Example 3.

[0081] Figure 4 This is the HIC-HPLC chromatogram of Example 3.

[0082] Figure 5Schematic diagram of hIMB1636-MMAE prepared in Example 3.

[0083] Figure 6 This is a graph showing the results of ELISA detection of the binding activity of hIMB1636-MMAE to human TROP2 antigen in Example 4.

[0084] Figure 7 This is a graph showing the results of flow cytometry detection of the affinity activity of hIMB1636-MMAE with cells in Example 6.

[0085] Figure 8 This is a graph showing the results of laser confocal microscopy to detect the localization of hIMB1636-MMAE in tumor cells in Example 7.

[0086] Fig. 9 This is a graph showing the results of detecting the bystander effect of hIMB1636-MMAE in Example 9.

[0087] Fig.10 This is a graph showing the results of detecting the ADCC effect of hIMB1636-MMAE in Example 10.

[0088] Fig.11 This is a graph showing the results of detecting the in vivo targeting of hIMB1636-MMAE using the in vivo imaging method in Example 11.

[0089] Fig.12 This is a graph showing the therapeutic effect of hIMB1636-MMAE on the T3M4 xenograft tumor model.

[0090] Fig.13 The graph shows the therapeutic effect of hIMB1636-MMAE on BxPc-3, FaDu and A431 xenograft tumor models. DETAILED DESCRIPTION

[0091] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0092] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. Unless otherwise specified, the quantitative tests in the following examples are set up with three repeated experiments, and the results are averaged. Unless otherwise specified, PBS in the examples refers to pH 7.4, 0.01M PBS buffer. Unless otherwise specified, the cell culture conditions in the examples are 37°C, 5% CO 2 The antibody concentrations in the examples are all measured as protein concentrations, and the protein concentrations are measured using the BCA method. The meaning of DAR is: the number of drug molecules coupled to one antibody molecule on average.

[0093] HOSu: N-hydroxysuccinimide, CAS number is 6066-82-6. THF: Tetrahydrofuran, CAS number is 109-99-9. DCC: N,N'-dicyclohexylcarbodiimide, CAS number is 538-75-0. EEDQ: 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, CAS number is 16357-59-8. DMF: N,N-dimethylformamide, CAS number is 68-12-2. DME: ethylene glycol dimethyl ether, CAS number is 110-71-4. Et 2 NH: diethylamine, CAS number is 107-15-3. MC-OSu: 6-(maleimido)hexanoic acid succinimidyl ester, CAS number is 55750-63-5. DIEA: N,N-diisopropylethylamine, CAS number is 7087-68-5. MMAE: Monomethyl auristatin E, CAS number is 474645-27-7. HOBt: 1-hydroxybenzotriazole, CAS number is 2592-95-2. TCEP: tris(2-carbonylethyl)phosphine hydrochloride, CAS number is 51805-45-9. DTPA: diethylenetriaminepentaacetic acid, CAS number is 67-43-6. TFA: trifluoroacetic acid, CAS number is 76-05-1. Fmoc-L-Val-OH (Fmoc-L-valine): CAS number is 68858-20-8. L-Cit (L-citrulline): CAS number is 372-75-8. PABOH: p-aminobenzyl alcohol, CAS number is 623-04-1. (PNP) 2 CO: di(p-nitrobenzene) carbonate, CAS number is 5070-13-3.

[0094] Human TROP2 antigen (Human TROP-2 / TACSTD2 Protein): Kaixia Biotechnology (Shanghai) Co., Ltd., product catalog number is TRP-HM221. CD20 IgG, namely rituximab (anti-CD20 monoclonal antibody), MabThera.

[0095] Example 1. Synthesis of MC-VC-PAB-MMAE

[0096] 1. Synthesis of MC-VC-PAB-PNP

[0097] Synthesis route such as Figure 1 As shown in a.

[0098] 1. Synthesis of Fmoc-Val-OSu

[0099] Dissolve Fmoc-L-Val-OH (10.18 g) and HOSu (3.8 g) in 100 mL THF, add DCC (6.8 g) in an ice bath, then remove the ice bath, and stir at room temperature to react overnight. After the reaction is completed, transfer to an ice bath until the solid is completely precipitated, discard the solid, collect the liquid phase and filter with a Buchner funnel, collect the filtrate and spin dry the solvent to obtain a dry substance, which is the product.

[0100] Product 1 H NMR (400 MHz, DMSO-d 6 )δ(ppm): 8.16(d,J=8.4Hz,1H),7.90(d,J=7.5Hz,2H),7.79–7.71(m,2H),7.43(t,J=7.4Hz,2H),7.38–7.30(m,2H),4 .40–4.32(m,2H),4.29(d,J=7.9Hz,1H),4.25(d,J=7.3Hz,1H),2.82(s,4H),2.21(h,J=6.8Hz,1H),1.06–0.97(m,6H).

[0101] The results showed that the product was C 24 H 24 N 2 O 6 , that is, Fmoc-Val-OSu.

[0102] 2. Synthesis of Fmoc-VC

[0103] The product of step 1 (7 g) was dissolved in 90 mL of DME, i.e., system 1. L-Cit (2.8 g) was dissolved in a 90 mL solution of NaHCO 3(1.3g) in 90mL water, i.e., system 2. System 2 was added to system 1, and then 50mL THF was added, and the reaction was stirred at room temperature overnight until it was clear. After the reaction was completed, 50mL 15g / 100mL citric acid aqueous solution was added to an ice bath, and after the white solid was completely precipitated, it was filtered with a Buchner funnel until the filtrate was clear, and the filter cake was dried in a vacuum drying oven. After drying, the white solid was ground, washed with ether, filtered, and then the solvent was naturally evaporated to obtain a white solid, i.e., the product.

[0104] Product 1 H NMR (400 MHz, DMSO-d 6 )δ(ppm): 12.52(s,1H),8.17(d,J=7.3Hz,1H),7.90(d,J=7.5Hz,2H),7.76(t,J=7.2Hz,2H), 7.41(q,J=8.8,8.1Hz,4H),7.36–7.30(m,2H),5.94(t,J=5.4Hz,1H),5.38(s,2H),4.24(dd,J =11.1,4.8Hz,3H),4.18–4.12(m,1H),3.93(dd,J=9.0,7.2Hz,1H),2.95(q,J=6.3Hz,2H),2. 03–1.93(m,1H),1.76–1.64(m,1H),1.63–1.51(m,1H),1.46–1.34(m,2H),0.90–0.85(m,6H).

[0105] The results showed that the product was C 26 H 32 N 4 O 6 , that is, Fmoc-VC.

[0106] 3. Synthesis of Fmoc-VC-PABOH

[0107] The product of step 2 (6.0 g) and PABOH (7.4 g) were dissolved in a mixed solvent (the mixed solvent consisted of 2 parts by volume of CH 2 Cl 2 and 1 part by volume CH 3 OH), add EEDQ (14.8 g) in dark, and stir at room temperature for 5 h. After the reaction, spin dry the solvent, collect the dry matter, wash with ether, filter, and then evaporate the solvent naturally to obtain a yellow solid, which is the product.

[0108] Product 1 H NMR (400 MHz, DMSO-d 6)δ(ppm): 9.98(s,1H),8.11(d,J=7.6Hz,1H),7.93–7.87(m,2H),7.75(t,J=7.9Hz,2H),7.5 7–7.51(m,2H),7.47–7.38(m,3H),7.36–7.29(m,2H),7.24(d,J=8.5Hz,2H),5.97(t,J=5.8 Hz,1H),5.41(s,2H),5.10(t,J=5.7Hz,1H),4.43(d,J=5.6Hz,3H),4.34–4.20(m,3H),3.94 (dd,J=9.0,7.0Hz,1H),3.08–2.88(m,2H),2.04–1.93(m,1H),0.87(dd,J=11.0,6.7Hz,6H).

[0109] The results showed that the product was C 33 H 39 N 5 O 6 , that is, Fmoc-VC-PABOH.

[0110] 4. Synthesis of VC-PABOH

[0111] The product of step 3 (1.95 g) was dissolved in DMF and Et 2 NH 1.72 mL, stirred at room temperature and reacted overnight. After the reaction was completed, the solvent was dried by spin drying, the dry matter was collected, washed with ether, and then the solvent was naturally evaporated to obtain a slightly yellow solid, which was the product.

[0112] Product 1 H NMR (400 MHz, DMSO-d 6 )δ(ppm): 10.04(s,1H),8.13(d,J=7.2Hz,1H),7.55(d,J=8.5Hz,2H),7.24(d,J= 8.5Hz,2H),5.98(t,J=5.7Hz,1H),5.41(s,2H),5.10(t,J=5.7Hz,1H),4.45(dd, J=15.9,5.5Hz,3H),3.34(s,4H),3.02(dd,J=9.6,5.7Hz,2H),2.95(dt,J=13.1, 6.8Hz, 1H), 2.00–1.89 (m, 1H), 1.50–1.29 (m, 2H), 0.84 (dd, J = 41.3, 6.8Hz, 6H).

[0113] The results showed that the product was C 18 H 29 N 5 O4 , that is VC-PABOH.

[0114] 5. Synthesis of MC-VC-PABOH

[0115] The product of step 4 (0.3 g) was dissolved in DMF, MC-OSu (0.3 g) was added, and the mixture was stirred at room temperature overnight. After the reaction was completed, the solvent was dried, the dry matter was collected, washed with ether, filtered, and then the solvent was naturally evaporated to obtain a yellow solid, which was the product.

[0116] Product 1 H NMR (400 MHz, DMSO-d 6 )δ(ppm): 9.90(s,1H),8.06(d,J=7.6Hz,1H),7.81(d,J=8.7Hz,1H),7.55(d,J=8.5Hz,2H),7 .23 (d, J = 8.5Hz, 2H), 7.00 (d, J = 4.3Hz, 2H), 5.97 (t, J = 5.8Hz, 1H), 5.41 (s, 2H), 5.09 (t, J = 5. 7Hz, 1H), 4.43 (d, J = 5.6Hz, 3H), 4.40–4.33 (m, 1H), 4.23–4.16 (m, 1H), 3.08–2.88 (m, 3H), 2. 25–2.05(m,3H),2.03–1.90(m,1H),1.54–1.42(m,6H),1.28–1.11(m,3H),0.90–0.78(m,6H).

[0117] The results showed that the product was C 28 H 40 N 6 O 7 , that is MC-VC-PABOH.

[0118] 6. Synthesis of MC-VC-PAB-PNP

[0119] The product of step 5 (0.3 g) was dissolved in DMF and (PNP) was added. 2 CO (0.6 g) and DIEA 0.4 mL were stirred at room temperature for 4 hours (nitrogen protection during the reaction). After the reaction was completed, the solvent was spin-dried to obtain a dry substance. The dry substance was dissolved in methanol and then loaded on a silica gel column (silica gel particle size 200-300 mesh, purchased from Qingdao Ocean Chemical Plant Branch), and separated and purified using a medium / low pressure preparative chromatography integrated machine (RF+UV-VIS, purchased from Teledyne Isco).

[0120] The mobile phase consisted of dichloromethane and methanol; the flow rate of the mobile phase was 18 mL / min.

[0121] Elution process: The volume fraction of methanol in the mobile phase increases linearly from 1% to 6%, at which time the target peak begins to appear, and the eluate after passing through the column of the target peak is collected.

[0122] The eluate after passing through the column was taken and the solvent was spin-dried to obtain a slightly yellow solid, which was the product.

[0123] Product 1 H NMR (400 MHz, DMSO-d 6 )δ(ppm): 10.04(s,1H),8.35–8.29(m,3H),8.16–8.10(m,2H),7.60–7.55(m,3H),7 .00(d,J=1.4Hz,3H),5.25(s,3H),4.33(t,J=7.2Hz,1H),4.19(dd,J=8.6,6.4Hz,1 H),2.09(t,J=7.3Hz,3H),1.98(dt,J=13.4,6.7Hz,2H),1.48(d,J=4.9Hz,2H),1.3 3–1.30(m,4H),1.18(d,J=7.0Hz,6H),0.87(d,J=6.7Hz,3H),0.83(d,J=6.8Hz,3H).

[0124] The results showed that the product was C 35 H 43 N 7 O 11 , that is, MC-VC-PAB-PNP.

[0125] 2. Synthesis of MC-VC-PAB-MMAE

[0126] Synthesis route such as Figure 1 As shown in b.

[0127] 1. Dissolve MC-VC-PAB-PNP (100 mg) and MMAE (90 mg) in 4 mL DMF, add HOBt (15 mg) and 44 μL DIEA in turn, and stir at room temperature to react overnight. Then, add 30 mL of water to quench the reaction, and then extract the aqueous phase with ethyl acetate 3 times (each time the amount of ethyl acetate added is 30 mL), and combine and collect the organic phase. Take the organic phase, first extract it twice with 15% citric acid aqueous solution (impurities are present in the aqueous phase), discard the aqueous phase, and retain the organic phase. Take the organic phase, extract it once with saturated sodium chloride aqueous solution (impurities are present in the aqueous phase), discard the aqueous phase, and retain the organic phase. Take the organic phase, spin-dry the solvent to obtain the product.

[0128] 2. The product of step 1 was dissolved in methanol and purified using a preparative liquid phase (chromatographic column: Shim-pack PREP-ODS, 250×20 mm ID, 10 μm; chromatograph: Shimadzu LC-20AT), and the eluate after passing the column of the target peak was collected.

[0129] The mobile phase consists of phase A and phase B. Phase A: 0.08% (volume ratio) TFA aqueous solution. Phase B: 0.08% (volume ratio) TFA acetonitrile solution. Elution process: 0-5min, the volume fraction of phase B in the mobile phase increases linearly from 20% to 60%; 5-30min, the volume fraction of phase B in the mobile phase is 60%. The flow rate of the mobile phase is 5mL / min. The retention time corresponding to the target peak is 24.99min.

[0130] 3. Take the eluate obtained in step 2, spin dry the solvent, and obtain the dry matter, which is the product.

[0131] Product 1 H NMR (400 MHz, DMSO-d 6 )δ(ppm): 9.98(d,J=6.9Hz,1H),8.09(d,J=7.6Hz,1H),7.81(d,J=8.6Hz,1H),7.59(d,J=8.3Hz,2H),7.37–7.23(m,6H),7.18(t,J=7.7Hz, 1H),7.01(s,2H),5.98(t,J=5.6Hz,1H),5.46–5.30(m,3H),5.15–4.93(m,2H),4.55–4.33(m,3H),4.31–4.15(m,2H),4.07–3.89(m,2H),3 .28–3.16(m,7H),3.12(s,2H),3.09–2.90(m,4H),2.87(d,J=14.6Hz,3H),2.42(d,J=15.9Hz,1H),2.32–2.21(m,1H),2.21–2.05(m,4H),2 .06–1.90(m,3H),1.55–1.40(m,7H),1.28–1.22(m,5H),1.22–1.13(m,2H),1.10(t,J=7.0Hz,1H),1.07–0.96(m,7H),0.92–0.72(m,26H).

[0132] The results showed that the product was C 68 H 105 N 11 O 15 , that is, MC-VC-PAB-MMAE.

[0133] Example 2. Preparation of hIMB1636 antibody

[0134] 1. Construction of recombinant plasmid

[0135] Vector pIZDHL is described in the following literature (i.e., “pIZDHL vector” in the literature): Liu XY, Pop LM, Tsai L, et al. Chimeric, divalent and tetravalent anti-CD19 monoclonal antibodies with potent in vitro and in vivo antitumor activity against human B-cell lymphoma and pre-Bacute lymphoblastic leukemia cell lines [J]. Int J Cancer, 2011, 129 (2): 497-506. Schematic diagram of the elements of vector pIZDHL is shown in Figure 2 .

[0136] Using vector pIZDHL as the starting vector, the small fragment between the MluI and BsiWI restriction sites was replaced with the coding gene of the hIMB1636 antibody light chain variable region (VL), and the small fragment between the XhoI and NheI restriction sites was replaced with the coding gene of the hIMB1636 antibody heavy chain variable region (VH), to obtain a recombinant plasmid, which was named recombinant plasmid pIZDHL-VL-VH. The coding gene of the hIMB1636 antibody heavy chain variable region is shown in SEQ ID NO: 2, positions 1-345. The coding gene of the hIMB1636 antibody light chain variable region is shown in SEQ ID NO: 4, positions 1-321.

[0137] The recombinant plasmid pIZDHL-VL-VH contains the DNA molecule shown in SEQ ID NO:2 (encoding the antibody heavy chain shown in SEQ ID NO:1) and the DNA molecule shown in SEQ ID NO:4 (encoding the antibody light chain shown in SEQ ID NO:3). The recombinant plasmid pIZDHL-VL-VH is a plasmid for expressing the hIMB1636 antibody. The hIMB1636 antibody (hIMB1636 for short) has a heavy chain as shown in SEQ ID NO:1 and a light chain as shown in SEQ ID NO:3.

[0138] 2. Preparation and purification of hIMB1636 antibody

[0139] 1. Digest the recombinant plasmid pIZDHL-VL-VH with restriction endonuclease pvuI and collect the linearized large fragment (about 11000 bp).

[0140] 2. The linear large fragment obtained in step 1 was transfected into CHO / dhFr- cells (ATCC number: CRL-9096), and then cultured in IMDM medium containing 0.2 mg / ml bleomycin and 10% Dialyzed FBS for 2-3 weeks.

[0141] 3. After completing step 2, collect the surviving cells (named as recombinant cells CHO-Trop2-IgG), culture them in T75 cell culture flasks using IMDM medium containing 10% Dialyzed FBS until the cell density reaches more than 90%, and then replace the medium with serum-free medium (CD Opti CHO TM Medium) and cultured for 10 days, and the culture supernatant was collected.

[0142] 4. Load the culture supernatant obtained in step 3 onto a Protein G affinity chromatography column, first wash with 10 column volumes of washing solution to remove impurities, then elute the antibody with 9 column volumes of elution solution, and collect the post-column eluate corresponding to the 2nd to 9th column volumes during the elution process.

[0143] Washing solution (pH 7.0): 0.02 M NaH 2 PO 4 -Na 2 HPO 4 Buffer.

[0144] Eluent: pH 2.5, 0.1 M Gly-HCl buffer.

[0145] 5. Take the eluate obtained in step 4 after column passing, use PD-10 desalting column (Sephadex G25 M, Cytiva) for desalting, and replace the buffer system with PBS buffer to obtain a product solution.

[0146] 6. Take the product solution obtained in step 5 and concentrate it using a 30 kDa ultrafiltration tube (4°C, 6600 rpm, 12 min), collect the concentrate, and adjust the antibody concentration to 7 mg / mL with PBS, i.e., the hIMB1636 antibody solution.

[0147] Example 3. Preparation of hIMB1636-MMAE

[0148] 1. Preparation

[0149] Synthesis route such as Figure 3 shown.

[0150] 1. Dissolve the MC-VC-PAB-MMAE prepared in Example 1 in dimethyl sulfoxide to a concentration of 1 mM, which is a MC-VC-PAB-MMAE stock solution. Dissolve TCEP in water to a concentration of 1 mM, which is a TCEP stock solution.

[0151] 2. Take the hIMB1636 antibody solution prepared in Example 2 (antibody concentration is 7 mg / mL), add borax buffer (pH 8, 0.025 mM) containing 1 mM DTPA, and then add TCEP stock solution, and react at 37°C, 100 rpm for 2 h (nitrogen protection during the reaction). The molar amount of TCEP added is 3.5 times the molar amount of hIMB1636 antibody.

[0152] 3. Add the MC-VC-PAB-MMAE stock solution dropwise to the system after completing step 2, and react at 100 rpm for 1 hour under ice bath (nitrogen protection during the reaction). The molar amount of MC-VC-PAB-MMAE added is 6 times the molar amount of hIMB1636 antibody.

[0153] 4. After the reaction is completed, desalting is performed using a PD-10 desalting column (Sephadex G25 M, Cytiva) and the buffer system is replaced with PBS buffer to obtain a product solution.

[0154] 5. Collect the product solution obtained in step 4, perform ultrafiltration centrifugation using a 30 kDa ultrafiltration tube (4°C, 6600 rpm, 12 min), collect the concentrate, filter with a sterile filter membrane and collect the filtrate, which is the hIMB1636-MMAE solution, and store at -80°C.

[0155] 2. Identification

[0156] The hIMB1636-MMAE solution prepared in step 1 was subjected to hydrophobic intercalation liquid chromatography (HIC-HPLC). HIC-HPLC separated the free thiol groups formed by reduction and connected to different numbers of hydrophobic small molecules. The antibody without drug conjugation had the lowest hydrophobicity and was the first to be eluted, while the antibody conjugated with 8 drugs had the highest hydrophobicity and was the last to be eluted.

[0157] Chromatographic system: Agilent 1200.

[0158] Chromatographic column: TSKgel Butul-NPR column (TOSOH), 2.5 μm; 4.6 mm × 36 mm.

[0159] Sample volume: 8 μL.

[0160] Column temperature: 25℃.

[0161] Detection wavelength: 280nm.

[0162] The mobile phase is phase A and / or phase B. Phase A (pH 6.99): contains 1.5 mol / L ammonium sulfate and 25 mmol / L sodium phosphate, and the balance is water. Phase B: consists of 75% (volume ratio) 25 mmol / L sodium phosphate aqueous solution (pH 6.99) and 25% (volume ratio) isopropanol. Mobile phase flow rate: 0.5 mL / min. Elution process: 0-15 min, the volume fraction of phase A in the mobile phase decreases linearly from 100% to 0%, and the corresponding volume fraction of phase B in the mobile phase increases linearly from 0% to 100%; 15.01-17.00 min, the volume fraction of phase A in the mobile phase is 100%.

[0163] Chromatogram see Figure 4 D0 represents an antibody not conjugated with MMAE, D2 represents an antibody conjugated with 2 MMAEs, D4 represents an antibody conjugated with 4 MMAEs, D6 represents an antibody conjugated with 6 MMAEs, and D8 represents an antibody conjugated with 8 MMAEs.

[0164] The peak areas were calculated based on the chromatograms and the results are shown in Table 1.

[0165] Table 1

[0166] Drug Load Retention time (min) Peak area percentage (%) Weighted peak area 0 7.662 4.351 0 1 7.901 1.791 1.791 2 8.528 24.444 48.888 3 9.208 2.418 7.254 4 9.895 39.843 159.372 6 11.19 21.275 127.65 8 12.189 5.877 47.016 total / / 391.971

[0167] The DAR of hIMB1636-MMAE prepared in step 1 was calculated according to Table 1. DAR = 391.971 / 100≈3.92≈4. Number average molecular weight: 155153.94.

[0168] Schematic diagram of hIMB1636-MMAE is shown in Figure 5 The Y type represents mAb, i.e. hIMB1636; n≈4.

[0169] Example 4, affinity determination of hIMB1636-MMAE

[0170] 1. ELISA to detect the binding activity of hIMB1636-MMAE and human TROP2 antigen

[0171] 1. Dilute human TROP2 antigen to 10 μg / mL with PBS, plate in a 96-well plate (100 μL / well), and coat overnight at 4°C.

[0172] 2. Take the 96-well plate prepared in step 1, wash it with PBS for 3 times, then add 2% BSA blocking solution (200 μL / well), and incubate at 37° C. for 2 h.

[0173] 3. Take the 96-well plate that has completed step 2, wash it with PBS for 3 times, then add the test solution (100 μL / well), and incubate at 37°C for 1 hour. The test solution is hIMB1636 dilution or hIMB1636-MMAE dilution. The hIMB1636 dilution is obtained by diluting the hIMB1636 solution prepared in Example 2 with PBS, and the antibody concentrations are 3 μg / mL, 1 μg / mL, 0.3 μg / mL, 0.1 μg / mL, 0.03 μg / mL, 0.01 μg / mL, 0.003 μg / mL or 0.001 μg / mL. The hIMB1636-MMAE dilutions were prepared by diluting the hIMB1636-MMAE solution prepared in Example 3 with PBS, and the antibody concentrations were 3 μg / mL, 1 μg / mL, 0.3 μg / mL, 0.1 μg / mL, 0.03 μg / mL, 0.01 μg / mL, 0.003 μg / mL or 0.001 μg / mL. Three parallel wells were set for each test solution.

[0174] 4. Take the 96-well plate that has completed step 3, wash it with PBS three times, add the secondary antibody working solution (100 μL / well), and incubate at 37°C for 1 hour. The secondary antibody working solution is obtained by diluting the secondary antibody (alkaline phosphatase-labeled goat anti-human IgG, Fab specific) to 1000 times the volume.

[0175] 5. Take the 96-well plate that has completed step 4, wash it with PBS three times, add TMB colorimetric solution (100 μL / well), incubate at room temperature for 30 min, and then add 2M H 2 The reaction was terminated with SO4 solution, and the absorbance at 450 nm was detected using a SpectraMax i3x multi-function microplate reader.

[0176] The binding curves of hIMB1636 and hIMB1636-MMAE with human TROP2 antigen are shown in Figure 6 The binding of hIMB1636-MMAE to human TROP2 antigen was concentration-dependent, and the coupling with MMAE had little effect on the affinity activity of the hIMB1636 antibody itself.

[0177] 2. Determination of the dissociation constant of hIMB1636-MMAE by SPR

[0178] Freshly prepared PBST solution was filtered through a 0.22 μm filter membrane, and the filtrate was ultrasonically degassed for 10 min.

[0179] Prepare a new uncoupled SAM chip, load it into the instrument, replace the freshly prepared PBST solution, set up two channels, with the reference channel upstream of the channel to be tested, start the Prime program, wait for 10 minutes, and calculate the coupling amount: K max ×RL ×S m ; R L =50; S m =1. Weigh 40mg EDC and 10mg NHS into a 1.5mL EP tube and dilute with 1mL water respectively. Inject as soon as possible after dilution, prepare it now, the purpose is to activate the two channels where the fixed sample is located. Dilute the human TROP2 antigen to 10μg / mL with a 10mM NaAc buffer solution at pH5.5, and the pre-enrichment binding value reaches 500RU. Start coupling, wait for 10min for the coupling to be uninterrupted, indicating that the calculated coupling amount has been reached, and wait for about 30min for the coupling to end. Inject 1M ethanolamine at pH8.5 to block the SAM chip for 8min. Use PBST solution to adjust the concentration of hIMB1636-MMAE to 500nM, and then start the surface binding test and regeneration. The regeneration reagent is a 10mM Gly-HCl solution at pH1.5, the regeneration time is 15s, and the neutralization time is 30s. The concentration of hIMB1636-MMAE was diluted to 15.85nM, 7.93nM, 3.96nM, 1.98nM, 0.99nM or 0.50nM with PBST solution, with a total of six concentration gradients; the concentration of hIMB1636 was diluted to 25nM, 12.5nM, 6.25nM, 3.13nM, 1.56nM or 0.78nM, with a total of six concentration gradients; the sample volume was 200μL, and the flow rate was 5μL / min. 200μL of 10mM Gly-HCl regeneration solution at pH1.5 was added to the sample holder for the corresponding sample wells, and it was ensured that the PBST solution was passed through at least once before injection. The program was started to allow the analysis sample to flow from low concentration to high concentration through the chip surface of the coupled antigen. The dissociation constant (K) was calculated using the SPR system. D ).

[0180] The results are shown in Table 2.

[0181] Table 2 K of hIMB1636-MMAE and hIMB1636 detected by SPR D value

[0182] ka(1 / (M*s)) kd(1 / s) <![CDATA[K D (M)]]> hIMB1636-MMAE <![CDATA[5.77×10 5 ]]> <![CDATA[3.96×10 -4 ]]> <![CDATA[6.87×10 -10 ]]> hIMB1636 <![CDATA[8.88×10 5 ]]> <![CDATA[5.88×10 -4 ]]> <![CDATA[6.6310 -10 ]]>

[0183] Example 5: Detection of the density of TROP2 antigen on the surface of tumor cells

[0184] Leveraging Quantum TM Simply (QSC) Antibody Titration Kit detects the density of TROP2 antigen on the surface of various tumor cells. Quantum TM Simply (QSC) Antibody Titration Kit: Bangs Laboratories, product catalog number 816.

[0185] 1. Take the hIMB1636 solution prepared in Example 2, label it with FITC, and then adjust the antibody concentration to 10 μg / mL with PBS to obtain a FITC-hIMB1636 solution.

[0186] 2. Set up 8 EP tubes, numbered 1-8, add 100μL FITC-hIMB1636 solution to tube 1, and add 50μL PBS to the other 7 tubes. Transfer 50μL of solution from tube 1 to tube 2, mix gently, then transfer 50μL of solution from tube 2 to tube 3, mix gently, and transfer / mix mode to tube 8 in sequence, and finally aspirate the excess 50μL solution from tube 8.

[0187] 3. Gently shake the QSC microspheres to resuspend the spheres. Add 50 μL of QSC microspheres to each tube that completed step 2, mix gently to make the total volume reach 100 μL, and incubate at room temperature for 30 min in the dark.

[0188] 4. After completing step 3, wash twice (each wash: add 500 μL PBS to each tube, centrifuge at 2500 rpm for 5 min, and discard the supernatant), then add 500 μL PBS to each tube.

[0189] 5. After completing step 4, perform standard flow cytometric analysis to determine the mean fluorescence intensity of each dilution of the liquid phase in each tube and enter it into the QuickCal spreadsheet.

[0190] 6. On the same day, using the same instrument and with the same instrument settings, test the cell staining samples and record the mean fluorescence intensity of each cell. According to the known template provided by the official website, enter the mean fluorescence intensity value to calculate the antibody binding capacity (ABC) value of each cell and obtain the cell surface antigen density value.

[0191] Cell staining samples: Tumor cells to be tested were collected, resuspended in FITC-hIMB1636 solution, and incubated for 0.5 h.

[0192] The test results of 16 types of tumor cells tested are shown in Table 3. The surface antigen density of different tumor cells varies greatly, ranging from 1717 to 483671. Even tumor cells of the same type may have large differences in cell surface antigen density: for example, for pancreatic cancer cells, the antigen densities of T3M4 cells, BxPc-3 cells, and AsPC-1 cells are 268963, 483671, and 7348, respectively. In the present invention, when qualitatively judging, the value of the average number of TROP2 molecules on the surface of each cell is greater than 50,000 and is judged as positive, and the value of the average number of TROP2 molecules on the surface of each cell is less than 50,000 and is judged as negative.

[0193] Table 3 Density of TROP2 antigen on cell surface detected by QSC antibody titration kit

[0194]

[0195]

[0196] Example 6: Flow cytometry detection of affinity activity of hIMB1636-MMAE and cells

[0197] The test cells were BxPc-3 cells, T3M4 cells or AsPC-1 cells.

[0198] 1. Resuspend the test cells in PBS containing 2% FBS to a cell concentration of 1×10 6 / mL, which is the cell suspension.

[0199] 2. Take a sterile 1.5mL EP tube and add 100μL of the cell suspension prepared in step 1 and 100μL of hIMB1636-MMAE diluent to each tube. Mix well and incubate at 4℃ for 1h. Then centrifuge at 4℃ and 2000rpm for 5min and discard the supernatant.

[0200] The hIMB1636-MMAE diluent was prepared by diluting the hIMB1636-MMAE solution prepared in Example 3 with PBS containing 2% FBS, and the antibody concentrations were 10 μg / mL, 1 μg / mL or 0.1 μg / mL. A 0 concentration control was set, i.e., an equal volume of PBS containing 2% FBS was used instead of the hIMB1636-MMAE diluent.

[0201] 3. After completing step 2, wash twice (each wash: add 500 μL PBS to each tube to resuspend the cells, then centrifuge at 4°C, 2000 rpm for 5 min, and discard the supernatant).

[0202] 4. After completing step 3, add 200 μL of secondary antibody working solution to each tube, incubate at 4°C in the dark for 1 hour, then centrifuge at 4°C, 2000 rpm for 5 minutes, and discard the supernatant. Secondary antibody working solution: Dilute the secondary antibody (FITC-labeled goat anti-human IgG) to 100 times the volume with PBS containing 2% FBS.

[0203] 5. After completing step 4, wash twice (each wash: add 500 μL PBS to each tube to resuspend the cells, then centrifuge at 4°C, 2000 rpm for 5 min, and discard the supernatant).

[0204] 6. After completing step 5, add 100 μL of PBS containing 2% FBS to each tube to resuspend the cells, filter with a 300-mesh filter, and use a flow cytometer to detect the fluorescence intensity.

[0205] Results Figure 7 hIMB1636-MMAE has high affinity and concentration-dependent binding to tumor cells with high TROP2 expression (BxPc-3 cells and T3M4 cells), but hardly binds to tumor cells with low TROP2 expression (AsPC-1 cells).

[0206] Example 7: Confocal laser scanning to detect the localization of hIMB1636-MMAE in tumor cells

[0207] The test cells were BxPc-3 cells, T3M4 cells or AsPC-1 cells.

[0208] 1. Place the sterilized slide in a 6-well plate and add 2 ml of cells with a concentration of 1×10 4 / mL of the test cell suspension was inoculated on a glass slide and cultured for 24h.

[0209] 2. After completing step 1, hIMB1636-MMAE prepared in Example 3 was added to a 6-well plate and the antibody concentration in the system was adjusted to 10 μg / mL, and incubated for 1 h (the incubation temperature was set to 4° C. or 37° C., respectively).

[0210] 3. After completing step 2, discard the supernatant, wash twice with PBS, then add 4% paraformaldehyde solution to fix for 10 minutes, then wash twice with PBS, then add 0.5% Triton X-100 solution to permeabilize at room temperature for 10 minutes, then wash twice with PBS, and then add 5% BSA solution to block at room temperature for 2 hours.

[0211] 4. After completing step 3, add rabbit anti-lysosomal associated membrane protein 1 (LAMP-1) antibody, incubate overnight at 4°C, then wash twice with PBS, and then add Alexa Fluor 555-labeled donkey anti-rabbit IgG (H+L) antibody (for labeling LAMP-1) and Alexa Fluor 488-labeled goat anti-human IgG (H+L) antibody (for labeling hIMB1636-MMAE), and incubate at room temperature for 1 hour in the dark.

[0212] 5. After completing step 4, wash twice with PBS, add anti-fluorescence quencher containing DAPI on the slide, cover with a coverslip, dry it, and capture the image using a confocal microscope.

[0213] Results Figure 8 hIMB1636-MMAE can enter cells through TROP2 antigen endocytosis and be transported to lysosomes.

[0214] Example 8: CCK-8 detection of the killing activity of hIMB1636-MMAE on tumor cells

[0215] The tumor cells tested are shown in Table 3. The following tumor cells were cultured with DMEM complete medium: MDA-MB-231 cells, MDA-MB-468 cells, A431 cells, Eca109 cells, SW480 cells, HT29 cells, HCT116 cells, T3M4 cells and BxPc-3 cells. The following tumor cells were cultured with RPMI-1640 complete medium: MCF-7 cells, A549, AsPC-1 cells, KYSE150 cells, HCC827 cells and H460 cells. The following tumor cells were cultured with MEM complete medium: FaDu cells.

[0216] The treatment method for the drug-added group is as follows:

[0217] 1. Resuspend the tumor cells in culture medium to obtain a cell concentration of 2×10 4 -8×10 4 / mL single cell suspension.

[0218] 2. Take a 96-well plate, add 100 μL of the single-cell suspension prepared in step 1 to each well, culture for 24 hours, and discard the supernatant.

[0219] 3. Take the 96-well plate that has completed step 2, add 100 μL of test solution to each well, and culture for 48 hours. The test solution is obtained by diluting the hIMB1636-MMAE solution prepared in Example 3 with culture medium, and different dilution concentrations (in terms of the molar concentration of hIMB1636) are set. Alternatively, the test solution is obtained by diluting MMAE with culture medium, and different dilution concentrations (in terms of the molar concentration of MMAE) are set. Three parallel replicates are set for each test solution.

[0220] 4. Take the 96-well plate prepared in step 3, add 10 μL of CCK-8 reagent to each well, shake to mix, and incubate for 30 min.

[0221] 5. Take the 96-well plate that has completed step 4 and use an ELISA reader to detect the OD value at 450nm.

[0222] Blank group: replace the single cell suspension in step 2 with an equal volume of culture medium and replace the test solution in step 3 with an equal volume of culture medium, and the rest is the same as the drug-added group. Set up 3 parallel replicates.

[0223] Control group: replace the test solution in step 3 with an equal volume of culture medium, and the rest is the same as the drug-added group. Set up 3 parallel replicate wells.

[0224]

[0225] The results are shown in Table 4. Free MMAE molecules can kill all types of tumor cells in a non-selective manner (IC 50 In combination with Table 3, the following conclusions can be drawn: hIMB1636-MMAE has a strong selective killing activity against cells with high expression of TROP2 (IC 50 at nanomolar or subnanomolar levels).

[0226] Table 4 IC of hIMB1636-MMAE against different tumor cells detected by CCK-8 50 Value (nM)

[0227]

[0228] Example 9: RTCA detection of the bystander effect of hIMB1636-MMAE

[0229] According to the results of Example 5, H460 cells are tumor cells with low expression of TROP2, and T3M4 cells are tumor cells with high expression of TROP2.

[0230] Control group: E-Plate 16 was taken, H460 cells were added, and cultured with RPMI-1640 complete medium for 12 hours, and then the cell growth curve was detected in real time using the xCELLigene system.

[0231] H460 cell group: E-Plate 16 was taken, H460 cells were added, and RPMI-1640 complete medium was used to culture for 12 hours, and then hIMB1636-MMAE prepared in Example 3 was added to make the concentration of the drug in the system 75nM, 50nM or 25nM, respectively, and the growth curve of the cells was detected in real time using the xCELLigene system.

[0232] H460 cell / T3M4 cell group: Take E-Plate 16, add H460 cells and T3M4 cells (the number of both cells is equal), culture for 12 hours with RPMI-1640 complete medium, then add hIMB1636-MMAE prepared in Example 3, so that the concentration of the drug in the system is 75nM, 50nM or 25nM, and use xCELLigene system to detect the growth curve of the cells in real time.

[0233] The initial total cell numbers in the above groups were equal. The results of real-time detection of total cell numbers are shown in Fig. 9 At concentrations of 25nM, 50nM, and 75nM, hIMB1636-MMAE had weak killing activity against H460 cells cultured alone, and the cell index curve still showed an upward trend; while it had strong killing activity against co-cultured H460 cells, the cell index curve showed a downward trend, and the killing activity was dose-dependent with the drug concentration. This indicates that hIMB1636-MMAE has a bystander effect.

[0234] Example 10: Analysis of ADCC effect of hIMB1636-MMAE

[0235] The target cells are: BxPc-3 cells (using DMEM culture medium), T3M4 cells (using DMEM culture medium) or AsPC-1 cells (using 1640 culture medium).

[0236] Effector cells: Jurkat-Lucia TM NFAT-CD16 cells.

[0237] 1. Collect the digested target cells and resuspend them in culture medium to make the cell concentration of target cells 1.1×10 6 / mL is the target cell suspension. Collect the digested effector cells and resuspend them in RPMI-1640 medium to make the cell concentration of the effector cells 2.2×10 6 / mL, which is the effector cell suspension.

[0238] 2. Take a 96-well plate, add the test solution (20 μL / well), and then add 90 μL of target cell suspension (1×10 5 After culturing for 1 h, 90 μL of effector cell suspension (2×10 5 / well) and cultured for 6 h.

[0239] The test solution is hIMB1636 dilution or hIMB1636-MMAE dilution. hIMB1636 dilution: Take the hIMB1636 solution prepared in Example 2 and adjust the antibody concentration to 0.16 μg / mL, 0.04 μg / mL, 0.01 μg / mL, 0.0025 μg / mL, 0.000625 μg / mL, 0.000156 μg / mL or 0.0000391 μg / mL with PBS. hIMB1636-MMAE dilution: Take the hIMB1636-MMAE solution prepared in Example 3 and adjust the antibody concentration to 0.16 μg / mL, 0.04 μg / mL, 0.01 μg / mL, 0.0025 μg / mL, 0.000625 μg / mL, 0.000156 μg / mL or 0.0000391 μg / mL with PBS.

[0240] Set up negative control wells with an equal volume of PBS instead of the test solution.

[0241] Three replicate wells were set for each test solution, and three replicate wells were set for the negative control well.

[0242] 3. After completing step 2, transfer 20 μL of supernatant to each well of a flat-bottomed opaque 96-well plate, and then add 50 μL of QUANTI-Luc TM Detection solution, and immediately read the relative luminescence units (RLU) of each well on the SpectraMax i3x Multi-Mode Microplate Reader.

[0243] Results Fig.10 . The fluorescence signal of drug-treated TROP2-high-expressing T3M4 cells and BxPc-3 cells gradually increased with the increase of drug concentration, and the fluorescence signal intensity of the conjugate and naked antibody was basically the same, indicating that both hIMB1636-MMAE and naked antibody hIMB1636 can induce ADCC effect, and the activities of the two are basically the same. The fluorescence signal of drug-treated antigen-negative AsPC-1 cells was very weak and did not change with concentration, and no ADCC effect was shown. The results show that ADC exerts cytotoxic effects in vivo by inducing ADCC effect by binding to antigen-positive cells, and the coupling reaction basically does not affect the ADCC activity of the antibody.

[0244] Example 11: In vivo targeting of hIMB1636-MMAE detected by in vivo imaging

[0245] 1. Label the test substance with Dylight 680 reagent.

[0246] 1. Take the test solution, dilute it with PBS to an antibody concentration of 2 mg / mL, and then divide it into several portions (500 μL each). Add 40 μL of 0.67 M borate buffer to each portion, mix well, and then add it to the reaction tube containing Dylight 680 reagent, mix it with a gun, centrifuge it for 1 minute, and react at room temperature in the dark for 1 hour.

[0247] 2. Place the column in a collection tube, then mix the resin filler and add it to the column, centrifuge at 1000 rpm for 1 min, and discard the collection tube.

[0248] 3. Replace the new collection tube, add the solution from step 1 to the column, mix it gently and quickly with the resin, centrifuge at 1000 rpm for 1 min, and the blue solution in the collection tube is the labeled test solution.

[0249] When the test solution is the hIMB1636 solution prepared in Example 2, the labeled hIMB1636 solution is prepared by the above steps.

[0250] When the test solution is the hIMB1636-MMAE solution prepared in Example 3, the labeled hIMB1636-MMAE solution is prepared by the above steps.

[0251] When the test solution is a CD20IgG-MMAE solution, the labeled CD20IgG-MMAE solution is prepared by the above steps. CD20IgG-MMAE is obtained by coupling CD20 IgG and MMAE through a linker, and the preparation method is basically the same as Example 3 (the only difference is that hIMB1636 is replaced by CD20 IgG).

[0252] When using, adjust the concentration with PBS.

[0253] 2. Targeted detection

[0254] 1. Resuspend BxPc-3 cells in PBS to obtain a cell concentration of 2×10 7 / ml cell suspension.

[0255] 2. Inoculate the cell suspension prepared in step 1 into the right axilla of BALB / c nude mice. Each mouse is inoculated with 100 μL of cell suspension (i.e., 2×10 6 The tumor growth was observed and the tumor volume reached 300-400mm after about 10 days. 3 , which are model mice.

[0256] 3. The model mice obtained in step 2 were divided into three groups (3 mice in each group). The first group was injected with labeled hIMB1636 solution through the tail vein, the second group was injected with labeled hIMB1636-MMAE solution through the tail vein, and the third group was injected with labeled CD20IgG-MMAE solution through the tail vein. The injection dose of the three groups was 20 mg antibody / kg body weight.

[0257] 4. After completing step 3, use XENGOEN (Caliper, USA) small animal live imaging device to take photos and observe.

[0258] Results Fig.11 The results showed that hIMB1636-MMAE exhibited good tumor targeting in mice.

[0259] Example 12: Therapeutic effect of hIMB1636-MMAE on pancreatic cancer T3M4 xenograft tumor model

[0260] Test tumor cells: T3M4 cells.

[0261] 1. Resuspend the tumor cells in PBS to a cell concentration of (1-2.5)×10 7 The cell suspension is 100 cells / mL.

[0262] 2. On the first day of the experiment, 6-8 week old BALB / c nude mice were subcutaneously injected with the cell suspension obtained in step 1 under the right armpit (100 μL per mouse) and fed normally for 8 days. At this time, the tumor volume reached about 100-200 mm 3 , which is the transplanted tumor model.

[0263] 3. The transplanted tumor models were randomly divided into 6 groups (6-8 mice in each group) and the drugs were administered as follows:

[0264] hIMB1636-MMAE low-dose group: hIMB1636-MMAE was administered once on the 8th, 12th, 16th, 20th and 24th days of the experiment, with a single administration volume of 100 μL and a single administration dose of 3 mg (in terms of antibody amount) / kg body weight.

[0265] hIMB1636-MMAE medium-dose group: hIMB1636-MMAE was administered once on the 8th, 12th, 16th, 20th and 24th days of the experiment, with a single administration volume of 100 μL and a single administration dose of 6 mg (in terms of antibody amount) / kg body weight.

[0266] hIMB1636-MMAE high-dose group: hIMB1636-MMAE was administered once on the 8th, 12th, 16th, 20th and 24th days of the experiment, with a single administration volume of 100 μL and a single administration dose of 12 mg (in terms of antibody amount) / kg body weight.

[0267] hIMB1636 group: hIMB1636 was administered once on the 8th, 12th, 16th, 20th and 24th days of the experiment, with a single administration volume of 100 μL and a single administration dose of 12 mg (in terms of antibody amount) / kg body weight.

[0268] CD20-MMAE group: CD20IgG-MMAE was administered once on the 8th, 12th, 16th, 20th and 24th days of the experiment, with a single administration volume of 100 μL and a single administration dose of 12 mg (in terms of antibody amount) / kg body weight.

[0269] Control group: PBS was administered once on the 8th, 12th, 16th, 20th and 24th day of the experiment, with a single administration volume of 100 μL.

[0270] The administration method was tail vein injection. hIMB1636-MMAE was provided by the hIMB1636-MMAE solution prepared in Example 3, and the concentration was adjusted using PBS. CD20IgG-MMAE was prepared in Example 11, and the concentration was adjusted using PBS.

[0271] Starting from the 9th day of the experiment, the tumor volume (0.5×length×width) was measured every 3 days. 2 ) and animal body weight.

[0272] Results Fig.12 . The results showed that hIMB1636-MMAE was able to inhibit tumor growth in a dose-dependent manner. At a high dose (12 mg / kg), hIMB1636-MMAE was able to partially eliminate tumors (5 out of 8 mice had tumors disappear), and no tumor recurrence was observed after drug withdrawal. It is worth noting that even at lower doses, hIMB1636-MMAE can still significantly inhibit tumor growth and significantly reduce tumor volume.

[0273] Example 13. Therapeutic effect of hIMB1636-MMAE on pancreatic cancer BxPc-3, pharyngeal cancer FaDu and skin squamous cell carcinoma A431 xenograft tumor models

[0274] The administration method was tail vein injection. hIMB1636-MMAE was provided by the hIMB1636-MMAE solution prepared in Example 3, and the concentration was adjusted using PBS.

[0275] 1. The therapeutic effect of hIMB1636-MMAE on BxPc-3 xenograft tumor model

[0276] 1. Resuspend BxPc-3 cells in PBS to a cell concentration of (1-2.5)×10 7 The cell suspension is 100 cells / mL.

[0277] 2. On the first day of the experiment, 6-8 week old BALB / c nude mice were subcutaneously injected with the cell suspension obtained in step 1 in the right axilla (100 μL per mouse) and fed normally until the tumor volume reached about 100-200 mm 3 , which is the transplanted tumor model.

[0278] 3. The transplanted tumor models were randomly divided into 2 groups (6-8 mice in each group) and administrated as follows:

[0279] hIMB1636-MMAE group: hIMB1636-MMAE was administered once on the 7th, 11th, 15th, 19th and 23rd days of the experiment, with a single administration volume of 100 μL and a single administration dose of 10 mg (in terms of antibody amount) / kg body weight.

[0280] Control group: PBS was administered once on the 7th, 11th, 15th, 19th and 23rd day of the experiment, with a single administration volume of 100 μL.

[0281] Starting from the 9th day of the experiment, the tumor volume (0.5×length×width) was measured every 4 days. 2 ) and animal body weight.

[0282] 2. The therapeutic effect of hIMB1636-MMAE on FaDu xenograft tumor model

[0283] 1. Resuspend FaDu cells in PBS to a cell concentration of (1-2.5)×10 7 The cell suspension is 100 cells / mL.

[0284] 2. On the first day of the experiment, 6-8 week old BALB / c nude mice were subcutaneously injected with the cell suspension obtained in step 1 in the right axilla (100 μL per mouse) and fed normally until the tumor volume reached about 100-200 mm 3 , which is the transplanted tumor model.

[0285] 3. The transplanted tumor models were randomly divided into 2 groups (6-8 mice in each group) and administrated as follows:

[0286] hIMB1636-MMAE group: hIMB1636-MMAE was administered once on the 6th, 10th, 14th, 18th and 22nd day of the experiment, with a single administration volume of 100 μL and a single administration dose of 10 mg (in terms of antibody amount) / kg body weight.

[0287] Control group: PBS was administered once on the 6th, 10th, 14th, 18th and 22nd day of the experiment, with a single administration volume of 100 μL.

[0288] Starting from the 9th day of the experiment, the tumor volume (0.5×length×width) was measured every 4 days. 2 ) and animal body weight.

[0289] III. The therapeutic effect of hIMB1636-MMAE on A431 xenograft tumor model

[0290] 1. Resuspend A431 cells in PBS to a cell concentration of (1-2.5)×10 7 The cell suspension is 100 cells / mL.

[0291] 2. On the first day of the experiment, 6-8 week old BALB / c nude mice were subcutaneously injected with the cell suspension obtained in step 1 in the right axilla (100 μL per mouse) and fed normally until the tumor volume reached about 100-200 mm 3 , which is the transplanted tumor model.

[0292] 3. The transplanted tumor models were randomly divided into 2 groups (6-8 mice in each group) and administrated as follows:

[0293] hIMB1636-MMAE group: hIMB1636-MMAE was administered once on the 10th, 14th, 18th, 22nd and 26th days of the experiment, with a single administration volume of 100 μL and a single administration dose of 10 mg (in terms of antibody amount) / kg body weight.

[0294] Control group: PBS was administered once on the 10th, 14th, 18th, 22nd and 26th days of the experiment, with a single administration volume of 100 μL.

[0295] Starting from the 13th day of the experiment, the tumor volume (0.5×length×width) was measured every 4 days. 2 ) and animal body weight.

[0296] Results Fig.13 The results showed that the tumors in the BxPc-3 and FaDu xenograft models were completely eliminated at 22 days and 34 days, respectively. hIMB1636-MMAE significantly inhibited the tumor growth of the A431 xenograft model and reduced the tumor volume.

[0297] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. An antibody-drug conjugate, which is a compound obtained by conjugating hIMB1636 and MMAE; hIMB1636 is an IgG antibody that specifically binds to human TROP2; MMAE is shown in formula (I); 2. The antibody-drug conjugate according to claim 1, characterized in that: In the antibody-drug conjugate, MC-VC-PAB-MMAE is coupled to the hIMB1636 via a carbon-sulfur bond; MC-VC-PAB-MMAE is a compound represented by formula (II); 3. The antibody-drug conjugate according to claim 1 or 2, characterized in that: The structural formula of the antibody-drug conjugate is shown in formula (III); Represents hIMB1636; n represents DAR; 2≤n≤8.

4. The antibody-drug conjugate according to any one of claims 1 to 3, characterized in that: The three CDRs of the heavy chain variable region of hIMB1636 are shown in SEQ ID NO:1 at positions 31-35, 50-66, and 99-104, respectively, and the three CDRs of the light chain variable region are shown in SEQ ID NO:3 at positions 24-34, 50-56, and 89-97, respectively.

5. The antibody-drug conjugate according to any one of claims 1 to 3, characterized in that: The heavy chain variable region of hIMB1636 is shown at positions 1-115 in SEQ ID NO:1, and the light chain variable region is shown at positions 1-107 in SEQ ID NO:

3.

6. The antibody-drug conjugate according to any one of claims 1 to 3, characterized in that: The hIMB1636 shown is shown in SEQ ID NO:1, and the light chain is shown in SEQ ID NO:

3.

7. A method for preparing an antibody-drug conjugate, comprising the following steps: breaking the interchain disulfide bond of hIMB1636, and then connecting it with MC-VC-PAB-MMAE through a carbon-sulfur bond to obtain an antibody-drug conjugate.

8. The antibody-drug conjugate prepared by the method of claim 7.

9. Use of the antibody-drug conjugate according to claim 1 or 2 or 3 or 4 or 5 or 6 or 8 in the preparation of a drug for targeted tumor therapy.

10. A drug for targeted tumor therapy, characterized in that: It contains the antibody-drug conjugate according to claim 1 or 2 or 3 or 4 or 5 or 6 or 8.

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