CN106467575B

CN106467575B - Cysteine engineered antibody-toxin conjugates

Info

Publication number: CN106467575B
Application number: CN201510507792.6A
Authority: CN
Inventors: 朱义; 王一茜; 卓识; 李�杰; 陈澜; 余永国
Original assignee: Sichuan Baili Pharmaceutical Co Ltd
Current assignee: Chengdu Bailidote Biological Pharmaceutical Co ltd
Priority date: 2015-08-18
Filing date: 2015-08-18
Publication date: 2020-07-31
Anticipated expiration: 2035-08-18
Also published as: CN106467575A

Abstract

According to the invention, valine (V) at the 205 th site of a light chain of a target antibody is transformed into cysteine (C), and fixed-point coupling is carried out on a mc-vc-PAB-OH linker coupled with small-molecule high-activity cytotoxin (Payload) through the free sulfydryl (-SH) of the transformed cysteine to form a cysteine-transformed antibody-toxin conjugate with excellent uniformity, wherein the toxin-to-antibody ratio (DAR) of the antibody-toxin conjugate is 1.6-2.0.

Description

Cysteine engineered antibody-toxin conjugates

Technical Field

The invention relates to a compound, a preparation method and application thereof, in particular to cysteine-modified antibody-toxin conjugates (TDC) and a preparation method and application thereof.

Technical background:

the epidermal Growth Factor receptor EGFR (Erdermal Growth Factor receptor) is a glycoprotein belonging to the ErbB receptor family, which includes EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her 3(ErbB-3) and Her 4 (ErbB-4). EGFR is a receptor for cell proliferation and signaling of Epithelial Growth Factor (EGF), penetrates the cell membrane, has a molecular weight of 170KDa, and is activated by ligand binding. After activation, EGFR is converted from monomer to dimer. EGFR may also be activated by aggregation with other members of the ErbB receptor family, such as ErbB2/Her 2/neu.

EGFR is usually expressed in low amounts in a variety of normal tissue cells, including skin, liver, etc., and is associated with normal physiological effects.

EGFR overexpression has been associated with the development and progression of a variety of tumors, including head and neck cancer, bladder cancer, ovarian cancer, non-small cell lung cancer, colorectal cancer, brain glioma, renal cancer, prostate cancer, pancreatic cancer, breast cancer (Atalay et al, 2003; Herbst and Shin, 2002).

Many tumors overexpress wild-type EGFR (egfrwt) and also express mutant EGFR, EGRFvIII (de2-7 EGFR). EGRFvIII is an EGFR mutant with EGFRwt amino acid deletion 6-273 (Sugawa et al, 1990) that mediates ligand-independent sustained cell activation. EGRFvIII has been reported in a variety of tumors including brain gliomas, breast cancer, non-small cell lung cancer, ovarian cancer and prostate cancer (Wikstrand et al, 1997; OlapideOlaopa et al, 2000). The appearance of EGRFvIII is often indicative of poor tumor prognosis.

EGRFvIII is specifically expressed in tumor cell tissues, and EGRFvIII is not expressed in normal tissue cells, so that a good target is provided for targeted therapy.

Antibody-toxin conjugates (ADCs) are a hot area of targeted therapy, two drugs, adsetris and kadcella, that have been approved for sale in the united states for good clinical efficacy, and over 50 ADC drugs are under clinical phase study.

The ADC drug ABT-414 which takes EGFRvIII as a target spot and is coupled through sulfydryl of an interchain disulfide bond at a non-fixed point is tested in the United states at the clinical II stage, and shows a certain clinical effect, but because the ADC drug ABT-414 adopts a non-fixed point coupling mode, the drug uniformity is poor, serious toxic and side effects are caused to patients, the clinical dosage is limited, and the clinical treatment effect is directly influenced.

The invention content is as follows:

the cysteine-modified antibody-toxin conjugate (TDC) of the compound disclosed by the invention has the general formula of 2C 1-L C-V205C-mc-vc-PAB-payload, wherein 2C1 is an antibody obtained by modifying valine (V) at the 205 th position of a light chain of a parent antibody 2A1 into cysteine (C). the amino acid sequence of a heavy chain variable region of 2C1 is SEQ ID NO:1, the light chain amino acid sequence of 2C1 is SEQ ID NO: 12.2C 1, the amino acid sequence of a heavy chain variable region of a parent antibody 2A1 is SEQ ID NO:1, and the light chain amino acid sequence of 2A1 is SEQ ID NO: 14.2C 1, the binding capacity (affinity) of the parent antibody 2A1 and an antigen is kept, the antigen is a human wild-type epidermal growth factor receptor (EGFRwt) and a human epidermal growth factor receptor (de2-7 EGFR/EGFRGFVIGFIII). 2C1, the antibody is a cysteine-binding site-binding protein conjugate (CTC) of a human epidermal growth factor receptor, a cysteine-toxin conjugate (CTC-modified antibody, a cysteine-toxin conjugate, a colorectal cancer antigen, a colorectal cancer-toxin, a colorectal cancer-toxin conjugate, a pancreatic cancer-treating drug, a colorectal cancer-treating drug, a colorectal cancer-targeting drug, a.

Compared with non-site-specific-coupled ADC, the antibody-toxin conjugate (TDC) modified by the brand-new cysteine has the advantages of good drug uniformity, small side effect and the like, and preclinical research results show that the TDC is obviously superior to the non-site-specific-coupled ADC.

Drawings

FIG. 1 is a schematic representation of HIC-HP L C assay 2C 1-L C-V205C-mc-vc-PAB-MMAE TDC DAR;

FIG. 2A is SEC-HP L C detecting 2C 1-L C-V205C-mc-vc-PAB-MMAE TDC aggregation;

FIG. 2B is a SEC-HP L C assay for 2A1-mc-vc-PAB-MMAE ADC samples conjugated cysteine non-site through the natural interchain disulfide bonds of the antibody;

FIG. 3A is a graph showing that 2C 1-L C-V205C-mc-vc-PAB-MMAE TDC and 2C1 retain the affinity of 2A1 to the antigen EGFRvIII;

FIG. 3B is a graph showing that 2C 1-L C-V205C-mc-vc-PAB-MMAE TDC, 2C 1-L C-V205C-mc-vc-PAB-MMAFTDC, 2C 1-L C-V205C-mc-vc-PAB-PBD TDC, 2C 1-L C-V205C-mc-vc-PAB-SN38TDC, and 2C 1-L C-V205C-mc-vc-PAB-DOX TDC retain the affinity of 2A1 for the antigen EGFRvIII;

FIG. 4A is the result of IC50 detection of 2C 1-L C-V205C-mc-vc-PAB-MMAE TDC and its corresponding 2A1-mc-vc-PAB-MMAE ADC on EGFRwt-overexpressed human skin squamous carcinoma cell A431;

FIG. 4B shows the results of IC50 detection of 2C 1-L C-V205C-mc-vc-PAB-MMAF TDC and its corresponding 2A1-mc-vc-PAB-MMAFADC on EGFRwt-overexpressed human skin squamous carcinoma cell A431;

FIG. 4C is the result of IC50 detection of EGFRwt-overexpressed human skin squamous carcinoma cell A431 by 2C 1-L C-V205C-mc-vc-PAB-PBD TDC and its corresponding 2A1-mc-vc-PAB-PBD ADC;

FIG. 4D is the results of IC50 detection of U87-EGFRvIII expressing EGFRvIII from 2C 1-L C-V205C-mc-vc-PAB-MMAE TDC and its corresponding 2A1-mc-vc-PAB-MMAE ADC;

FIG. 4E is the result of IC50 detection of U87-EGFRvIII expressing EGFRvIII by 2C 1-L C-V205C-mc-vc-PAB-MMAF TDC and its corresponding 2A1-mc-vc-PAB-MMAF ADC;

FIG. 4F is the result of IC50 detection of U87-EGFRvIII expressing EGFRvIII from 2C 1-L C-V205C-mc-vc-PAB-PBD TDC and its corresponding 2A1-mc-vc-PAB-PBD ADC;

FIG. 4G is the result of IC50 detection of U87-EGFRvIII expressing EGFRvIII by 2C 1-L C-V205C-mc-vc-PAB-SN38TDC and its corresponding 2A1-mc-vc-PAB-SN38 ADC;

FIG. 4H is the result of IC50 detection of U87-EGFRvIII expressing EGFRvIII by 2C 1-L C-V205C-mc-vc-PAB-Dox TDC and its corresponding 2A1-mc-vc-PAB-Dox ADC;

FIG. 5A shows the single intravenous administration results of 1mg/kg dose for drug effect detection in tumor-bearing mice;

FIG. 5B shows the result of drug effect test of 6mg/kg dose administration in tumor-bearing mice;

FIG. 6A shows the neutrophil count of the toxicity test in rats;

FIG. 6B shows AST detection results of rat toxicity detection;

figure 6C monitors body weight change daily for rat toxicity testing.

Example 1 Synthesis of mc

Adding 3.9g (0.03mol) of 6-aminocaproic acid and 3.5g (0.036mol) of 1.2eq maleic anhydride into 30ml glacial acetic acid, stirring the reaction solution at 120 ℃ for 4-6H, after the reaction is completed, stopping heating, naturally cooling to room temperature, concentrating under reduced pressure at 60 ℃ to remove most of acetic acid, pouring the obtained brown yellow viscous liquid into water, adding 20ml of ethyl acetate for extraction, combining organic layers, washing the organic layers with water and saturated brine in sequence, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure to obtain brown yellow oily substance, adding 50ml of water, stirring, precipitating as white solid, filtering, and drying at 50 ℃ under reduced pressure to obtain the target product 5.08g, yield 80%, mp: 89-92 ℃, M/z: 212.2[ M + H ] +.1 HNMR (400Mz, DMSO): 13.21(br,1H, COOH), 6.75(s,2H, COCH 3.63, NCH 2J ] +.7H, NCH (7.42H), NCH 2H 2, 3642.42H, NCH 2H 2, 3668, NCH 2H, 364.42 Hz), NCH 2H, 368 Hz, 368.42 Hz.

Example 2 Synthesis of Mc-OSu

Under the protection of nitrogen, adding 4.7g (22mmol) of MC and 25g (22mmol) of HOSu into 50ml of acetonitrile, dissolving 4.5g (22mmol) of DCC in 25ml of acetonitrile, keeping the internal temperature at about 0 ℃, slowly dropping the DCC into the reaction solution, reacting the reaction solution at 0 ℃ for 2 hours, reacting at room temperature overnight, filtering, washing the filter cake with 10ml of acetonitrile × 3, concentrating the filtrate under reduced pressure to dryness, drying the obtained oily substance at room temperature under reduced pressure for 6 hours to obtain light brown solid 6.4g, yield 95% ("the oily substance is directly put into the next reaction) M/z, 309.2[ M + H ] +, 1HNMR (400Mz, CDCl3), 1-2 (M,6H, CCH2CH2CH2C), 2.68(t,2H, CH2CO), 2.95(s,4H, COCH2CH 2CH2CO), 3.68(t,2H, CH2CH 2H, 2N, CH 2H 38, CH 2H N, CH.

EXAMPLE 3 Synthesis of Fmoc-Val-OSu

Fmoc-Val 10g and HOSu 3.4g were added to 100ml of THF. DCC6g was dissolved in 50ml of acetonitrile, and the internal temperature was kept at about 0 ℃ and slowly dropped into the reaction mixture. The reaction mixture was stirred at room temperature for 24 hours. Filtration, washing of the filter cake with THF, and concentration of the filtrate under reduced pressure gave a clear oil. The oil was directly subjected to the next reaction without purification. m/z: 437.4[ M + H ] +

Example 4 Synthesis of Fmoc-vc

To 20ml of THF were added 4.0g of Cit (1.05eq) and 60ml of aqueous sodium bicarbonate solution (NaHCO 32 g,1.05 eq). Another 22.35mmol of Fmoc-Val-OSu was dissolved in 60ml of DME and added to the reaction mixture. The reaction mixture was stirred at room temperature for 24 hours. After the reaction, 110ml of 15% citric acid aqueous solution was added to the system, followed by extraction twice with EA, and the organic layers were combined and concentrated under reduced pressure to give a white solid. And adding 100ml of methyl tert-butyl ether into the white solid, stirring, washing, filtering, and drying the filter cake at 40 ℃ under reduced pressure for 4 hours to obtain 4.83g of a product with the yield of 65%. m/z: 497.6(M + H) +. 1HNMR (400Mz, DMSO) is 0.92(6H, m), 1.35-1.65 (4H, m), 2.10(1H, m), 3.01(2H, q), 3.99(1H, t), 4.01-4.45(2H, m), 4.45(2H, t), 5.46(2H, br), 6.03(1H, t), 7.20-8.02(8H, m) and 8.25(1H, d).

Example 5 Synthesis of Fmoc-vc-PABOH

After 60ml of DCM/MeOH 2/1 mixed solvent was added to the reaction flask, 2g (4.2mmol) of Fmoc-vc and 1.04g (2eq) of PABOH were added, and EEDQ was added thereto after stirring the dissolved fractions to 2.0g (2 eq). The reaction system is stirred for reaction for 2.0d under the condition of room temperature and protection from light. After the reaction was completed, the reaction mixture was concentrated under reduced pressure at 40 ℃ to obtain a white solid. The white solid was collected, 100ml of methyl t-butyl ether was added, stirred, filtered, and the filter cake was washed with methyl t-butyl ether, and the resulting white solid was dried at 40 ℃ under reduced pressure to give 2.2g, with a yield of about 88%. m/z: 602.6(M + H) +. 1HNMR (400Mz, DMSO) is 0.95(6H, m), 1.45-1.69 (4H, m), 2.10(1H, m), 3.11(2H, m), 3.99(1H, m), 4.30(2H, d), 4.05-4.66 (2H, m), 4.55(2H, d), 5.21(1H, t), 5.51(2H, br), 6.11(1H, t), 7.09-8.10(12H, m), 8.21(1H, d), 10.51(1H, br).

Example 6 Synthesis of vc-PABOH

Fmoc-vc-PABOH 490mg (0.815mmol) was added to NMP in an amount of 10ml, and dissolved by stirring, followed by addition of diethylamine in an amount of 2 ml. The reaction was stirred at room temperature for 24 h. After the reaction was completed, the reaction mixture was concentrated under reduced pressure at 40 ℃ to obtain an oil, 20ml of DCM was added to the oil, followed by crystallization with stirring, filtration, washing of the cake with DCM, and drying of the obtained solid under reduced pressure to obtain 277mg, yield 90%. m/z: 380.2(M + H) +. 1HNMR (400Mz, DMSO):0.89(6H, m), 1.31-1.61 (4H, m),1.82(1H, m),2.86(1H, m),2.89(2H, d),4.38(2H, d),4.44(1H, m),5.01(1H, br),5.35(2H, br),5.84(1H, br),7.14(2H, d),7.42(2H, d),8.08(1H, br),9.88(1H, br).

Example 7 Synthesis of mc-vc-PABOH

To 10ml of NMP were added vc-PABOH 205mg (0.54mmol) and MC-OSu 184mg (1.1eq), and the reaction was stirred at room temperature for 24 hours. After the reaction, the mixture was concentrated under reduced pressure at 40 ℃ to obtain an oil, and 20ml of methyl t-butyl ether was added to the oil and the mixture was stirred for crystallization. Filtration and washing of the filter cake with methyl tert-butyl ether gave 310mg of product in 100% yield. M/z 573.3(M + H) +. 1HNMR (400Mz, DMSO) 0.89(6H, m), 1.15-1.99(10H, m), 2.11(1H, m), 2.31(2H, t), 3.21(2H, m), 3.53(2H, t), 4.32(1H, t), 4.51(1H, m), 4.59(2H, br), 5.24(1H, br), 5.56(2H, br), 6.20(1H, br), 7.12(2H, s), 7.23(2H, d),7.58(2H, d), 7.94(1H, d),8.17(1H, d), 10.21(1H, br).

Example 8 Synthesis of mc-vc-PAB-PNP

Under the protection of nitrogen, 168.6mg (0.294mmol) of mc-vc-PABOH is dissolved in 5ml of anhydrous pyridine, and the reaction system is cooled to about 0 ℃. Another PNP179mg (3eq) was dissolved in 5ml DCM and slowly added to the reaction. Keeping the temperature at about 0 ℃ for 10min, removing the ice bath, and stirring the mixture at room temperature for reaction for 3 h. After the reaction, 70ml of EA and 100ml of a 15% citric acid aqueous solution were added to the reaction solution, and the organic layer was separated. The organic layer was washed with citric acid, water and saturated brine in this order, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to give a pale yellow oil, which was crystallized by addition of methyl t-butyl ether to give an off-white solid of 86mg in a yield of 40%. M/z 738(M + H) +. 1HNMR (400Mz, CDCl3/CD3OD) 0.84(6H, m), 1.11-1.84(10H, m), 2.05(1H, m), 2.15(2H, t), 3.09(2H, m), 3.32(2H, t), 4.12(1H, m), 4.38(1H, m), 5.15(2H, s), 6.61(2H, s), 6.84(1H, d),7.61(1H, d), 7.21(2H, d),7.50(2H, d),7.61 (2H, d),8.18(2H, d), 9.59(1H, br).

Example 9 Synthesis of mc-vc-PAB-MMAE

20mg of mc-vc-PAB-PNP (1.5eq) and 3mg of HOBT were added to 2ml of DMF. After stirring at room temperature for a while, 13mg of MMAE, 0.5ml of pyridine and 25ul of DIEA were added. The reaction mixture was stirred at room temperature for 2 d. After the reaction is finished, the reaction solution is directly purified by a preparation column, the required components are collected, concentrated and freeze-dried to obtain about 10mg of the product, and the yield is about 42%. M/z 1317.1(M + H) +.

Example 10 Synthesis of mc-vc-PAB-MMAF

By following the procedure of example 9, approximately 12.5mg of mc-vc-PAB-MMAF were obtained in 45.2% yield, M/z:1331.7(M + H) +.

Example 11 Synthesis of mc-vc-PAB-PBD

By following the procedure of example 9, approximately 9.5mg of mc-vc-PAB-PBD was obtained in 32.5% yield, M/z:1325.4(M + H) +.

Example 12 Synthesis of mc-vc-PAB-DOX

By following the procedure of example 9, approximately 11.2mg of mc-vc-PAB-DOX was obtained in 38.9% yield, M/z:1143.2(M + H) +.

Example 14 Synthesis of mc-vc-PAB-SN-38

Dissolving 100mg of 10-O-Boc-SN-38 with 10ml of dried dichloromethane, adding 25.6mg (1eq) of DMAP, dropwise adding a dichloromethane solution of triphosgene at 0 ℃ (62mg of triphosgene is dissolved with 2ml of dichloromethane), continuing to react at 0 ℃ for 12H after dropwise adding, removing dichloromethane under reduced pressure, dissolving with 10ml of dried DMF, adding 144mg of mc-vc-PABOH, stirring at room temperature for 24H, and separating a prepared liquid phase to obtain mc-vc-PAB-SN-3841 mg, wherein the total yield of the two steps is 19.7%, and M/z is 992.1(M + H) +.

Example 15, 2C1 antibody expression and purification

Using Freestyle^TM293-F (Invitrogen) suspension cells expressing 2C1 antibody one day before transfection, cells were seeded at 6 × 105/m L density in 1L shake flasks containing 300m L F17 complete medium (Freestyl F17 expression medium, Gibco) at 37 ℃, 5% CO2, overnight on a cell culture shaker at 120rpm, and on the next day, transfection of an antibody expression plasmid was performed with PEI, wherein the PEI is added at a ratio of 2:1 one day after transfection, TN1 feed medium is added at 2.5% (v/v), and the supernatant is collected by centrifugation after further 4 days of culture.

The resulting cell expression supernatant was collected, eluted with 0.1M citric acid (pH3.0) through a Protein A affinity column (Mabselect Sure L X, GE Co.), the captured antibody was adjusted to pH7.0 with 1M Tris-HCl (pH9.0) at 1/10(v/v), and then purified by gel filtration chromatography (Superdex 200, GE Co.) to remove impurities such as multimer and endotoxin while replacing the antibody buffer with PBS (pH7.4), and a UV280nm target peak sample was collected and concentrated to 5mg/ml through an ultrafiltration tube (30KD, Pall Co.).

The 2C1 antibody obtained by this method, at a concentration of 5mg/ml, was greater than 98% of the antibody monomer of interest (POI%) for subsequent testing.

Example 16 preparation of 2C 1-L C-V205C-mc-vc-PAB-MMAE by coupling 2C1 antibody and mc-vc-PAB-MMAE 2C1 antibody expressed by cells of sample is purified by Mabselect Sure, eluted at low pH immediately neutralized with Tris solution and changed to Tris-HCl buffer solution of pH7.5 compound mc-vc-PAB-MMAE, white powder, dissolved in DMA for use in order to remove the shielding on the mutated cysteine residues, antibody reduction is first required, 1M aqueous solution of DTT is added to 2C1 antibody solution according to 20 fold molecular ratio, after mixing, reaction is carried out for 4 hours at 20 ℃, pH of sample is adjusted to Sepharose 5.0 after reaction time, DTT and shielding in sample is removed by SP harose f.f.f. cation exchange chromatography, DHAA solution is added to sample according to 10 fold molecular ratio, dhhc sample is reacted for 3 hours, after reaction is carried out again for 4 hours at 25 ℃ with Sepharose pH, mmf-mhe exchange reaction is carried out for removing the mutated chains of mmc-pac-PAB-MMAE after complete exchange reaction with MMAE, mmcs are added to obtain mmcs chain exchange antibody coupling, mmcs-spc, MMAE, mmcs-25, mmcs-aa-25, mmcs are added to obtain the mixture.

Example 17 preparation of 2C 1-L C-V205C-mc-vc-PAB-MMAF TDC samples by coupling 2C1 antibody and mc-vc-PAB-MMAF 2C 1-L C-V205C-mc-vc-PAB-MMAF TDC by coupling 2C1 antibody and mc-vc-PAB-MMAF following the procedure of example 16.

Example 18 preparation of a sample of 2C 1-L C-V205C-mc-vc-PAB-PBDTDC by coupling 2C1 antibody and mc-vc-PAB-PBD 2C 1-L C-V205C-mc-vc-PAB-PBDC was prepared by coupling 2C1 antibody and mc-vc-PAB-PBD according to the procedure of example 16.

Example 19 preparation of 2C 1-L C-V205C-mc-vc-PAB-SN38TDC samples by coupling 2C1 antibody and mc-vc-PAB-SN38 2C 1-L C-V205C-mc-vc-PAB-SN38TDC by coupling 2C1 antibody and mc-vc-PAB-SN38 according to the procedure of example 16.

Example 20 preparation of 2C 1-L C-V205C-mc-vc-PAB-DoxTDC samples by coupling 2C1 antibody and mc-vc-PAB-Dox 2C 1-L C-V205C-mc-vc-PAB-Dox TDC by coupling 2C1 antibody and mc-vc-PAB-Dox according to the procedure of example 16.

Example 21 preparation of 2A1-mc-vc-PAB-MMAE ADC samples by coupling 2A1 antibody and mc-vc-PAB-Dox

The 2A1 antibody expressed by the cells is purified by Mabselect Sure, and is neutralized by adding a Tris solution immediately after low pH elution, and the solution is changed into a Tris-HCl buffer solution with pH 7.5. Mc-vc-PAB-MMAE, white powder, which was dissolved in DMA for use. In order to open the interchain disulfide bonds of an antibody, the antibody needs to be reduced first. 1M DTT aqueous solution was added to 2A1 antibody solution at a molecular ratio of 20 times, and after mixing, the reaction was carried out at 20 ℃ for 4 hours. After the reaction time had elapsed, the pH of the sample was adjusted to 5.0 and DTT in the sample was removed by means of cation exchange chromatography on SPSepharose f.f. Then adding the mc-vc-PAB-MMAE solution to couple the mc-vc-PAB-MMAE with the cysteine residues of the opened interchain disulfide bonds, fully mixing uniformly, and reacting for 1 hour at 25 ℃. After the reaction was completed, mc-vc-PAB-MMAE not coupled with the antibody molecule was removed using SP Sepharose f.f. cation exchange chromatography. A2A 1-mc-vc-PAB-MMAE ADC sample was obtained.

Example 22 preparation of 2A1-mc-vc-PAB-MMAF ADC samples by coupling 2A1 antibody and mc-vc-PAB-MMAF

2A1-mc-vc-PAB-MMAF ADC was prepared by coupling 2A1 antibody and mc-vc-PAB-MMAF following the procedure of example 21.

Example 23 preparation of 2A1-mc-vc-PAB-PBD ADC samples by coupling 2A1 antibody and mc-vc-PAB-PBD

Following the procedure of example 21, 2A1-mc-vc-PAB-PBD ADC was prepared by coupling 2A1 antibody and mc-vc-PAB-PBD.

Example 24 preparation of 2A1-mc-vc-PAB-SN38ADC samples by coupling 2A1 antibody and mc-vc-PAB-SN38

Following the procedure of example 21, 2A1-mc-vc-PAB-SN38ADC was prepared by coupling 2A1 antibody and mc-vc-PAB-SN 38.

Example 25 preparation of 2A1-mc-vc-PAB-Dox ADC samples by coupling 2A1 antibody and mc-vc-PAB-Dox

2A1-mc-vc-PAB-Dox ADC was prepared by coupling 2A1 antibody and mc-vc-PAB-Dox according to the procedure of example 21.

Example 26 detection of toxin-to-antibody ratio of HIC-HP L C DAR

And analyzing TDC and ADC samples by a high performance liquid chromatography hydrophobic chromatography method, and calculating DAR according to corresponding peak areas. The specific method comprises the following steps:

a chromatographic column:

HICBu‐NP5(5μm,4.6x 35mm)；

mobile phase: a is 2M ammonium sulfate and 0.025M, pH7 phosphate buffer; b: 0.025M, pH7 phosphate buffer; c: 100% isopropyl alcohol;

buffer a equilibrated, gradient elution with buffer B and buffer C, detection at 25 ℃, 214nm DAR calculated as DAR ═ (0D area × 0+1D area × 1+2D area × 2)/(0D area +1D area +2D area).

In the attached figure 1, the HIC-HP L C detects 2C 1-L C-V205C-mc-vc-PAB-MMAE TDC DAR, the fixed-point coupling DAR is calculated according to the attached figure 1 and is 1.9, and the compound has good uniformity.

Attached table 1, 2C 1-L C-V205C-mc-vc-PAB-payload TDC and 2A1-mc-vc-PAB-payload ADC coupling efficiency DAR table

The attached table 1 shows that the TDC compounds subjected to site-specific coupling by cysteine modification have high coupling efficiency (the theoretical maximum value is 2.0), DAR is not less than 1.7, and the product uniformity is significantly better than the ADC compounds subjected to cysteine non-site-specific coupling by the disulfide bonds between the natural chains of the antibody (the DAR theoretical maximum value is 8.0).

Example 27 detection of TDC accumulation by SEC-HP L C

TDC samples were stored at 37 deg.C and analyzed for aggregation on

days

0, 7, and 21 by SEC-HP L C as follows:

column TSKgel SuperSW mAb HR (7.8mm × 30cm)

Mobile phase: 0.1M sodium sulfate, 0.1M phosphate buffer pH 6.7.

And detecting at 25 ℃ and 280 nm.

2A, SEC-HP L C detected 2C 1-L C-V205C-mc-vc-PAB-MMAE TDC aggregation, and the aggregate content was not substantially changed when the sample was stored at 37 ℃ for 3 weeks;

FIG. 2B, SEC-HP L C detects 2A1-mc-vc-PAB-MMAE ADC samples that were cysteine non-site coupled via the natural interchain disulfide bonds of the antibody, and the samples were detected immediately after coupling, with approximately 30% aggregates, including a large number of high molecular weight aggregates.

Appendix 2, 2C 1-L C-V205C-mc-vc-PAB-payload TDC, 2A1-mc-vc-PAB-payload ADC target monomer content list

The attached table 2 shows that the target monomer content of the ADC compound subjected to cysteine non-site-specific coupling by the natural interchain disulfide bond of the antibody is significantly lower than that of the TDC compound subjected to site-specific coupling by cysteine modification.

Example 28, TDC retains the affinity of the backbone antibody 2C1 to the original antibody 2A1 for EGFRvIII the relative affinity of TDC, 2C1 and 2A1 to EGFRvIII was compared by the indirect E L ISA method. the specific steps are as follows, recombinant EGFRvIII-His 6 antigen coating, fish skin gelatin blocking, dilution of 2A1, 2C1, 2C 1-L C-V205C-mc-vc-PAB-MMAE TDC, 2C 1-L C-V205C-mc-vc-PAB-MMAF-TDC, 2C 1-L C-V205C-mc-vc-PAB-PBD, 2C C-C C-C-V C-mc-vc-PAB-SN38, 2 EGFR72-C-mc-72-pac-PBD, 2C C-mc-pac-PAB-SN 38, 2C C-hcc-200-mC-hcc-PAPC-SN 38, the affinity of which is similar to the concentration of the antigen detected after incubation with the highest staining affinity of the antibody 2A, the absorbance gradient of the antibody 2A 5872 nm, the absorbance of the antibody, the absorbance of the antibody is similar to the absorbance of the antigen after the absorbance of the antibody is detected by the absorbance of the antibody 2A 50nm, the absorbance of the antibody.

FIGS. 3A, 2C 1-L C-V205C-mc-vc-PAB-MMAE TDC and 2C1 maintained the affinity of 2A1 for the antigen EGFRvIII.

FIG. 3B, 2C 1-L C-V205C-mc-vc-PAB-MMAE TDC, 2C 1-L C-V205C-mc-vc-PAB-MMAFTDC, 2C 1-L C-V205C-mc-vc-PAB-PBD TDC, 2C 1-L C-V205C-mc-vc-PAB-SN38TDC, 2C 1-L C-V205C-mc-vc-PAB-DOX TDC retains the affinity of 2A1 to the antigen EGFRvIII, and 2C 1-L C-V205C-mc-vc-PAB-DOX TDC can be coupled to a variety of small molecule cytotoxins at a site and retain antigen-antibody affinity.

Example 29 detection of cytotoxic drug Effect

The cytotoxic activity of TDC and ADC was determined by the following experimental procedure: TDC and ADC were added to EGFR-over-expressed or EGFRVIII-expressed human tumor cell culture medium, respectively, and cell viability was determined after 72 hours of cell culture. Cell-based in vitro experiments were used to determine cell viability, cytotoxicity and TDC-induced apoptosis of the invention.

The in vitro potency of the antibody-toxin conjugates was determined by a cell proliferation assay. CellTiter

The aquousOneresolution Cell promotion Assay is commercially available (Promega Corp., Madison, Wis.). CellTiter

The AQueous One Solution Cell Proliferation Assay (a) is a reagent for measuring the number of living cells in Cell Proliferation and cytotoxicity experiments by colorimetry. The reagent contains a novel tetrazolium compound [3- (4, 5-dimethylthiozol-2-yl) -5- (3-carboxymethyloxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt; MTS]And an electron coupling agent (PES). PES has enhanced chemical stability which allows it to be mixed with MTS to form a stable solution. This convenient "single solution" model was in the first generation CellTiter

Improvement on the basis of AQueous Assay, CellTiter

The electron coupling agent PMS used in the AQueous Assay is provided separately from the MTS solution. MTS (Owen's reagent) is bioreduced by cells into a colored formazan product that dissolves directly in the culture medium (lower panel). This conversion is most likely accomplished by the action of NADPH or NADH produced by dehydrogenases in metabolically active cells. When in detection, only a small amount of CellTiter is needed

The AQueous One Solution Reagent is directly added into the culture medium of a culture plate hole, incubated for 1-4 hours and then read the absorbance value of 490nm by a microplate reader.

The amount of formazan product detected at 490nm is directly proportional to the number of viable cells in culture. Since the formazan product of MTS is soluble in tissue culture medium, CellTiter

The AQueous One Solution Assay has fewer steps than the MTT or INT methods.

In the invention, A431(EGFR overexpression cells) and U87-EGFRVIII (EGFR mutant stable cell line) are adopted as a research system for in vitro drug effect detection. In 96-well plates, 6000/well cell plating was performed, and 24 hours later, antibody dosing was performed. The drug concentration for A431 was 2uM-3.8nM, two-fold dilution, and for U87-EGFRVIIII 2nM-0.4 pM. MTS measures cell viability 72 hours after treatment.

FIGS. 4A-4C, 2C 1-L C-V205C-mc-vc-PAB-MMAE TDC, 2C 1-L C-V205C-mc-vc-PAB-MMAFTDC, 2C 1-L C-V205C-mc-vc-PAB-PBD TDC and its corresponding 2A1-mc-vc-PAB-MMAE ADC, 2A1-mc-vc-PAB-MMAF ADC, and 2A1-mc-vc-PAB-PBD ADC have better IC50 detection results on EGFRwt over-expressed human skin squamous carcinoma cell A431.

FIG. 4D-4H, 2C 1-L C-V205C-mc-vc-PAB-MMAE TDC, 2C 1-L C-V205C-mc-vc-PAB-MMAFTDC, 2C 1-L C-V205C-mc-vc-PAB-PBD TDC, 2C 1-L C-V205C-mc-vc-PAB-SN38TDC, 2C 1-L C-V205C-mc-vc-PAB-Dox TDC and its corresponding 2A1-mc-vc-PAB-MMAE ADC, 2A1-mc-vc-PAB-MMAFADC, 2A 1-mc-pac-PAB-PBD ADC, 2A 1-mc-EGFB-PAB-38, 2A 5842-PAB-MMADC III, and the result of detecting the toxicity of the cells is better than the result of the PAvRvADC III.

Appendix 3, TDC, ADC on EGFRwt over-expression cell line A431 and EGFRvIII expression stability strain U87-EGFRvIII cytotoxicity IC₅₀And (6) detecting the result.

The results of the attached table 3 show that 2C 1-L C-V205C-mc-vc-PAB-MMAE TDC, 2C 1-L C-V205C-mc-vc-PAB-MMAF TDC, 2C 1-L C-V205C-mc-vc-PAB-PBD TDC, 2C 1-L C-V205C-mc-vc-PAB-SN38TDC, 2C 1-L C-V205C-mc-vc-PAB-Dox TDC have cytotoxicity activity superior to that of the corresponding 2A1-mc-vc-PAB-MMAE ADC, 2A 1-mc-vc-PAEGFB-ADC, 2A 5-mc-PAc-PAB-MMAE 23-PAB-PAC-PAB-24-PAc-PAB-24-PAC ADC, and PAC-PAB-24-PAC ADC, and PAC-24-PAC-PAB-24-PAB-ADC, and the test results of the drug effect of mice

A U87-EGFRvIII tumor-bearing mouse model is established in the invention to evaluate the in vivo efficacy of TDC and ADC conjugate drugs, namely 3 × U87-EGFRvIII cells are injected subcutaneously to two sides of a BA L B/C nude mouse aged 4-6 weeks, when the average size of tumors of the mouse grows to 65-88 mm3, the mice are randomly grouped, 5 mice in each group are subjected to single administration of 2C 1-L C-V205C-mc-vc-PAB-MMAE, 2C 1-L C-V205C-mc-vc-PAB-MMAF, 2C 2-L C-V205C-mc-vc-PAB-PBD, 2A 1-mc-vc-PAB-MMB-PBD, 2A 1-mc-pac-PAB-MMAF, 2A 1-mc-pac-PAB-PBD, 2A 87472-mc-L-PAC L-72-5-mSN 5C-L C L-6C-V-III cells are respectively subjected to single administration of tumor volume (PAA-L-mC-L-C-L-C-L-PAB-L-C-V-3-mC administration.

FIG. 5A, 2C 1-L C-V205C-mc-vc-PAB-MMAE, 2C 1-L C-V205C-mc-vc-PAB-MMAF, 2C 1-L C-V205C-mc-vc-PAB-PBD, 2A1-mc-vc-PAB-MMAE, 2A1-mc-vc-PAB-MMAF, 2A1-mc-vc-PAB-PBD on day 0, single intravenous administration with 1mg/kg dose respectively, TDC group (2C 1-L C-V205C-mc-vc-PAB-MMAE, 2C 1-L C-V205-C-mc-vc-PAB-MMAF, 2C 1-L C-V205-2-mc-PAB-MMAE-PBD) and 2C-MAC 848653-MMAE-PBD have more significant effect than PAvAF-PAB-PBA-PBD (PAv43-MMAD group) and PAvNO-PBB-PBD-2C-1).

FIG. 5B, 2C 1-L C-V205C-mc-vc-PAB-SN38, 2C 1-L C-V205C-mc-vc-PAB-DOX, 2A1-mc-vc-PAB-SN38, 2A1-mc-vc-PAB-DOX were administered in single intravenous administration at 6mg/kg dose on day 0, respectively, the tumor suppression effect of the TDC group (2C 1-L C-V205C-mc-vc-PAB-SN38, 2C 1-L C-V205C-mc-vc-PAB-DOX) was significantly better than that of the ADC group (2A1-mc-vc-PAB-SN38, 2A 1-mc-vc-PAB-DOX).

Example 31 toxicity testing in rats

In order to evaluate the cysteine engineered antibody-toxin conjugate tolerance, normal Sprague-Dawley rats were injected with TDC or ADC (day one) intravenously in a single dose, wherein 2C 1-L C-V205 2-mc-vc-PAB-MMAE, 2C 1-L C-V205C-mc-vc-PAB-MMAF, 2C 7378-6C-V205C-mc-vc-PAB-PBD, 2A1-mc-vc-PAB-MMAE, 2A1-mc-vc-PAB-MMAF, 2A1-mc-vc-PAB-PBD were administered at a dose of 50mg/kg, 2C 1-L C-V C-mc-PAB-SN 38, 2C 585-L C-V68624-pac-PAB-C-PAB-SN 5-596 mg/kg, 2C 596-D was sampled after the administration of PAC 596-CD, 2C 596-CD-PAB-596-PAB cells were collected and examined on the following day 100 (day 12) and the change of the blood count (FIG. 5-CD 596, 2A, 2C 596-CD-V6866, 2A, 2C 596-CD-D, 2A, the test was performed.

FIG. 6A, TDC shows the toxicity profile of ADC in rats, neutrophil change. On day 12, the neutrophil count was significantly higher in the ADC group than the control and TDC groups, showing that the ADC group was significantly more toxic than the TDC group.

FIG. 6B, TDC shows the change in AST levels in the liver in the ADC rat toxicity test. At day 12, AST levels in the liver of ADC group were significantly higher than those of control and TDC group, ADC group showed significant liver damage, and TDC group was significantly safer than ADC group.

FIG. 6C, TDC shows toxicity test in ADC rats and changes in body weight. In the first 5 days, the weight average of the ADC body continuously decreases, and gradually recovers in 5 days; the TDC group is significantly different from the control group, indicating that the TDC group is significantly safer than the ADC group.

The present invention is not to be limited in scope by the specific embodiments disclosed in the examples which are intended as illustrations of several aspects of the invention, any embodiment which is functionally equivalent being within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the claims appended hereto.

Sequence listing

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ACCTGCACTG TCACTGGCTA CTCAATCACC AGTGATTTTG CCTGGAACTG GATTCGGCAG

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TTTCCAGGAA ACAAGCTGGA GTGGATGGGC TACATAAGTT ATAGTGGTAA CACTAGGTAC

180

AACCCATCTC TCAAAAGTCG AATCTCTATC ACTCGCGACA CATCCAAGAA CCAATTCTTC

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CTGCAGTTGA ACTCTGTGAC TATTGAGGAC ACAGCCACAT ATTACTGTGT AACGGCGGGA

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CGCGGGTTTC CTTATTGGGG CCAAGGGACT CTGGTCACTG TCTCTGCA

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DVQLQESGPS LVKPSQSLSL TCTVTGYSITSDFAWNWIRQ FPGNKLEWMGYISYSGNTRY

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NPSLKSRISI TRDTSKNQFF LQLNSVTIED TATYYCVTAG RGFPYWGQGT LVTVSA

116

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GACATCCTGA TGACCCAATC TCCATCCTCC ATGTCTGTAT CTCTGGGAGA CACAGTCAGC

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ATCACTTGCC ATTCAAGTCA GGACATTAAC AGTAATATAG GGTGGTTGCA GCAGAGACCA

120

GGGAAATCAT TTAAGGGCCT GATCTATCAT GGAACCAACT TGGACGATGA AGTTCCATCA

180

AGGTTCAGTG GCAGTGGATC TGGAGCCGAT TATTCTCTCA CCATCAGCAG CCTGGAATCT

240

GAAGATTTTG CAGACTATTA CTGTGTACAG TATGCTCAGT TTCCGTGGAC GTTCGGTGGA

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GGCACCAAGC TGGAAATCAAA CGT

323

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DILMTQSPSS MSVSLGDTVS ITCHSSQDIN SNIGWLQQRP GKSFKGLIYH GTNLDDEVPS

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RFSGSGSGAD YSLTISSLES EDFADYYCVQ YAQFPWTFGG GTKLEIKR

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GACATCCTGA TGACCCAATC TCCATCCTCC ATGTCTGTAT CTCTGGGAGA CACAGTCAGC

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ATCACTTGCC ATTCAAGTCA GGACATTAAC AGTAATATAG GGTGGTTGCA GCAGAGACCA

120

GGGAAATCAT TTAAGGGCCT GATCTATCAT GGAACCAACT TGGACGATGA AGTTCCATCA

180

AGGTTCAGTG GCAGTGGATC TGGAGCCGAT TATTCTCTCA CCATCAGCAG CCTGGAATCT

240

GAAGATTTTG CAGACTATTA CTGTGTACAG TATGCTCAGT TTCCGTGGAC GTTCGGTGGA

300

GGCACCAAGC TGGAAATCAA ACGTACGGTG GCTGCACCAT CTGTCTTCAT CTTCCCGCCA

360

TCTGATGAGC AGTTGAAATC TGGAACTGCC TCTGTTGTGT GCCTGCTGAA TAACTTCTAT

420

CCCAGAGAGG CCAAAGTACA GTGGAAGGTG GATAACGCCC TCCAATCGGG TAACTCCCAG

480

GAGAGTGTCA CAGAGCAGGA CAGCAAGGAC AGCACCTACA GCCTCAGCAG CACCCTGACG

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CTGAGCAAAG CAGACTACGA GAAACACAAA GTCTACGCCT GCGAAGTCAC CCATCAGGGC

600

CTGAGCTCGC CCTGCACAAA GAGCTTCAAC AGGGGAGAGT GTTAG

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<213>2C1 light chain (L C) amino acid sequence

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DILMTQSPSS MSVSLGDTVS ITCHSSQDIN SNIGWLQQRP GKSFKGLIYH GTNLDDEVPS

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RFSGSGSGAD YSLTISSLES EDFADYYCVQ YAQFPWTFGG GTKLEIKRTV AAPSVFIFPP

120

SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT

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LSKADYEKHK VYACEVTHQG LSSPCTKSFN RGEC

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GACATCCTGA TGACCCAATC TCCATCCTCC ATGTCTGTAT CTCTGGGAGA CACAGTCAGC

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ATCACTTGCC ATTCAAGTCA GGACATTAAC AGTAATATAG GGTGGTTGCA GCAGAGACCA

120

GGGAAATCAT TTAAGGGCCT GATCTATCAT GGAACCAACT TGGACGATGA AGTTCCATCA

180

AGGTTCAGTG GCAGTGGATC TGGAGCCGAT TATTCTCTCA CCATCAGCAG CCTGGAATCT

240

GAAGATTTTG CAGACTATTA CTGTGTACAG TATGCTCAGT TTCCGTGGAC GTTCGGTGGA

300

GGCACCAAGC TGGAAATCAA ACGTACGGTG GCTGCACCAT CTGTCTTCAT CTTCCCGCCA

360

TCTGATGAGC AGTTGAAATC TGGAACTGCC TCTGTTGTGT GCCTGCTGAA TAACTTCTAT

420

CCCAGAGAGG CCAAAGTACA GTGGAAGGTG GATAACGCCC TCCAATCGGG TAACTCCCAG

480

GAGAGTGTCA CAGAGCAGGA CAGCAAGGAC AGCACCTACA GCCTCAGCAG CACCCTGACG

540

CTGAGCAAAG CAGACTACGA GAAACACAAA GTCTACGCCT GCGAAGTCAC CCATCAGGGC

600

CTGAGCTCGC CCGTCACAAA GAGCTTCAAC AGGGGAGAGT GTTAG

645

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<211>214

<212>PRT

<213>2A1 light chain (L C) amino acid sequence

<400>2

DILMTQSPSS MSVSLGDTVS ITCHSSQDIN SNIGWLQQRP GKSFKGLIYH GTNLDDEVPS

60

RFSGSGSGAD YSLTISSLES EDFADYYCVQ YAQFPWTFGG GTKLEIKRTV AAPSVFIFPP

120

SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT

180

LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

214

Claims

1. The cysteine-modified antibody-toxin conjugate has the following general formula 2C 1-L C-V205C-mc-vc-PAB-payload, wherein 2C1 is the antibody obtained by modifying valine (V) at position 205 of a target antibody light chain into cysteine (C), the amino acid sequence of a 2C1 heavy chain variable region is SEQ ID NO. 2, and the amino acid sequence of a light chain is SEQ ID NO. 12;

wherein the Payload is a small molecule highly active cytotoxin, including MMAE, MMAF, PBD, SN-38 or Dox;

the molecular formulas of MMAE, MMAF, PBD, SN-38 or Dox are respectively as follows:

。

2. the cysteine engineered antibody-toxin conjugate of claim 1, wherein: wherein the mc-vc-PAB-OH linker is:

。

3. the cysteine engineered antibody-toxin conjugate of claim 1, wherein: wherein the toxin-to-antibody ratio (DAR) is 1.6-2.0.

4. The cysteine engineered antibody-toxin conjugate of claim 2 wherein the amino acid sequence of the site-directed conjugation of the mc-vc-PAB linker to the antibody is G L SSPCTKSFN, wherein C is valine engineered cysteine at position 205 of the light chain of the antibody of interest.

5. The cysteine engineered antibody-toxin conjugate of claim 2, wherein: the 2C1 antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, Fab, and f (ab) 2.

6. The cysteine engineered antibody-toxin conjugate of claim 1, wherein: the indications are tumors expressing EGFRvIII and EGFRwt, including glia cerebri cancer, non-small cell lung cancer, head and neck cancer, colorectal cancer, bladder cancer, colorectal cancer, kidney cancer, prostate cancer, pancreatic cancer or breast cancer.

7. The cysteine engineered antibody-toxin conjugate of claim 1, wherein: the main fixed-point coupling steps are as follows: firstly, reducing an antibody by using DTT, removing shielding on a modified cysteine residue on the antibody, and removing the DTT and a shielding substance by cation exchange chromatography; the antibody is then oxidized using DHAA to reconnect the interchain disulfide bonds of the antibody; and finally adding mc-vc-PAB-payload to couple with the modified cysteine residue of the antibody, and removing the mc-vc-PAB-payload which is not coupled with the antibody molecule by cation exchange chromatography.