Antibody drug conjugate and application thereof

Abstract

The invention aims to provide an antibody drug conjugate and application of the antibody drug conjugate in preparation of drugs for treating cancers.

Description

Technical Field

[0001] The present invention relates to the field of chemical medicine, and in particular to an antibody-drug conjugate and uses thereof. Background Art

[0002] As a new type of targeted therapeutic drug, antibody-drug conjugate (ADC) utilizes the specificity of antibodies binding to surface antigens of normal cells and tumor cells and the high efficiency of cytotoxins. It can effectively deliver cytotoxins to the diseased site to exert its efficacy, while reducing the defect of excessive side effects of cytotoxins in traditional drug forms. It is gradually playing an increasingly important role in the treatment of tumors and other fields.

[0003] ADC drugs are composed of three parts: monoclonal antibodies, linkers, and cytotoxic drugs (cytotoxins). Cytotoxic substances are essential for ADC drugs to work. Camptothecin drugs are widely used in ADCs. Currently, many clinical and marketed products use camptothecin drugs (such as exotecan or its analogs) as toxin molecules. There is still a wide demand for the development of ADC drugs of other toxins. Summary of the invention

[0004] One aspect of the present invention is to provide an antibody drug conjugate having the formula Ab-[H-L1-L2-L3-D] x The structure shown, wherein:

[0005] Ab is an antibody or its antigen-binding fragment;

[0006] H is a linker site with the antibody or antigen-binding fragment thereof;

[0007] L1 is a connecting unit connecting the joint parts H and L2;

[0008] L2 is the linker between L1 and L3;

[0009] L3 is the spacer between L2 and D;

[0010] D is a toxin having the following structural formula:

[0011]

[0012] X Select from 1-10.

[0013] In some embodiments, the H is linked to Ab via a sulfhydryl, azide, or amide group.

[0014] In some embodiments, H is selected from:

[0015]

[0016] In some preferred embodiments, H is

[0017] In some embodiments, the L1 comprises -L1'-C(O)-, wherein L1' is selected from optionally substituted alkylene, optionally substituted polyethylene glycol, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted alicyclic group, optionally substituted alicyclic heterocyclic group, optionally substituted arylene, optionally substituted heteroarylene, or a combination thereof.

[0018] In some embodiments, the L1' is selected from -(CH2)m-, -(PEG)n- or -(CH2) m -(PEG) n -(CH2) z -, where m, n, z are integers from 0 to 10.

[0019] In some preferred embodiments, m, n, and z are integers of 0-8, for example, integers of 0, 1, 2, 3, 4, 5, 6, 7, and 8.

[0020] In other preferred embodiments, the L1' is selected from -(CH2)5-, -(PEG)4- or -(CH2)2-(PEG)4-.

[0021] In some embodiments, L2 is a cleavable linker, a non-cleavable linker, a hydrophilic linker, a hydrophobic linker, a charged linker, or an uncharged linker.

[0022] In some specific embodiments, the L2 is a non-cleavable linker. In some preferred embodiments, the L2 is -(PEG) k - or -NH-(CH2) j -(PEG) k -, wherein j, k are integers of 0-10; preferably, j, k are integers of 2-8, for example, integers of 2, 3, 4, 5, 6, 7, 8.

[0023] In other specific embodiments, the L2 is a cleavable peptide chain consisting of 2-10 amino acids. In some preferred embodiments, the L2 is selected from Gly-Gly-Phe-Gly, Val-Cit, Val-Ala, Val-Lys, Val-Arg, AcLys-Val-Cit, AcLys-Val-Ala, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Glu-Val-Ala, Glu-Val-Cit, Ala-Lys, Leu-Cit, lle-Cit, Trp-Cit, Ala-Ala-Asn, Ala-Ala-Ala, Ala-Asn, Lys. In other preferred embodiments, L2 is selected from Gly-Gly-Phe-Gly, Val-Cit, Val-Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, Ala-Ala-Asn.

[0024] In some embodiments, the L3 is selected from:

[0025]

[0026]

[0027] In some preferred embodiments, the L3 is selected from In some embodiments, the -H-L1-L2-L3-D structure is as follows:

[0028]

[0029]

[0030] In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of binding to carbonic acid tincture enzyme IX, alpha-fetoprotein, alpha-actinin, A3, A33, ART4, B7, B7H3, B7H4, BAGE, BrE3 antigen, CA125, CAMEL, CAP", CASP-8 / m, CCL19, CCL21, CD1, CD1a.CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD 45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4, CDKN2A, HIF-Ια, colon-specific antigen p (CSAp), CEA, CEACAM5, CEACAM6, oMet, DAM, EGFR, EGFRvIII, cMet, EGP-1 (Trop-2), EGP-2.ELF2-M, Ep-CAM, Her2, Her3, Claudin 18.2, ROR1, ROR2, dll3, marcl7, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gpl00, GRO-β, HLA-DR.HML24, HMGB-1, HSP70-2M., IGF-1R, IGR1R, MAGE, MAGE-3, MART-1.MART-2.NY-ESO-1, TRAG3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16 , MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, PD-1 receptor, PD-L1 receptor, placental growth factor, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-1R, RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAG, TAG-72, tenascin, TRAIL receptor, Tn antigen, ED-B, WT-1, 17-1A antigen; preferably, the antibody or antigen-binding fragment binds to Her2, Her3, B7H3, Claudin 18.2. DLL-3, EGP-1 (Trop-2). .

[0031] In some specific embodiments, the antibody or antigen-binding fragment is selected from epratuzumab, veltuzumab, sacituzumab, patritumab, trastuzumab, pertuzumab, abciximab, alemtuzumab, cetuximab, gemtuzumab, ibritumomab, panitumumab, rituximab, tositumomab, abagovomab, atlizumab, benralizumab, obinutuzumab, basiliximab, d adizumab, efalizumab, muromomab, natlizumab, omalizumab, gaiitenemmab, solanezumab, tisotumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, enfortumab, belantamab, cetuximab, loncastuximab, daratumumab, nimotuzumab, zolbetuximab, omburtamab, rovalpituzumab.

[0032] In some preferred embodiments, the antibody or antigen-binding fragment is selected from sacituzumab, daratumumab, zolbetuximab, omburtamab, and rovalopituzumab.

[0033] In some embodiments, the antibody or antigen-binding fragment is selected from Sacituzumab antibody or its antigen-binding fragment, and the Sacituzumab antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions HCDR1-3, respectively comprising the amino acid sequences described in SEQ ID NOs: 1-3; wherein the light chain variable region comprises three complementarity determining regions LCDR1-3, respectively comprising the amino acid sequences described in SEQ ID NOs: 4-6.

[0034] In some more specific embodiments, the heavy chain variable region of the Sacituzumab antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:7, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:8.

[0035] In some more specific embodiments, the heavy chain of the Sacituzumab antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:9, and the light chain comprises the amino acid sequence of SEQ ID NO:10.

[0036] In some embodiments, the antibody or antigen-binding fragment is selected from a daratumumab antibody or an antigen-binding fragment thereof, the daratumumab antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions HCDR1-3, each comprising the amino acid sequence described in SEQ ID NO: 11-13; wherein the light chain variable region comprises three complementarity determining regions LCDR1-3, each comprising the amino acid sequence described in SEQ ID NO: 14-16.

[0037] In some more specific embodiments, the heavy chain variable region of the daratumumab antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:17, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:18.

[0038] In some more specific embodiments, the heavy chain of the daratumumab antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO: 19, and the light chain comprises the amino acid sequence of SEQ ID NO: 20.

[0039] Another aspect of the present invention is to provide a pharmaceutical composition, which comprises the antibody-drug conjugate of the present invention and a pharmaceutically acceptable carrier, excipient or diluent.

[0040] Another aspect of the present invention is to provide a use of the antibody-drug conjugate of the present invention in the preparation of a drug for treating and / or preventing tumors.

[0041] In some preferred embodiments, the tumor is selected from tumors associated with the following group of expressions: Trop-2, Her2, Her3, B7H3, Claudin 18.2, CD30, CD38, CD33, CD70 and EGFR.

[0042] In some specific embodiments, the tumor comprises a solid tumor or a hematological tumor.

[0043] In some more specific embodiments, the tumor is selected from: lung cancer, kidney cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, leukemia, malignant lymphoma, cervical cancer, squamous cell carcinoma, penile cancer, esophageal cancer, salivary gland cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Attached Figure 1 Test results of the inhibitory effect of toxin molecules on MDA-MB-468 cells;

[0045] Attached Figure 2 Test results of the inhibitory effect of ADC molecules on MDA-MB-468 cells;

[0046] Attached Figure 3 Test results of the inhibitory effect of ADC molecules on multiple myeloma RPMI 8226 cells;

[0047] Attached Figure 4 The test results of the inhibitory effect of toxin molecules on the proliferation of human papillary ovarian adenocarcinoma cell Caov3;

[0048] Attached Figure 5 The test results of the inhibitory effect of ADC molecules on the proliferation of human papillary ovarian adenocarcinoma cell Caov3;

[0049] Attached Figure 6 Test results of the inhibitory effect of toxin molecules on the proliferation of human colorectal adenocarcinoma cells HCT-15;

[0050] Attached Figure 7 Test results of the inhibitory effect of ADC molecules on the proliferation of human colorectal adenocarcinoma cells HCT-15;

[0051] Attached Figure 8 Results of ADC endocytosis test;

[0052] Attached Fig. 9 Results of the ADC killing effect test. DETAILED DESCRIPTION

[0053] The present invention is further described below in conjunction with specific embodiments.

[0054] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, the following examples do not represent that the following experiments are all experiments or the only experiments of the present invention.

[0055] One of the exemplary antibodies used in the present invention is Sacituzumab (also known as certolizumab / gosartuzumab / hRS7), which is described in detail in WO2014057687A. Specifically, the heavy chain variable region of the Sacituzumab antibody includes three complementary determining regions HCDR1-3, each comprising the amino acid sequence described in SEQ ID NO: 1-3; the light chain variable region includes three complementary determining regions LCDR1-3, each comprising the amino acid sequence described in SEQ ID NO: 4-6. More specifically, the heavy chain variable region of the Sacituzumab antibody includes the amino acid sequence described in SEQ ID NO: 7, and the light chain variable region includes the amino acid sequence described in SEQ ID NO: 8; more specifically, the heavy chain of the Sacituzumab antibody includes the amino acid sequence described in SEQ ID NO: 9, and the light chain includes the amino acid sequence described in SEQ ID NO: 10. The construction of the antibody can be constructed by conventional biological methods in the art or purchased commercially.

[0056] One of the exemplary antibodies used in the present invention is Daratumumab (also known as Daratumumab), which is described in detail in WO2006099875A. Specifically, the heavy chain variable region of Daratumumab antibody includes three complementary determining regions HCDR1-3, which respectively include the amino acid sequences described in SEQ ID NO: 11-13; the light chain variable region includes three complementary determining regions LCDR1-3, which respectively include the amino acid sequences described in SEQ ID NO: 14-16. More specifically, the heavy chain variable region of Sacituzumab antibody includes the amino acid sequence described in SEQ ID NO: 17, and the light chain variable region includes the amino acid sequence described in SEQ ID NO: 18; more specifically, the heavy chain of Sacituzumab antibody includes the amino acid sequence described in SEQ ID NO: 19, and the light chain includes the amino acid sequence described in SEQ ID NO: 20. The construction of the antibody can be constructed by conventional biological methods in the art or purchased commercially.

[0057] The toxin used in the present invention is triptolide purchased from China Food and Drug Inspection Institute, CAT#111567-201404, ID: DP03-BYVB, M.wt: 360.4

[0058] Dxd is an exotecan derivative, the structure of which is described in detail in WO2014057687A, and can be prepared by conventional methods in the art or purchased commercially. For example, Dxd in the embodiments of the present invention is purchased from MCE, CAT#HY-13631D / CS-0044752, Lot#247301, M.wt: 493.48.

[0059] The reference drug (positive control drug) is dato-DXd (datopotamab deruxtecan, DS-1062a), which is an antibody-drug conjugate targeting trop-2 antibody (datopotamab) and topoisomerase inhibitor (Dxd) developed based on the Dxd-ADC platform developed by Daiichi Sankyo. Its structure and preparation method are described in detail in WO2014057687A and can be prepared or commercially purchased using the patented method.

[0060] Other raw materials of the present invention are commercially available or prepared by methods known in the art. Among them, DCM is dichloromethane, EDCI is carbodiimide, DMF is N,N-dimethylformamide, ACN is acetonitrile, HCl is hydrochloric acid, H2O is water, THF is tetrahydrofuran, DMSO is dimethyl sulfoxide, TCEP is tris(2-carboxyethyl)phosphine hydrochloride, and EA is ethyl acetate.

[0061] Example 1 Preparation of Compound 1

[0062]

[0063] Compound 1-1 (70 mg, 0.13 mmol) and compound 1-2 (16 mg, 0.15 mmol) were dissolved in 7 mL DCM, and EDCI (50 mg, 0.15 mmol) was added under nitrogen protection and stirred at room temperature for 2 h. The reaction solution was then spin-dried, dissolved in DMF and directly purified by reverse phase column with H2O:ACN=70:30%, and freeze-dried to obtain 47 mg of white powder with a yield of 56%.

[0064]

[0065] Triptolide (28 mg, 0.07 mmol) and triphosgene (27 mg, 0.09 mmol) were placed in a 25 mL three-necked flask under nitrogen protection. DCM was added to dissolve the mixture. Pyridine (41 mg, 0.52 mmol) was then added and stirred at room temperature for 1 h. Compound 1-3 (45 mg, 0.07 mmol) was then added and stirred at room temperature for 1 h. The reaction solution was then spin-dried and dissolved in DMF and directly sent to reverse phase HPLC for preparation. After freeze-drying, 16 mg of white powder (Compound 1) was obtained with a yield of 20%.

[0066]

[0067] Compound 1 NMR: 1HNMR(400MHz, CDCl3)δ7.70(s,2H),7.39(s,2H),7.24–7.06(m,4H),6.69(s,2H),5.21(s,2H),4.85(s ,1H),4.68(s,3H),4.11(d,J=146.9Hz,3H),3.82(d,J=2.9Hz,1H),3.55(d,J=2.5Hz,1H),3.51(d,J=5. 2Hz,1H),3.41(s,2H),3.24(s,1H),2.69(s,1H),2.45(s,11H),2.38–2.03(m,4H),1.93(dd,J=18.8,1 1.6Hz,2H),1.81–1.39(m,5H),1.18(s,3H),1.04(s,3H),0.97(d,J=6.9Hz,3H),0.84(d,J=6.8Hz,3H).

[0068] Example 2 Preparation of Compound 2

[0069]

[0070] Compound 2-1 (200 mg, 0.37 mmol) was dissolved in HCl (2 mL, 1 MinTHF) and stirred at room temperature overnight; the reaction solution was then adjusted to pH = 8 with saturated sodium bicarbonate, the layers were separated, and the aqueous phase was directly purified on a reverse phase column with ACN = 30%. It was lyophilized to obtain 85 mg of white powder (Compound 2-2) with a yield of 52%.

[0071]

[0072] Compound 2-2 (80 mg, 0.18 mmol) and compound 2-3 (88 mg, 0.19 mmol) were dissolved in 2 mL DMF and stirred at room temperature for 1 h. The reaction solution was then directly purified by reverse phase column and freeze-dried to obtain 85 mg of a white hygroscopic solid (compound 2-4) with a yield of 61%.

[0073]

[0074] Triptolide (40 mg, 0.11 mmol) and triphosgene (39 mg, 0.13 mmol) were placed in a 25 mL three-necked flask under nitrogen protection. DCM was added to dissolve the mixture, followed by pyridine (60 mg, 0.75 mmol). The mixture was stirred at room temperature for 1 h, and then compound 2-4 (80 mg, 0.09 mmol) was added and stirred at room temperature for 1 h. The reaction solution was then spin-dried and directly sent to reverse phase HPLC for purification. After freeze-drying, 15 mg of a white hygroscopic solid (compound 2) was obtained with a yield of 11%.

[0075]

[0076] Compound 2 NMR: 1 HNMR (400MHz, CDCl3) δ8.74 (s, 1H), 7.71 (d, J = 5.7Hz, 1H), 7.66 (d, J = 8.5Hz, 2H ),7.60(s,1H),7.50(d,J=6.3Hz,1H),7.32(d,J=8.4Hz,2H),7.25(s,2H),7.22 –7.18(m,1H),6.67(s,2H),6.17(s,3H),5.14(q,J=12.1Hz,2H),4.83(s,1H),4 .64(d,J=18.5Hz,3H),4.12–4.01(m,2H),3.83(dd,J=18.7,10.3Hz,5H),3.66(t ,J=5.2Hz,4H),3.55(dd,J=14.3,5.9Hz,15H),3.47(d,J=5.6Hz,1H),3.37–3.3 1(m,1H),3.11(dd,J=14.2,9.8Hz,1H),2.67(d,J=13.1Hz,1H),2.49(s,2H),2.2 8(s,1H),2.20–2.09(m,2H),1.96–1.86(m,2H),1.55(dd,J=12.4,4.8Hz,1H),1 .19(d,J=5.7Hz,1H),1.04(s,3H),0.95(d,J=7.0Hz,3H),0.82(d,J=6.9Hz,3H).

[0077] Example 3 Preparation of Compound 3

[0078]

[0079] Triptolide (36 mg, 0.1 mmol) and compound 3-1 (62 mg, 0.1 mmol) were added to pyridine (1.5 mL dry), stirred at -10 ° C under nitrogen protection, and then POCl3 (46 mg, 0.3 mmol) was added, and then the mixture was stirred at -10 ° C under N2 atmosphere for 0.5 h. After the mixture was purified by HPLC, a white solid (compound 3) (20 mg, yield 21%) was obtained.

[0080]

[0081] 1HNMR (400MHz, DMSO) δ8.62(d,J=6.8Hz,1H),8.32(t,J=5.7Hz,1H),8.18–8.05(m,2H),8.01(t,J=5.6Hz,1H),7.35-7.15(m,5H),7.02(s,2H),5 .06(s,1H),4.92–4.77(m,2H),4.72–4.58(m,2H),4.52(td,J=9.2,4.6 Hz,1H),4.19(s,2H),3.97(d,J=3.2Hz,1H),3.83–3.53(m,8H),3.40(d, J=7.0Hz,2H),3.08(dd,J=13.8,4.5Hz,1H),2.82(dd,J=13.7,9.8Hz,1H),2.62(s,1H),2.31–2.20(m,1H),2.18–2.06(m,3H),1.88(ddd,J=33. 7,27.6,17.1Hz,3H),1.50(dq,J=14.6,7.3Hz,4H),1.39–1.28(m,2H),1.21(dt,J=15.1,7.6Hz,2H),0.94–0.85(m,6H),0.77(t,J=5.9Hz,3H).

[0082] Example 4 Preparation of Compound 4

[0083]

[0084] Compound 4-1 (300 mg, 0.58 mmol) was dissolved in 20% pyridine in DMF solution (2 mL) and stirred at room temperature for 1 h. The reaction solution was then directly purified using a reverse phase column, H2O:ACN = 95%:5% (volume ratio), and lyophilized to obtain 130 mg of white powder, with a yield of 76%.

[0085]

[0086] 4-2 (130 mg, 0.44 mmol) and 4-3 (145 mg, 0.44 mmol) were dissolved in 3 mL DMF and stirred at room temperature overnight; the reaction solution was then diluted with EA and washed three times with water, then washed once with saturated brine, and then dried over anhydrous sodium sulfate. After mixing, the sample was passed through a silica gel column, DCM:MeOH = 100%-94%:6%, and 160 mg of a slightly yellow solid (compound 4) was obtained after spin drying, with a yield of 58%.

[0087]

[0088] Triptolide (29 mg, 0.08 mmol) and triphosgene (30 mg, 0.1 mmol) were placed in a 25 mL three-necked flask under nitrogen protection. DCM was added to dissolve the mixture, followed by pyridine (47 mg, 0.6 mmol). The mixture was stirred at room temperature for 1 h, and then compound 4-4 (50 mg, 0.08 mmol) was added and stirred at room temperature for 1 h. After the reaction, the reaction solution was spin-dried and directly purified by reverse phase HPLC. After freeze-drying, 12 mg of an off-white solid (compound 4) was obtained with a yield of 15%.

[0089]

[0090] Compound 4 NMR: 1 HNMR(400MHz,DMSO)δ9.97(s,1H),8.18(d,J=6.9Hz,1H),7.87(d,J=8.7Hz,1H ),7.61(d,J=8.5Hz,2H),7.35(d,J=8.6Hz,2H),7.02(s,2H),5.11(q,J=12.0H z,2H),4.82(dd,J=31.1,13.6Hz,3H),4.38(dd,J=13.9,6.9Hz,1H),4.24–4.1 7(m,1H),3.95(d,J=3.1Hz,1H),3.69(d,J=2.8Hz,1H),3.64–3.54(m,5H),3.51 (d,J=5.1Hz,2H),3.46(t,J=4.9Hz,12H),2.63(d,J=27.6Hz,1H),2.40(ddd,J =27.3,14.2,6.8Hz,2H),2.23(dt,J=14.8,5.8Hz,1H),2.15–2.06(m,1H),1.9 6(dd,J=13.5,6.7Hz,2H),1.87–1.73(m,2H),1.28(dd,J=22.8,8.5Hz,5H),0. 90(s,3H),0.86(d,J=6.9Hz,6H),0.83(d,J=6.8Hz,3H),0.76(d,J=6.8Hz,3H).

[0091] Example 5 Preparation of ADC 1-4

[0092] Preparation method of ADC1, ADC2 and ADC3:

[0093] First, the antibody was reduced, and the antibody (Sacituzumab) was replaced with 20mmol / L PB, 500mmol / L NaCl, pH 6.5 buffer, and the antibody concentration was adjusted to 5-10mg / ml. 0.05mol / L TCEP was added to make the molar ratio of TCEP to antibody 20, and the antibody was reduced in a 30°C water bath for 2h for use. The second step was to couple the antibody and the drug, and the drug molecules with linkers (compounds 1, 2, and 3) were added to the reduced antibody solution to make the molar ratio of the drug molecules to the antibody 20, and the drugs were coupled in a 30°C water bath for 2h to obtain ADC1, ADC2, and ADC3, respectively. The DAR value of ADC1 was 8.1, the DAR value of ADC2 was 8.0, and the DAR value of ADC3 was 7.9 as measured by HPLC.

[0094] Preparation method of ADC1-d, ADC2-d and ADC3-d:

[0095] First, the antibody was reduced, and the antibody (Daratumumab) was replaced with 20mmol / LPB, 500mmol / LNaCl, pH6.5 buffer, and the antibody concentration was adjusted to 5-10mg / ml. 0.05mol / L TCEP was added to make the molar ratio of TCEP to antibody 20, and the antibody was reduced in a water bath at 30°C for 2h for standby use. The second step was to couple the antibody and the drug, and the drug molecules with linkers (compounds 1, 2, and 3) were added to the reduced antibody solution to make the molar ratio of the drug molecules to the antibody 20, and the drugs were coupled in a water bath at 30°C for 2h to obtain ADC1-d, ADC2-d, and ADC3-d, respectively. The DAR values ​​of ADC1-d, ADC2-d, and ADC3-d were measured by HPLC to be 8.1, 8.0, and 7.9 for ADC3-d.

[0096] Preparation method of ADC4:

[0097] First, the antibody was reduced, and the antibody (Sacituzumab) was replaced with 20mmol / L PB, 500mmol / L NaCl, pH 6.5 buffer, and the antibody concentration was adjusted to 5-10mg / ml. 0.05mol / L TCEP was added to make the molar ratio of TCEP to antibody 8, and the antibody was reduced in a 20°C water bath for 2h for use. The second step was to couple the antibody and the drug, and the drug molecule with a linker (Compound 4) was added to the above reduced antibody solution to make the molar ratio of the drug molecule to the antibody 8, and the drug was coupled in a 20°C water bath for 1h. Finally, the solution was ultrafiltrated and replaced in DPBS to obtain ADC4. The DAR value of ADC4 was measured by HPLC to be 4.0.

[0098] Example 6 Inhibitory effect of ADC molecules on MDA-MB-468 cells

[0099] MDA-MB-468 cells in the logarithmic growth phase were taken, digested with 0.25% Trypsin-EDTA, resuspended in 1640 medium containing 10% FBS, counted by trypan blue staining, and the cell density was adjusted to 5×10 4 The cells were inoculated into 96-well cell culture plates, 100 μl per well, and allowed to adhere to the plate for 4 h at 37°C and 5% CO2 incubator.

[0100] Small molecule toxin (triptolide, Dxd) sample detection process: The highest concentration of the drug detected is 20μg / ml. The small molecule toxin is diluted 5 times from 20μg / ml in 1640 medium containing 10% FBS, with a total of 9 dilution gradients, and the 10th gradient is blank. The diluted small molecule toxin is transferred to the culture plate inoculated with MDA-MB-468 cells, 100μl / well, and continuously cultured at 37℃, 5% CO2. After 70h of culture, 20μl CCK-8 solution is added to each well, placed in a 37℃, 5% CO2 incubator, color development for 4h, and the absorbance value at 450nm is read with a SpectraMaxi3x microplate reader, and the IC50 is calculated.

[0101] ADC sample detection process: The highest concentration of the test sample is 300μg / ml (the positive control is dato-DXd, and the negative control is Sacituzumab). The test sample is diluted 3 times from 300μg / ml using 1640 culture medium containing 10% FBS, for a total of 9 dilution gradients, and the 10th gradient is blank. The diluted test sample is transferred to the culture plate inoculated with MDA-MB-468 cells, 100μl / well, and cultured continuously at 37°C and 5% CO2. After 70h of culture, 20μl of CCK-8 solution is added to each well, placed in a 37°C, 5% CO2 incubator, color developed for 4h, and the absorbance at 450nm is read using a SpectraMaxi3x microplate reader, and the IC50 is calculated. The experimental results are as follows Figure 1 and Figure 2 shown.

[0102] Example 6 Inhibitory Effect of ADC on Multiple Myeloma RPMI8226 Cells

[0103] Cell plating: Take RPMI8226 cells in the logarithmic growth phase, collect the cells by centrifugation, resuspend the cells in 1640 medium containing 10% FBS, count the cells by trypan blue staining, and adjust the cell density to 2×10 5 The cells were inoculated into 96-well cell culture plates, 100 μl per well, and cultured overnight in a 37°C, 5% CO2 incubator.

[0104] Sample testing process: Use 1640 culture medium containing 10% FBS to dilute ADC samples to 100μg / ml, 30μg / ml, 10μg / ml, 3μg / ml, 1μg / ml, and 0.3μg / ml, respectively. Transfer the diluted test sample to the culture plate inoculated with RPMI8226 cells, 100μl / well, and culture continuously at 37°C and 5% CO2. After 72 hours of culture, add 20μl CCK-8 solution to each well, place in a 37°C, 5% CO2 incubator, color develop for 2 hours, read the absorbance at 450nm with a SpectraMaxi3x microplate reader, and calculate the IC50. The experimental results are as follows: Figure 3 shown.

[0105] Example 7 Inhibitory effect of ADC on proliferation of human papillary ovarian adenocarcinoma cell line Caov3

[0106] Caov3 cells in the logarithmic growth phase (purchased from Wuhan Pronocell Life Science Co., Ltd.) were digested with 0.25% Trypsin-EDTA (purchased from Hyclone), resuspended in DMEM medium (purchased from Gibco) containing 10% FBS (purchased from Cellmax), counted by trypan blue staining, and the cell density was adjusted to 6×10 4 The cells were seeded in 96-well cell culture plates at 100 μl per well and allowed to adhere to the plate for 4 h at 37°C and 5% CO2 incubator.

[0107] Small molecule toxin (triptolide, Dxd) concentration setting: The highest detected drug concentration is 20μg / ml. Use DMEM culture medium containing 10% FBS to dilute the small molecule toxin from 20μg / ml in 5-fold gradient dilutions, for a total of 9 dilution gradients, and the 10th gradient is blank. Transfer the diluted small molecule toxin to the culture plate inoculated with Caov3 cells, 100μl / well, and culture continuously at 37°C and 5% CO2. After 70h of culture, add 20μl CCK-8 solution (purchased from Dojindo) to each well, place in a 37°C, 5% CO2 incubator, color develop for 4h, use SpectraMaxi3x microplate reader to read the absorbance at 450nm, and calculate IC50. The experimental results are as follows Figure 4 shown.

[0108] The concentration of the test drugs (ADC3, ADC4 and positive control drug) was set as follows: the highest detected drug concentration was 2000nM. The test samples were diluted three times from 2000nM using DMEM culture medium containing 10% FBS, for a total of 9 dilution gradients, and the 10th gradient was blank. The diluted test samples were transferred to the culture plates inoculated with Caov3 cells and HCT-15 cells, 100μl / well, and cultured continuously at 37°C and 5% CO2. After 70h of culture, 20μl of CCK-8 solution was added to each well, and the plates were placed in a 37°C, 5% CO2 incubator for 4h of color development. The absorbance at 450nm was read using a SpectraMax i3x microplate reader. The experimental results are as follows: Figure 5 shown.

[0109] Example 8 Inhibitory effect of ADC on proliferation of human colorectal adenocarcinoma cell HCT-15

[0110] Human colorectal adenocarcinoma cells HCT-15 (purchased from Wuhan Punosai Life Science Co., Ltd.) in the logarithmic growth phase were digested with 0.25% Trypsin-EDTA (purchased from Hyclone), and the cells were resuspended in DMEM medium (purchased from Gibco) containing 10% FBS (purchased from Cellmax), and the cells were counted by trypan blue staining, and the cell density was adjusted to 4×10 4 The cells were seeded in 96-well cell culture plates at 100 μl per well and allowed to adhere to the plate for 4 h at 37°C and 5% CO2 incubator.

[0111] Small molecule toxin (triptolide, Dxd) concentration setting: The highest detected drug concentration is 20μg / ml. Use DMEM culture medium containing 10% FBS to dilute the small molecule toxin from 20μg / ml in 5-fold gradient dilutions, for a total of 9 dilution gradients, and the 10th gradient is blank. Transfer the diluted small molecule toxin to the culture plate inoculated with HCT-15 cells, 100μl / well, and culture continuously at 37°C and 5% CO2. After 70h of culture, add 20μl CCK-8 solution (purchased from Dojindo) to each well, place in a 37°C, 5% CO2 incubator, color develop for 4h, use SpectraMax i3x microplate reader to read the absorbance at 450nm, and calculate IC50. The experimental results are as follows Figure 6 shown.

[0112] The concentration of the test drugs (ADC3, ADC4 and positive control) was set as follows: the highest detected drug concentration was 2000nM. The test samples were diluted three times from 2000nM using DMEM culture medium containing 10% FBS, for a total of 9 dilution gradients, and the 10th gradient was blank. The diluted test samples were transferred to the culture plate inoculated with HCT-15 cells, 100μl / well, and cultured continuously at 37°C and 5% CO2. After 70h of culture, 20μl of CCK-8 solution was added to each well, placed in a 37°C, 5% CO2 incubator, and color developed for 4h. The absorbance at 450nm was read using a SpectraMax i3x microplate reader, and the IC50 was calculated. The experimental results are as follows: Figure 7 shown.

[0113] Example 9 ADC Endocytosis-Killing Experiment

[0114] pHrodo labeled ADC (ADC3, ADC4 and positive control) drugs: dilute the ADC drug to 1 mg / ml (the diluent is 0.1 mol / L NaHCO3); take 500ul of the diluted ADC, add 0.5ul pHrodo (thermo P36600), and incubate at room temperature in the dark for 1h; wash the labeled ADC with a 10Kd ultrafiltration tube (wash with PBS), measure A280, calculate the concentration value, and finally dilute to 33μg / ml with 1640 culture medium + 10% FBS.

[0115] MDA-MB-468-GFP cells were plated in 96-well plates at 2000 cells / well and allowed to adhere overnight. The culture medium was removed and 150ul of ADC drug gradient dilutions (culture medium dilutions) were added to the wells in sequence. Incucyte was used to continuously take photos for 4 days for data analysis.

[0116] The results are as follows Figure 8 and 9 As shown in the figure, the amount of green light is equivalent to the number of cells, and the amount of red light is equivalent to the amount of endocytosis. It can be seen from the figure that ADC4 is better than the positive control drug in both endocytosis and inhibition of cell proliferation.

Claims

1. An antibody-drug conjugate having a structure shown in the formula Ab-[H-L1-L2-L3-D]x, wherein: Ab is an antibody or its antigen-binding fragment; H is a linker site with the antibody or antigen-binding fragment thereof; L1 is a connecting unit connecting the joint parts H and L2; L2 is the linker between L1 and L3; L3 is the spacer between L2 and D; D is a toxin having the following structural formula: X Select from 1-10.

2. The antibody-drug conjugate according to claim 1, characterized in that: The H is linked to Ab via a sulfhydryl, azide or amide group.

3. The drug conjugate according to claim 1, characterized in that The H is selected from: Preferably, H is 4. The antibody-drug conjugate according to claim 1, characterized in that: The L1 comprises -L1'-C(O)-, wherein L1' is selected from an optionally substituted alkylene group, an optionally substituted polyethylene glycol group, an optionally substituted alkenylene group, an optionally substituted alkynylene group, an optionally substituted alicyclic group, an optionally substituted aliphatic heterocyclic group, an optionally substituted arylene group, an optionally substituted heteroarylene group, or a combination thereof.

5. The antibody-drug conjugate according to claim 1, characterized in that: The L1' is selected from -(CH2)m-, -(PEG)n- or -(CH2) m -(PEG) n -(CH2) z -, wherein m, n, z are integers of 0-10, preferably, m, n, z are integers of 0-8; preferably, L1' is selected from -(CH2)5-, -(PEG)4- or -(CH2)2-(PEG)4-.

6. The antibody-drug conjugate according to claim 1, characterized in that: The L2 is a cleavable linker, a non-cleavable linker, a hydrophilic linker, a hydrophobic linker, a charged linker, or an uncharged linker.

7. The antibody-drug conjugate according to claim 1, characterized in that: The L2 is a non-cleavable linker; preferably, the L2 is -(PEG) k - or -NH-(CH2) j -(PEG) k -, wherein j, k are integers of 0-10; preferably, j, k are integers of 2-8.

8. The antibody-drug conjugate according to claim 1, characterized in that: The L2 is a cleavable peptide chain consisting of 2-10 amino acids; preferably, the L2 is selected from Gly-Gly-Phe-Gly, Val-Cit, Val-Ala, Val-Lys, Val-Arg, AcLys-Val-Cit, AcLys-Val-Ala, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Glu-Val-Ala, Glu-Val-Cit, Ala-Lys, Leu-Cit, lle-Cit, Trp-Cit, Ala-Ala-Asn, Ala-Ala-Ala, Ala-Asn, Lys; preferably, the L2 is selected from Gly-Gly-Phe-Gly, Val-Cit, Val-Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, Ala-Ala-Asn.

9. The antibody-drug conjugate according to claim 1, characterized in that: The L3 is selected from: Preferably, said L3 is selected from 10. The antibody-drug conjugate according to claim 1, characterized in that: The -H-L1-L2-L3-D structure is shown below:

11. The antibody-drug conjugate according to claim 1, characterized in that: The antibody or antigen-binding fragment is selected from the group consisting of binding to carbonic acid tincture enzyme IX, α-fetoprotein, α-actinin, A3, A33, ART 4, B7, B7H3, B7H4, BAGE, BrE3 antigen, CA125, CAMEL, CAP", CASP-8 / m, CCL19, CCL21, CD1, CD1a.CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59 、CD64、CD66a-e、CD67、CD70、CD70L、CD74、CD79a、CD80、CD83、CD95、CD126、CD132、CD133、CD138、CD147、CD154、CDC27、CDK-4、CDKN2A、HIF-Ια、colon-specific antigen p(CSAp)、CEA、CEACAM5、CEACAM6、oMet、DAM、EGFR、EGFRvIII、cMet、EGP-1(Trop-2)、EGP-2.ELF2-M、Ep-CAM、Her2、Her3、Claudin 18.2, ROR1, ROR2, dll3, marcl7, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gpl00, GRO-β, HLA-DR.HML24, HMGB-1, HSP70-2M., IGF-1R, IGR1R, MAGE, MAGE-3, MART-1.MART-2.NY-ESO-1, TRAG3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16 , MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, PD-1 receptor, PD-L1 receptor, placental growth factor, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-1R, RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAG, TAG-72, tenascin, TRAIL receptor, Tn antigen, ED-B, WT-1, 17-1A antigen; preferably, the antibody or antigen-binding fragment binds to Her2, Her3, B7H3, Claudin 18.

2. DLL-3, EGP-1 (Trop-2); Preferably, the antibody or antigen-binding fragment is selected from epratuzumab, veltuzumab, sacituzumab, patritumab, trastuzumab, pertuzumab, abciximab, alemtuzumab, cetuximab, gemtu zumab, ibritumomab, panitumumab, rituximab, tositumomab, abagovomab, atlizumab, benralizumab, obinutuzumab, basiliximab, dadizumab, efalizumab, muromomab, nat lizumab, omalizumab, gaiitenemmab, solanezumab, tisotumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, enfortumab, belantamab, cetuximab, loncastuximab, daratumumab, nimotuzumab, zolbetuximab, omburtamab, rovalvpituzumab; preferably, the antibody or antigen-binding fragment is selected from sacituzumab, daratumumab, zolbetuximab, omburtamab, rovalvpituzumab. .

12. The antibody-drug conjugate according to claim 1, characterized in that: The antibody or antigen-binding fragment is selected from the group consisting of sacituzumab antibody or antigen-binding fragment thereof, the sacituzumab antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementary determining regions HCDR1-3, comprising the amino acid sequences described in SEQ ID NOs: 1-3, respectively; wherein the light chain variable region comprises three complementary determining regions LCDR1-3, comprising the amino acid sequences described in SEQ ID NOs: 4-6, respectively; preferably, the heavy chain variable region of the sacituzumab antibody or antigen-binding fragment thereof comprises the amino acid sequence described in SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence described in SEQ ID NO: 8; more preferably, the heavy chain of the sacituzumab antibody or antigen-binding fragment thereof comprises the amino acid sequence described in SEQ ID NO: 9, and the light chain comprises the amino acid sequence described in SEQ ID NO: 10; or The antibody or antigen-binding fragment is selected from the daratumumab antibody or its antigen-binding fragment, the daratumumab antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementary determining regions HCDR1-3, respectively comprising the amino acid sequences described in SEQ ID NOs: 11-13; wherein the light chain variable region comprises three complementary determining regions LCDR1-3, respectively comprising the amino acid sequences described in SEQ ID NOs: 14-16; preferably, the heavy chain variable region of the daratumumab antibody or its antigen-binding fragment comprises the amino acid sequence described in SEQ ID NO: 17, and the light chain variable region comprises the amino acid sequence described in SEQ ID NO: 18; more preferably, the heavy chain of the daratumumab antibody or its antigen-binding fragment comprises the amino acid sequence described in SEQ ID NO: 19, and the light chain comprises the amino acid sequence described in SEQ ID NO:

20.

13. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody-drug conjugate according to any one of claims 1 to 12, and a pharmaceutically acceptable carrier, excipient or diluent.

14. Use of the antibody drug conjugate according to any one of claims 1 to 12 in the preparation of a medicament for treating and / or preventing a tumor; preferably, the tumor is selected from the group consisting of tumors associated with the expression of Trop-2, Her2, Her3, B7H3, Claudin 18.2, CD30, CD38, CD33, CD70 and EGFR; preferably, the tumor comprises a solid tumor or a blood tumor; more preferably, the tumor is selected from the group consisting of lung cancer, kidney cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, leukemia, malignant lymphoma, cervical cancer, squamous cell carcinoma, penile cancer, esophageal cancer, and salivary gland cancer.

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