Cysteine modified antibody-drug conjugate and preparation method thereof
11484605 · 2022-11-01
Assignee
Inventors
- Yi Zhu (Chengdu, CN)
- Yixi Wang (Chengdu, CN)
- Shi Zhuo (Chengdu, CN)
- Jie Li (Chengdu, CN)
- Lan Chen (Chengdu, CN)
- Weili Wan (Chengdu, CN)
- Yongguo Yu (Chengdu, CN)
Cpc classification
C07K16/2863
CHEMISTRY; METALLURGY
A61K47/6803
HUMAN NECESSITIES
A61K47/6849
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
C07K16/00
CHEMISTRY; METALLURGY
C07K2317/94
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
By inserting cysteine (C) into a heavy chain and/or a light chain of a target antibody at specific insertion site, and performing a site-specific conjugation through a free thiol group (—SH) from the site-specific inserted cysteine and a linker conjugated with a highly potent small molecule cytotoxin, a cysteine modified antibody-drug conjugate with good homogeneity is provided. The specific insertion sites of cysteine are position 205 and/or position 206 (Kabat numbering scheme) of the light chain of the antibody, and/or position 439 (Kabat numbering scheme) of the heavy chain.
Claims
1. A cysteine modified antibody-cytotoxin conjugate, comprising an antibody and a cytotoxin, wherein the antibody comprises an inserted cysteine at a cysteine insertion site such that the antibody comprises a light chain comprising the amino acid sequence GLSSPCVTKSF (SEQ ID NO: 13), a light chain comprising the amino acid sequence GLSSPVCTKSF (SEQ ID NO: 14), or a heavy chain comprising the amino acid sequence TQKSLSCLSPGK (SEQ ID NO: 15), and wherein C is the inserted cysteine, wherein the inserted cysteine comprises a thiol group, w herein the cytotoxin is conjugated to the thiol group through a linker.
2. The cysteine modified antibody-cytotoxin conjugate of claim 1, w herein the antibody comprises a light chain, w herein the light chain comprises the amino acid sequence of SEQ ID NO: 13.
3. The cysteine modified antibody-cytotoxin conjugate of claim 1, wherein the antibody comprises a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 14.
4. The cysteine modified antibody-cytotoxin conjugate of claim 1, wherein the antibody comprises a heavy chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 15.
5. The cysteine modified antibody-cytotoxin conjugate of claim 1, wherein the cytotoxin is selected from Monomethyl auristatin E (MMAE), Monomethyl Auristatin F (MMAF), Pyrrolobenzodiazepines (PBD), antineoplastic drug SN-38, Doxycycline (Dox), or a derivative thereof, wherein the formulae of MMAE, MMAF, PBD, SN-38 and Dox are respectively: ##STR00016##
6. A method for preparing the cysteine modified antibody-cytotoxin conjugate of claim 1, comprising, reducing the antibody with a reducing agent to provide a reduced antibody, wherein the antibody comprises the inserted cysteine having a shielded thiol group, wherein the shielded thiol group comprises a shielding group coupled to the thiol group on the inserted cysteine, wherein the reducing the antibody with a reducing agent comprises removing the shielding group from the shielded thiol group to provide the reduced antibody having a free thiol group on the inserted cysteine and a decoupled shielding group, and wherein the decoupled shielding group and the reducing agent are removed through cation exchange chromatography or ultrafiltration, and oxidizing the reduced antibody to reconnect inter-chain disulfide bonds to provide an oxidized antibody, contacting mc-vc-PAB-payload comprising a cytotoxin moiety with the oxidized antibody to conjugate the free thiol group on the inserted cysteine with the cytotoxin moiety to provide the cysteine modified antibody-cytotoxin conjugate, and removing the unconjugated mc-vc-PAB-payload by cation exchange chromatography or ultrafiltration.
7. The cysteine modified antibody-cytotoxin conjugate of claim 1, wherein the cytotoxin and the antibody has a ratio of 1.6 to 2.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The foregoing and other features of this disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments arranged in accordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings, in which:
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
(26)
(27)
(28)
(29)
DETAILED DESCRIPTION OF THE EMBODIMENTS
(30) In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the FIGUREs, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.
Example 1: Synthesis and Preparation of mc
(31) ##STR00002##
(32) 6-aminocaproic acid (3.9 g, 0.03 mol) and maleic anhydride (3.5 g, 0.036 mol) were added to glacial acetic acid (30 ml). After stirring at 120° C. for 4-6 h, the reaction was cooled to room temperature. Most of the acetic acid was removed by concentration in vacuum at 60° C. The obtained brownish yellow viscous liquid was poured into water, and then extracted with ethyl acetate (20 ml×3), and the organic layers were combined. The organic layers were washed with water and brine, dried over anhydrous sodium sulfate, filtered and evaporated in vacuo to yield a brown-yellow oil, which was stirred in 50 ml of water, and white solid materials precipitated out of the solution, the white solid materials is filtered, and the product was dried under reduced pressure at 50° C., 5.08 g, yield 80%. Mp: 89-92° C. m/z: 212.2 [M+H]+. 1H NMR (400 Mz, DMSO): 13.21 (br, 1H, COOH), 6.75 (s, 2H, COCH═CHCO), 3.63 (t, 2H, J=7.2 Hz, NCH2CH2), 2.42 (t, 2H, J=7.4 Hz, CH2COOH), 1.52-1.68 (m, 4H, NCH2CH2CH2CH2), 1.30-1.42 (m, 2H, NCH2CH2CH2CH2).
Example 2: Synthesis and Preparation of Mc-OSu
(33) ##STR00003##
(34) Under nitrogen atmosphere, to a solution of a mixture of MC (4.7 g, 22 mmol) and HOSu (25 g, 22 mmol) in acetonitrile (50 mL) at 0° C. was slowly added DCC (4.5 g, 22 mmol) dissolved in 25 ml acetonitrile. The reaction solution was reacted at 0° C. for 2 hours and then allowed to reacted at room temperature overnight. After filtering, the filter cake was washed with acetonitrile (10 ml×3). The filtrate was concentrated to dry under reduced pressure. The obtained oil was dried under reduced pressure at room temperature for 6 h to afford 6.4 g of pale brown solid, and yield 95%. (To be used directly in the next step without purification) m/z: 309.2 [M+H]+. 1HNMR (400 Mz, CDCl3): 1˜2 (m, 6H, CCH2CH2CH2C), 2.68 (t, 2H, CH2CO, 2.95 (s, 4H, COCH2CH2CO), 3.68 (t, 2H, CH2N), 6.81 (s, 2H, CH═CH)
Example 3: Synthesis and Preparation of Fmoc-Val-OSu
(35) ##STR00004##
(36) To a solution of a mixture of Fmoc-Val (10 g) and HOSu (3.4 g) in THF (100 mL) at 0° C. was slowly added DCC (6 g) dissolved in 50 ml acetonitrile. The reaction solution was stirred at room temperature for 24 hours. Perform filtration, and the filter cake was washed with THF. A transparent oil was obtained by concentrating the filtrates under reduced pressure. The oil was directly used in the next step directly without further purification. m/z: 474.4 [M+H]+.
Example 4: Synthesis and Preparation of Fmoc-vc
(37) ##STR00005##
(38) To a solution of Cit (4.0 g) in THF (20 mL) was added a solution of 60 ml aqueous sodium hydrogencarbonate (containing NaHCO.sub.3 2 g, 1.05 eq). A solution of Fmoc-Val-OSu (22.35 mmol) in DME (60 mL) was added to the mixture. After stirred at room temperature for 24 hours, the reaction was added a solution of 15% aqueous citric acid solution (110 ml), and then extracted with EtOAc twice. The combined organic phases were concentrated in vacuum to get a white solid. 100 ml of methyl tert-butyl ether was added to the white material, the mixture was stirred, filtered, and the filter cake was dried under reduced pressure at 40° C. for 4 h to obtain the product 4.83 g, and yield 65%. m/z: 497.6 (M+H)+. 1HNMR (400 Mz, DMSO): 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), 8.25 (1H, d).
Example 5: Synthesis and Preparation of Fmoc-vc-PABOH
(39) ##STR00006##
(40) To a solution of Fmoc-vc (2 g, 4.2 mmol) and PABOH (1.04 g, 2 eq) in DCM/MeOH=2/1 (60 mL) was added EEDQ (2.0 g, 2 eq) at 0° C. After stirred for 10 min, a solution of (S)-1-phenylethanamine (17.5 g, 144.2 mmol) in MeOH (200 mL) was added slowly to the mixture after partial dissolution. The reaction system was stirred at room temperature for 2 days in the dark. After completion of the reaction, the mixture was concentrated in vacuum at 40° C. to yield a white solid. The white solid was collected, washed with methyl tert-butyl ether (100 ml), and filtered. The filter cake was washed with methyl tert-butyl ether, and the obtained white solid was dried under reduced pressure at 40° C. to give the white solid 2.2 g, and yield 88%. m/z: 602.6 (M+H)+. .sup.1HNMR (400 Mz, DMSO): 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 and Preparation of vc-PABOH
(41) ##STR00007##
(42) To a solution of Fmoc-vc-PABOH (490 mg, 0.815 mmol) in THF (10 mL) was added diethylamine (2 ml). The reaction mixture was stirred at room temperature for 24 h. 20 ml of DCM was added to the obtained product, the mixture was stirred, and crystalline was precipitated out of reaction solution. Filter the crystalline and the filter cake was washed with DCM, and the obtained solid was dried under reduced pressure to yield 277 mg. The yield was 90%. m/z: 380.2 (M+H)+. 1HNMR (400 Mz, 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 and Preparation of mc-vc-PABOH
(43) ##STR00008##
(44) VP-PABOH (205 mg, 0.54 mmol) and MC-OSu (184 mg, 1.1 eq) were added to 10 ml of NMP, and the reaction was stirred at room temperature for 24 h. After completion of the reaction, the mixture was concentrated in vacuo at 40° C. Methyl tert-butyl ether (20 ml) was added to the obtained oil and stirred to crystallization. After filtering the crystalline and washing the filter cake with methyl tert-butyl ether, the product was yielded at 310 mg. The yield is 100%. m/z: 573.3 (M+H)+. 1HNMR (400 Mz, 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 and Preparation of mc-vc-PAB-PNP
(45) ##STR00009##
(46) Under nitrogen, to a solution of mc-vc-PABOH (166.0 mg, 0.294 mmol) in anhydrous pyridine (5 ml) was added PNP (179 mg, 3 eq) dissolved in DCM (5 ml) at 0° C. slowly. After stirring at about 0° C. for 10 min, the ice bath was removed, and the reaction was stirred at room temperature for 3 h. After completion of the reaction, EA (70 ml) and a 15% aqueous citric acid solution (100 ml) were added, and the organic layer was separated and recovered. The organic layer was sequentially washed with citric acid, water, brine, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to yield light yellowish oily product. Adding methyl tert-butyl ether for crystallization resulted in the white-like solid (86 mg). The yield was 40%. m/z: 738 (M+H)+. 1HNMR (400 Mz, 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 and Preparation of mc-vc-PAB-MMAE
(47) ##STR00010##
(48) 20 mg of mc-vc-PAB-PNP (1.5 eq) and 3 mg of HOBT were added to 2 ml of DMF. After stirring at room temperature for a moment, 13 mg of MMAE, 0.5 ml of pyridine, and 25 ul of DIEA were added. The reaction solution was stirred at room temperature for 2 d. After the reaction is completed, the reaction solution is directly purified by a preparative column, and the desired components are collected, concentrated, and lyophilized to obtain about 10 mg of a product, and the yield is about 42%. m/z: 1317.1 (M+H)+.
Example 10: Synthesis and Preparation of mc-vc-PAB-MMAF
(49) ##STR00011##
(50) Operate according to the steps of Example 9, about 12.5 mg of mc-vc-PAB-MMAF was obtained, and the yield was 45.2%.
Example 11: Synthesis and Preparation of mc-vc-PAB-PBD
(51) ##STR00012##
(52) Operate according to the steps of Example 9, about 9.5 mg of mc-vc-PAB-PBD was obtained. The yield was 32.5%. m/z: 1325.4 (M+H)+.
Example 12: Synthesis and Preparation of mc-vc-PAB-DOX
(53) ##STR00013##
(54) Operate according to the steps of Example 9, about 11.2 mg of mc-vc-PAB-DOX was obtained. The yield was 38.9%. m/z: 1143.2 (M+H)+.
Example 14: Synthesis and Preparation of mc-vc-PAB-SN-38
(55) ##STR00014##
(56) 100 mg of 10-O-Boc-SN-38 was dissolved in 10 ml of dry dichloromethane, 25.6 mg (1 eq) of DMAP was added to the solvent, and a solution of triphosgene in dichloromethane was added dropwise at 0° C. (62 mg of triphosgene was dissolved in 2 ml of Dichloromethane), and the reaction was continued at 0° C. for 12 h. The dichloromethane was removed under reduced pressure. The crude products were dissolved in 10 ml of dry DMF, 144 mg of mc-vc-PABOH was then added, and the mixture was stirred at room temperature for 24 h. 41 mg of mc-vc-PAB-SN-38 was isolated by preparation liquid phase separation, and the total yield in two steps was 19.7%. m/z: 1063.2 (M+H)+.
Example 15: Target Antibody Expression and Purification
(57) The target antibody was expressed using Freestyle™ 293-F (Invitrogen) suspension cells. One day before transfection, cells were seeded at a density of 6×10.sup.5 cells/mL in a 1 L shake flask containing 300 mL of F17 complete medium (Freestyle™ F17 expression medium, Gibco), grew overnight by shaken at 37° C., 5% CO.sub.2, 120 rpm at cell incubator. The next day, transfection of the antibody expression plasmid was carried out with PEI, wherein the ratio of plasmid:PEI was 2:1. One day after the transfection, the TN1 feed medium was added at 2.5% (v/v), and the culture was continued for 4 days, and the supernatant was collected by centrifugation.
(58) The collected cell expression supernatant was eluted by a Protein An affinity chromatography column (Mabselect Sure LX, GE) eluting with 0.1 M citric acid (pH 3.0), and the captured antibody was treated with 1 M Tris-HCl (pH 9.0) and adjusted to pH 7.0 at 1/10 (v/v). Remove impurities such as multimers and endotoxin by gel filtration column SEC (Superdex 200, GE), and replace the antibody buffer with PBS (pH 7.4) at the same time, a sample of the target peak of UV280 nm was collected and concentrated to 2 mg/ml through an ultrafiltration centrifuge tube (30 KD, Pall Corporation). The target antibody monomer (PO %) obtained by this method was greater than 90% and was stored for subsequent experiments.
Example 16: Synthesis and Preparation of 2A1-HC-Cys474ins-mc-vc-PAB-MMAE TDC by Conjugating/Coupling 2A1-HC-Cys474ins Antibody and mc-vc-PAB-MMAE
(59) The 2A1-HC-Cys474ins antibody expressed by the cells was purified by Protein A resin such as Mabselect Sure, eluted with low pH solution and neutralized by adding Tris solution immediately after the low pH elution, and the solution was changed to a pH 7.5 Tris-HCl buffer. The mc-vc-PAB-MMAE compound, being a white powder, was dissolved in DMA for use. In order to remove the masking group on the mutant cysteine residue, the antibody was reduced first. A 1 M aqueous solution of DTT was added to the 2A1-HC-Cys474ins antibody solution at a molecular ratio of 1:40, and the mixture was mixed evenly and reacted at 20° C. for 2 hours. After the reaction time was reached, the pH of the sample was adjusted to 5.0, and the DTT and the masking group in the mixture were removed by cation exchange chromatography such as SP Sepharose F.F. resin. Subsequently, a DHAA solution was added to the sample at a molecular ratio of 1:20 and reacted at 25° C. for 4 hours in the dark to re-connect the interchain disulfide bonds. subsequently, mc-vc-PAB-MMAE solution was added to couple the mc-vc-PAB-MMAE with the inserted or mutant cysteine in the antibody, and the mixture was thoroughly mixed and reacted at 25° C. for 2 hours. After the end of the reaction, mc-vc-PAB-MMAE to which the antibody was not coupled was removed using cation exchange chromatography such as SP Sepharose F.F. to obtain a 2A1-HC-Cys474ins-mc-vc-PAB-MMAE TDC sample.
Example 17: Synthesis and Preparation of 2A1-LC-Cys205ins-mc-vc-PAB-MMAE TDC Sample by Conjugating/Coupling 2A1-LC-Cys205ins Antibody and mc-vc-PAB-MMAE
(60) The 2A1-LC-Cys205ins antibody expressed by the cells was purified by Protein A resin such as Mabselect Sure, eluted with low pH solution and neutralized by adding Tris solution immediately after the low pH elution, and the solution was changed to a pH 7.5 Tris-HCl buffer. The mc-vc-PAB-MMAE compound, being a white powder, was dissolved in DMA for use. In order to remove the masking group on the mutant cysteine residue, the antibody was reduced first. A 1 M aqueous solution of DTT was added to the 2A1-LC-Cys205ins antibody solution at a molecular ratio of 1:40, and the mixture was mixed evenly and reacted at 20° C. for 2 hours. After the reaction time was reached, the pH of the sample was adjusted to 5.0, and the DTT and the masking group in the mixture were removed by cation exchange chromatography such as SP Sepharose F.F. resin. Subsequently, a DHAA solution was added to the sample at a molecular ratio of 1:20 and reacted at 25° C. for 4 hours in the dark to re-connect the interchain disulfide bonds. subsequently, mc-vc-PAB-MMAE solution was added to couple the mc-vc-PAB-MMAE with the inserted or mutant cysteine in the antibody, and the mixture was thoroughly mixed and reacted at 25° C. for 2 hours. After the end of the reaction, mc-vc-PAB-MMAE to which the antibody was not coupled was removed using cation exchange chromatography such as SP Sepharose F.F. to obtain a 2A1-LC-Cys205ins-mc-vc-PAB-MMAE TDC sample.
Example 18: Synthesis and Preparation of 2A1-LC-Cys206ins-mc-vc-PAB-MMAE TDC Sample by Conjugating/Coupling 2A1-LC-Cys206ins Antibody and mc-vc-PAB-MMAE
(61) The 2A1-LC-Cys206ins antibody expressed by the cells was purified by Protein A resin such as Mabselect Sure, eluted with low pH solution and neutralized by adding Tris solution immediately after the low pH elution, and the solution was changed to a pH 7.5 Tris-HCl buffer. The mc-vc-PAB-MMAE compound, being a white powder, was dissolved in DMA for use. In order to remove the masking group on the mutant cysteine residue, the antibody was reduced first. A 1 M aqueous solution of DTT was added to the 2A1-LC-Cys206ins antibody solution at a molecular ratio of 1:40, and the mixture was mixed evenly and reacted at 20° C. for 2 hours. After the reaction time was reached, the pH of the sample was adjusted to 5.0, and the DTT and the masking group in the mixture were removed by cation exchange chromatography such as SP Sepharose F.F. resin. Subsequently, a DHAA solution was added to the sample at a molecular ratio of 1:20 and reacted at 25° C. for 4 hours in the dark to re-connect the interchain disulfide bonds. subsequently, mc-vc-PAB-MMAE solution was added to couple the mc-vc-PAB-MMAE with the inserted or mutant cysteine in the antibody, and the mixture was thoroughly mixed and reacted at 25° C. for 2 hours. After the end of the reaction, mc-vc-PAB-MMAE to which the antibody was not coupled was removed using cation exchange chromatography such as SP Sepharose F.F. to obtain a 2A1-LC-Cys206ins-mc-vc-PAB-MMAE TDC sample.
Example 19: Synthesis and Preparation of 4D3-HC-Cys474ins-mc-vc-PAB-MMAE TDC Sample by Conjugating/Coupling 4D3-HC-Cys474ins Antibody and mc-vc-PAB-MMAE
(62) The 4D3-HC-Cys474ins antibody expressed by the cells was purified by Protein A resin such as Mabselect Sure, eluted with low pH solution and neutralized by adding Tris solution immediately after the low pH elution, and the solution was changed to a pH 7.5 Tris-HCl buffer. The mc-vc-PAB-MMAE compound, being a white powder, was dissolved in DMA for use. In order to remove the masking group on the mutant cysteine residue, the antibody was reduced first. A 1 M aqueous solution of DTT was added to the 4D3-HC-Cys474ins antibody solution at a molecular ratio of 1:40, and the mixture was mixed evenly and reacted at 20° C. for 2 hours. After the reaction time was reached, the pH of the sample was adjusted to 5.0, and the DTT and the masking group in the mixture were removed by cation exchange chromatography such as SP Sepharose F.F. resin.
(63) Subsequently, a DHAA solution was added to the sample at a molecular ratio of 1:20 and reacted at 25° C. for 4 hours in the dark to re-connect the interchain disulfide bonds. subsequently, mc-vc-PAB-MMAE solution was added to couple the mc-vc-PAB-MMAE with the inserted or mutant cysteine in the antibody, and the mixture was thoroughly mixed and reacted at 25° C. for 2 hours. After the end of the reaction, mc-vc-PAB-MMAE to which the antibody was not coupled was removed using cation exchange chromatography such as SP Sepharose F.F. to obtain a 4D3-HC-Cys474ins-mc-vc-PAB-MMAE TDC sample.
Example 20: Synthesis and Preparation of 4D3-LC-Cys205ins-mc-vc-PAB-MMAE TDC Sample by Conjugating/Coupling 4D3-LC-Cys205ins Antibody and mc-vc-PAB-MMAE
(64) The 4D3-LC-Cys205ins antibody expressed by the cells was purified by Protein A resin such as Mabselect Sure, eluted with low pH solution and neutralized by adding Tris solution immediately after the low pH elution, and the solution was changed to a pH 7.5 Tris-HCl buffer. The mc-vc-PAB-MMAE compound, being a white powder, was dissolved in DMA for use. In order to remove the masking group on the mutant cysteine residue, the antibody was reduced first. A 1 M aqueous solution of DTT was added to the 4D3-LC-Cys205ins antibody solution at a molecular ratio of 1:40, and the mixture was mixed evenly and reacted at 20° C. for 2 hours. After the reaction time was reached, the pH of the sample was adjusted to 5.0, and the DTT and the masking group in the mixture were removed by cation exchange chromatography such as SP Sepharose F.F. resin. Subsequently, a DHAA solution was added to the sample at a molecular ratio of 1:20 and reacted at 25° C. for 4 hours in the dark to re-connect the interchain disulfide bonds. subsequently, mc-vc-PAB-MMAE solution was added to couple the mc-vc-PAB-MMAE with the inserted or mutant cysteine in the antibody, and the mixture was thoroughly mixed and reacted at 25° C. for 2 hours. After the end of the reaction, mc-vc-PAB-MMAE to which the antibody was not coupled was removed using cation exchange chromatography such as SP Sepharose F.F. to obtain a 4D3-LC-Cys205ins-mc-vc-PAB-MMAE TDC sample.
Example 21: Synthesis and Preparation of 4D3-LC-Cys206ins-mc-vc-PAB-MMAE TDC Sample by Conjugating/Coupling 4D3-LC-Cys206ins Antibody and mc-vc-PAB-MMAE
(65) The 4D3-LC-Cys206ins antibody expressed by the cells was purified by Protein A resin such as Mabselect Sure, eluted with low pH solution and neutralized by adding Tris solution immediately after the low pH elution, and the solution was changed to a pH 7.5 Tris-HCl buffer. The mc-vc-PAB-MMAE compound, being a white powder, was dissolved in DMA for use. In order to remove the masking group on the mutant cysteine residue, the antibody was reduced first. A 1 M aqueous solution of DTT was added to the 4D3-LC-Cys206ins antibody solution at a molecular ratio of 1:40, and the mixture was mixed evenly and reacted at 20° C. for 2 hours. After the reaction time was reached, the pH of the sample was adjusted to 5.0, and the DTT and the masking group in the mixture were removed by cation exchange chromatography such as SP Sepharose F.F. resin. Subsequently, a DHAA solution was added to the sample at a molecular ratio of 1:20 and reacted at 25° C. for 4 hours in the dark to re-connect the interchain disulfide bonds. subsequently, mc-vc-PAB-MMAE solution was added to couple the mc-vc-PAB-MMAE with the inserted or mutant cysteine in the antibody, and the mixture was thoroughly mixed and reacted at 25° C. for 2 hours. After the end of the reaction, mc-vc-PAB-MMAE to which the antibody was not coupled was removed using cation exchange chromatography such as SP Sepharose F.F. to obtain a 4D3-LC-Cys206ins-mc-vc-PAB-MMAE TDC sample.
Example 22: Synthesis and Preparation of 4E1-HC-Cys474ins-mc-vc-PAB-MMAE TDC Sample by Conjugating/Coupling 4E1-HC-Cys474ins Antibody and mc-vc-PAB-MMAE
(66) The 4E1-HC-Cys474ins antibody expressed by the cells was purified by Protein A resin such as Mabselect Sure, eluted with low pH solution and neutralized by adding Tris solution immediately after the low pH elution, and the solution was changed to a pH 7.5 Tris-HCl buffer. The mc-vc-PAB-MMAE compound, being a white powder, was dissolved in DMA for use. In order to remove the masking group on the mutant cysteine residue, the antibody was reduced first. A 1 M aqueous solution of DTT was added to the 4E1-HC-Cys474ins antibody solution at a molecular ratio of 1:40, and the mixture was mixed evenly and reacted at 20° C. for 2 hours. After the reaction time was reached, the pH of the sample was adjusted to 5.0, and the DTT and the masking group in the mixture were removed by cation exchange chromatography such as SP Sepharose F.F. resin. Subsequently, a DHAA solution was added to the sample at a molecular ratio of 1:20 and reacted at 25° C. for 4 hours in the dark to re-connect the interchain disulfide bonds. subsequently, mc-vc-PAB-MMAE solution was added to couple the mc-vc-PAB-MMAE with the inserted or mutant cysteine in the antibody, and the mixture was thoroughly mixed and reacted at 25° C. for 2 hours. After the end of the reaction, mc-vc-PAB-MMAE to which the antibody was not coupled was removed using cation exchange chromatography such as SP Sepharose F.F. to obtain a 4E1-HC-Cys474ins-mc-vc-PAB-MMAE TDC sample.
Example 23: Synthesis and Preparation of 4E1-LC-Cys205ins-mc-vc-PAB-MMAE TDC Sample by Conjugating/Coupling 4E1-LC-Cys205ins Antibody and mc-vc-PAB-MMAE
(67) The 4E1-LC-Cys205ins antibody expressed by the cells was purified by Protein A resin such as Mabselect Sure, eluted with low pH solution and neutralized by adding Tris solution immediately after the low pH elution, and the solution was changed to a pH 7.5 Tris-HCl buffer. The mc-vc-PAB-MMAE compound, being a white powder, was dissolved in DMA for use. In order to remove the masking group on the mutant cysteine residue, the antibody was reduced first. A 1 M aqueous solution of DTT was added to the 4E1-LC-Cys205ins antibody solution at a molecular ratio of 1:40, and the mixture was mixed evenly and reacted at 20° C. for 2 hours. After the reaction time was reached, the pH of the sample was adjusted to 5.0, and the DTT and the masking group in the mixture were removed by cation exchange chromatography such as SP Sepharose F.F. resin. Subsequently, a DHAA solution was added to the sample at a molecular ratio of 1:20 and reacted at 25° C. for 4 hours in the dark to re-connect the interchain disulfide bonds. subsequently, mc-vc-PAB-MMAE solution was added to couple the mc-vc-PAB-MMAE with the inserted or mutant cysteine in the antibody, and the mixture was thoroughly mixed and reacted at 25° C. for 2 hours. After the end of the reaction, mc-vc-PAB-MMAE to which the antibody was not coupled was removed using cation exchange chromatography such as SP Sepharose F.F. to obtain a 4E1-LC-Cys205ins-mc-vc-PAB-MMAE TDC sample.
Example 24: Synthesis and Preparation of 4E1-LC-Cys206ins-mc-vc-PAB-MMAE TDC Sample by Conjugating/Coupling 4E1-LC-Cys206ins Antibody and mc-vc-PAB-MMAE
(68) The 4E1-LC-Cys206ins antibody expressed by the cells was purified by Protein A resin such as Mabselect Sure, eluted with low pH solution and neutralized by adding Tris solution immediately after the low pH elution, and the solution was changed to a pH 7.5 Tris-HCl buffer. The mc-vc-PAB-MMAE compound, being a white powder, was dissolved in DMA for use. In order to remove the masking group on the mutant cysteine residue, the antibody was reduced first. A 1 M aqueous solution of DTT was added to the 4E1-LC-Cys206ins antibody solution at a molecular ratio of 1:40, and the mixture was mixed evenly and reacted at 20° C. for 2 hours. After the reaction time was reached, the pH of the sample was adjusted to 5.0, and the DTT and the masking group in the mixture were removed by cation exchange chromatography such as SP Sepharose F.F. resin. Subsequently, a DHAA solution was added to the sample at a molecular ratio of 1:20 and reacted at 25° C. for 4 hours in the dark to re-connect the interchain disulfide bonds. subsequently, mc-vc-PAB-MMAE solution was added to couple the mc-vc-PAB-MMAE with the inserted or mutant cysteine in the antibody, and the mixture was thoroughly mixed and reacted at 25° C. for 2 hours. After the end of the reaction, mc-vc-PAB-MMAE to which the antibody was not coupled was removed using cation exchange chromatography such as SP Sepharose F.F. to obtain a 4E1-LC-Cys206ins-mc-vc-PAB-MMAE TDC sample.
Example 25: Measurement of Toxin:Antibody Ratio (DAR, Drug Antibody Ratio) by HIC-HPLC
(69) The TDC sample was analyzed by high performance liquid chromatography with hydrophobic chromatography, and drug:antibody ratio (DAR, also known as toxin:antibody ratio) was calculated from the corresponding peak area. One specific method is described in detail as follows:
(70) Column: Proteomix® HICBu-NP5 (5 μm, 4.6×35 mm);
(71) Mobile phase: Buffer A: 2M ammonium sulfate, 0.025 M, pH 7 phosphate buffer; Buffer B: 0.025 M, pH 7 phosphate buffer; Buffer C: 100% isopropanol;
(72) Buffer A was used for equilibration, Buffer B and buffer C were used for gradient elution, detection was performed at 25° C., 214 nm and 280 wavelengths. Based on data gathered from
Example 26: Measurement of Toxin:Antibody Ratio (DAR, Drug Antibody Ratio) by RP-HPLC
(73) The ratio of toxin to antibody was measured by RP-HPLC. The samples treated with DTT were analyzed by reversed-phase hydrophobic high-performance liquid chromatography, and DAR was calculated from the corresponding peak area. One specific method is described in detail as follows:
(74) Column: Proteomix RP-1000 (5 μm, 4.6×100 mm)
(75) Mobile phase: Buffer A: 0.1% TFA aqueous solution; Buffer B: 0.1% acetonitrile solution.
(76) Mobile phase A and mobile phase B were used to elute in a proportional gradient at 80° C., measurement was performed at 214 nm and 280 wavelengths. Based on data gathered in
(77) TABLE-US-00006 TABLE I Coupling Efficiency DAR List for ADRs: 2A1-LC-V205C- mc-vc-PAB-MMAE TDC, 2A1-LC-Cys205ins-mc-vc-PAB- MMAE TDC, 2A1-LC-Cys206ins--mc-vc-PAB-MMAE TDC, 2A1-HC-Cys439ins-mc-vc-PAB-MMAE, 4E1-LC-Cys205ins- mc-vc-PAB-MMAE, 4E1-LC-Cys206ins-mc-vc-PAB-MMAE, 4E1-HC-Cys439ins-mc- vc-PAB-MMAE TDC, 4D3-LC-Cys205ins- mc-vc-PAB-MMAE, 4D3-LC-Cys206ins-mc-vc-PAB-MMAE, 4D3-HC-Cys439ins-mc-vc-PAB-MMAE Compounds DAR Site- 2A1-LC-V205C-mc-vc-PAB-MMAE TDC 1.81 specific 2A1-LC-Cys205ins-mc-vc-PAB-MMAE TDC 1.72 coupling 2A1-LC-Cys206ins-mc-vc-PAB-MMAE TDC 1.65 (TDC) 2A1-HC-Cys439ins-mc-vc-PAB-MMAE TDC 1.74 4E1-LC-Cys205ins-mc-vc-PAB-MMAE TDC 1.92 4E1-LC-Cys206ins-mc-vc-PAB-MMAE TDC 1.64 4E1-HC-Cys439ins-mc-vc-PAB-MMAE TDC 1.75 4D3-LC-Cys205ins-mc-vc-PAB-MMAE TDC 1.81 4D3-LC-Cys206ins-mc-vc-PAB-MMAE TDC 1.74 4D3-HC-Cys439ins-mc-vc-PAB-MMAE TDC 1.82
(78) TABLE 1 shows that the coupling efficiency of site-directed TDC compounds by cysteine insertion mutation modification is uniformly high (theoretical maximum is 2.0), with DAR≥1.6.
Example 27: Measurement of TDC Antibody Skeleton Aggregation by SEC-HPLC
(79) TDC antibody skeleton samples were stored at 37° C., and their aggregation was analyzed by SEC-HPLC on days 0, 7, 21, and 29, respectively. One specific method is described in detail as follows:
(80) Chromatography columns: TSKgel SuperSW mAb HR (7.8 mm×30 cm),
(81) Mobile phase: 0.1 M sodium sulfate, 0.1 M, pH 6.7 phosphate buffer,
(82) Measurements were performed at 25° C., 280 nm.
(83) As shown in
(84) Using the same detecting and measurement method, the aggregations of the following TDCs are measured: 2A1-LC-V205C-mc-vc-PAB-MMAE TDC, 2A1-LC-Cys205ins-mc-vc-PAB-MMAE TDC, 2A1-LC-Cys206ins-mc-vc-PAB-MMAE TDC, 2A1-HC-Cys474ins-mc-vc-PAB-MMAE, 4E1-LC-Cys205ins-mc-vc-PAB-MMAE, 4E1-LC-Cys206ins-mc-vc-PAB-MMAE 4E1-HC-Cys474ins-mc-vc-PAB-MMAE TDC, 4D3-LC-Cys205ins-mc-vc-PAB-MMAE, 4D3-LC-Cys206ins-mc-vc-PAB-MMAE, 4D3-HC-Cys474ins-mc-vc-PAB-MMAE TDC. The results are shown in TABLE II
(85) TABLE-US-00007 TABLE II TDS target monomer content list for 2A1-LC-V205C- mc-vc-PAB-MMAE TDC, 2A1-LC-Cys205ins-mc-vc-PAB-MMAE TDC, 2A1-LC-Cys206ins--mc-vc-PAB-MMAE TDC and 2A1- HC-Cys439ins-mc-vc-PAB-MMAE, 4E1-LC-Cys205ins-mc- vc-PAB-MMAE, 4E1-LC-Cys206ins-mc-vc-PAB-MMAE, 4E1- HC-Cys439ins-mc-vc-PAB-MMAE TDC, 4D3-LC-Cys205ins- mc-vc-PAB-MMAE, 4D3-LC-Cys206ins-mc-vc-PAB-MMAE, 4D3-HC-Cys439ins-mc-vc-PAB-MMAE Compound POI % Site- 2A1-LC-V205C-mc-vc-PAB-MMAE TDC 96.0% specific 2A1-LC-Cys205ins-mc-vc-PAB-MMAE TDC 90.0% coupling 2A1-LC-Cys206ins-mc-vc-PAB-MMAE TDC 90.4% (TDC) 2A1-HC-Cys439ins-mc-vc-PAB-MMAE TDC 90.0% 4E1-LC-Cys205ins-mc-vc-PAB-MMAE TDC 98.18% 4E1-LC-Cys206ins-mc-vc-PAB-MMAE TDC 94.34% 4E1-HC-Cys439ins-mc-vc-PAB-MMAE TDC 95.77% 4D3-LC-Cys205ins-mc-vc-PAB-MMAE TDC 97.27% 4D3-LC-Cys206ins-mc-vc-PAB-MMAE TDC 96.06% 4D3-HC-Cys439ins-mc-vc-PAB-MMAE TDC 96.98%
(86) As shown by TABLE II, the target monomer content of the TDC compound coupled by the inserted cysteine is above 90%.
Example 28: Measurement of Affinities Between Skeletal Antibodies Undergoing Cysteine Site-Directed Mutagenesis and Insertional Mutagenesis and Parental Antibodies and EGFRvIII, Affinities Between 4E1 Antibodies and c-Met, Affinities Between 4D3 Antibodies and Trop2
(87) The relative affinities of 2A1-LC-V205C, 2A1-LC-Cys205ins, 2A1-LC-Cys206ins, 2A1-HC-Cys474ins and 2A1 for EGFRvIII were compared by indirect ELISA. The specific steps are as follows: Recombinant EGFRvIII-His*6 antigen-coated plate was blocked by fish skin gelatin; Antibodies 2A1, 2A1-LC-V205C, 2A1-LC-Cys205ins, 2A1-LC-Cys206ins and 2A1-HC-Cys474ins were respectively diluted by 4 folds gradient with a total of 11 concentrations with the highest concentration being 10 ug/ml; HRP-labeled secondary antibody incubation were performed; after TMB coloration, absorption was detected and measured at 450 nm. The absorption measurement results at A450 were plotted against concentration. The cysteine site-directed mutagenesis or insertion of the mutant antibodies 2A1-LC-V205C, 2A1-LC-Cys205ins, 2A1-LC-Cys206ins and 2A1-HC-Cys474ins retained affinities to EGFRvIII similar to 2A1, as shown by the close EC.sub.50 values; these results indicate that the site-directed mutagenesis of the light chain V205C on 2A1 antibody, the insertional mutation at position 205 of the light chain of the antibody, the insertional mutation at position 206 of the light chain of the antibody, or the insertion mutation at position 474 of the heavy chain of the antibody does not affect their affinity for the EGFRvIII antigen.
(88) As shown in the
Example 29: Measurement of Affinities of Skeletal Antibodies Undergoing Cysteine Site-Directed Mutagenesis and Insertional Mutagenesis and Linked to Toxin/Drug Towards Connate Antigens, Affinities of 4E1 Antibodies to c-Met, Affinities of 4D3 Antibodies to Trop2
(89) The relative affinities of 4E1-LC-Cys205ins-MVPM, 4E1-LC-Cys206ins-MVPM, 4E1-HC-Cys474ins-MVPM and 4E1 for C-met were compared by indirect ELISA. The specific steps are as follows: Recombinant C-met-His*6 antigen-coated plate was blocked by fish skin gelatin; TDC 4E1-LC-Cys205ins-MVPM, 4E1-LC-Cys206ins-MVPM, 4E1-HC-Cys474ins-MVPM and antibody 4E1 were respectively diluted by 4 folds gradient with a total of 11 concentrations with the highest concentration being 10 ug/ml; HRP-labeled secondary antibody incubation were performed; after TMB coloration, absorption was detected and measured at 450 nm. The absorption measurements at A450 were plotted against concentration, and the result shows that the antibodies harboring cysteine site-directed insertion mutation, TDC 4E1-LC-Cys205ins-MVPM, 4E1-LC-Cys206ins-MVPM, and 4E1-HC-Cys474ins-MVPM, retained their binding affinities to C-met similar to 4E1, as indicated by the close EC.sub.50 values; which indicates that the insertional mutation at the position 205 or 206 of 4E1 light chain or at the position 474 of 4E1 heavy chain does not affect the binding affinity of the corresponding TDC to the c-met antigen.
(90) The relative affinities of 4D3-LC-Cys205ins-MVPM, 4D3-LC-Cys206ins-MVPM, 4D3-HC-Cys474ins-MVPM and 4D3 for Trop2 were compared by indirect ELISA. The specific steps are as follows:
(91) Recombinant Trop2-His*6 antigen-coated plate was blocked by fish skin gelatin; TDC 4D3-LC-Cys205ins-MVPM, 4D3-LC-Cys206ins-MVPM, 4D3-HC-Cys474ins-MVPM and antibody 4D3 were respectively diluted by 4 folds gradient with a total of 11 concentrations with the highest concentration being 10 ug/ml; HRP-labeled secondary antibody incubation were performed; after TMB coloration, absorption was detected and measured at 450 nm. The absorption measurements at A450 were plotted against concentration. TDC 4D3-LC-Cys205ins-MVPM, 4D3-LC-Cys206ins-MVPM, and 4D3-HC-Cys474ins-MVPM retained their binding affinities to Trop2 similar to that of 4D3, as shown by the close EC.sub.50 values; which indicates that the insertional mutation at the position 205 or 206 of 4D3 light chain or at the position 474 of 4D3 heavy chain does not affect the binding affinity of the corresponding TDC to the Trop2 antigen.
(92) As shown in
(93) As shown in
Example 30: Cytotoxicity Pharmaceutical Efficacy Test
(94) TDC cytotoxic activity was determined by the following experimental procedures: TDC was separately added to culture media of human tumor cells in which EGFR was overexpressed or EGFRVIII was expressed, and cell viability was measured after 72 hours of cell culture. Cell-based in vitro assays were used to determine cell viability, cytotoxicity, and TDC-induced apoptosis in the present disclosure.
(95) The in vitro efficacy of the antibody-cytotoxin conjugate was determined by a cell proliferation assay. In one embodiment, the CellTiter 96® Aqueous One Solution Cell Proliferation Assay is commercially available (Promega Corp., Madison, Wis.). The Cell Proliferation Assay (a) is a detection reagent that uses colorimetry to detect the number of viable cells in cell proliferation and cytotoxicity experiments. This reagent contains a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electronic coupling agent (phenazine ethosulfate; PES). PES has enhanced chemical stability, which allows it to be mixed with MTS to form a stable solution. This convenient “single solution” mode is based on the first generation CellTiter 96® AQueous Assay, in which the electronic coupling agent PMS and MTS solution are supplied separately. MTS (Owen's reagent) is biologically reduced by cells to a colored formazan product that is directly soluble in the medium (
(96) ##STR00015##
(97) The amount of formazan product detected at 490 nm is directly proportional to the number of viable cells in the culture. Since the MTS hyperthyroid product is soluble in tissue culture media, the CellTiter 96® AQueous One Solution Assay has fewer steps than the MTT or INT method.
(98) In the present disclosure, A431 (EGFR overexpressing cells), U87-EGFRVIII (EGFR mutant stable cell line), U87-MG (glioblastoma cell line with highly expressed c-Met) and BXPC-3 (pancreatic cancer cell line with high expression of Trop2) are used as research systems for in vitro drug efficacy testing. In a 96-well plate, cell plating was performed at a concentration of 6000/well, and after 24 hours, antibody administration was performed. The initial concentrations of various TDCs corresponding to A431, U87-EGFRVIII cell lines were 10 μM, which were sequentially diluted according to a 5-fold gradient. The initial concentrations of various TDCs corresponding to U87-MG and BXPC-3 cell lines were 1 μM, which were sequentially diluted according to a 5-fold gradient. MTS assay for cell viability were performed after 72 hours of treatment
(99) TABLE-US-00008 TABLE III Cytotoxicity IC.sub.50 detection results of TDC, ADC on EGFRwt overexpressing cell line A431 and EGFRvIII expression stable strain U87-EGFRVIII MTS U87MG- Compounds A431 EGFRvIII U87-MG BXPC-3 Antibody 2A1 >10 μM >10 μM / / 2A1 2A1-LC-V205C >10 μM >10 μM / / 2A1-LC-Cys205ins >10 μM >10 μM / / 2A1-LC-Cys206ins >10 μM >10 μM / / 2A1-HC-Cys439ins >10 μM >10 μM / / Site- 2A1-LC-V205C-mc- 92.16 nM 296.11 nM / / directed vc-PAB-MMAE coupling 2A1-LC-Cys205ins- 133.67 nM 492.14 nM / / (TDC) mc-vc-PAB-MMAE 2A1-LC-Cys206ins- 179.26 nM 457.48 nM / / mc-vc-PAB-MMAE 2A1-HC-Cys439ins- 26.62 nM 118.52 nM / / mc-vc-PAB-MMAE Antibody 4E1 / / >1 μM / 4E1 Site- 4E1-LC-Cys205ins- / / 120.50 nM / directed mc-vc-PAB-MMAE coupling 4E1-LC-Cys206ins- / / 79.57 nM / (TDC) mc-vc-PAB-MMAE 4E1-HC-Cys439ins- / / 7.41 nM / mc-vc-PAB-MMAE Antibody 4D3 / / / >1 μM 4D3 Site- 4D3-LC-Cys205ins- / / / 0.38 nM directed mc-vc-PAB-MMAE coupling 4D3-LC-Cys206ins- / / / 0.45 nM (TDC) mc-vc-PAB-MMAE 4D3-HC-Cys439ins- / / / 0.23 nM mc-vc-PAB-MMAE
(100) The data from the TABLE III shows that, 2A1-LC-V205C-mc-vc-PAB-MMAE TDC, 2A1-LC-Cys205Cins-mc-vc-PAB-MMAE TDC, 2A1-LC-Cys206ins-mc-vc-PAB-MMAE TDC, and 2A1-HC-Cys474ins-mc-vc-PAB-MMAE TDC have comparable cytotoxic activity to EGFRwt overexpressing cell line A431 and EGFRvIII expression stable strain U87-EGFRVIII, and 474 inserted mutant TDC's activity is slightly higher than 205 and 206 insertion mutant TDC.
(101) There was a certain correlation between cytotoxic activity and coupling position for the 4E1-LC-Cys205ins-mc-vc-PAB-MMAE TDC, 4E1-LC-Cys206ins-mc-vc-PAB-MMAE TDC and 4E1-HC-Cys474ins-mc-vc-PAB-MMAE TDC in U87-MG cells. The TDC activity of the 474 inserted mutant was slightly better than those of the 205 and 206 insertion mutants. The activity of TDC was significantly better than that of the parental antibody.
(102) The cytotoxic activity of 4D3-LC-Cys205ins-mc-vc-PAB-MMAE TDC, 4D3-LC-Cys206ins-mc-vc-PAB-MMAE TDC and 4D3-HC-Cys474ins-mc-vc-PAB-MMAE TDC in pancreatic cancer cell line BXPC-3 was comparable or similar to each other, and the TDC activity of the 474 inserted mutant was slightly better than those of the 205 and 206 insertion mutant TDCs, and the activity of TDC was significantly better than that of the parental antibody.
Example 31: Plasma Stability Test
(103) Take a certain amount of ADC sample, add it to human plasma from which human IgG has been removed, repeat 2 tubes for each ADC, incubate in a 37° C. water bath, incubate for 0 h, 72 h, take ADC samples, add 100 μl ProteinA (MabSelect SuRe™ LX Lot: #10221479 GE washed with PBS), shaken for 2 h with a vertical mixer, and subjected to a washing and elution step to obtain an ADC after incubation. The samples, which had undergone incubation for a certain time, were subjected to HIC-HPLC and RP-HPLC to determine the plasma stability of the samples.
(104)
(105)
(106) TABLE-US-00009 TABLE IV TDC plasma stability test result (calculated by the change of DAR) DAR 37° C. 37° C. Compound 0 h 72 h Site- 4E1-LC-Cys205ins-mc- 1.89 1.77 directed vc-PAB-MMAE TDC coupling 4E1-LC-Cys206ins-mc- 1.81 1.62 (TDC) vc-PAB-MMAE TDC 4E1-HC-Cys439ins-mc- 1.85 1.83 vc-PAB-MMAE TDC 4D1-LC-Cys205ins-mc- 1.86 1.71 vc-PAB-MMAE TDC 4D3-LC-Cys206ins-mc- 1.76 1.52 vc-PAB-MMAE TDC 4D3-HC-Cys439ins-mc- 1.81 1.80 vc-PAB-MMAE TDC
(107) The above TDCs were stable after being incubated at 37° C. for 72 hours in human plasma samples and had good drug-forming properties. In comparison, TDC with 474 insertion mutations had the best stability, followed by TDC with 205 and 206 insertion mutations.
Example 32: Tumor-Bearing Mice Pharmaceutical Efficacy Test
(108) In the present disclosure, a BXPC-3 tumor-bearing mouse model was established to evaluate the in vivo efficacy of TDC and parental antibodies. In one embodiment, 3×10.sup.6 BXPC-3 cells were subcutaneously injected into the back side of 4-8 weeks old BALB/c nude mice, and the average tumor size of the mice was grown to 400-500 mm.sup.3, the mice were randomly grouped, 5 mice in each group. On Day 0 and Day 7, 4D3-LC-Cys205ins-mc-vc-PAB-MMAE TDC, 4D3-LC-Cys206ins-mc-vc-PAB-MMAE TDC and 4D3-HC-Cys474ins-mc-vc-PAB-MMAE TDC were administered in a single intravenous dose at a dose of 5 mg/kg, and the parental antibody 4D3 was administered at a dose of 5 mg/kg. Data A shows the mean tumor volume±SE at the time of measurement, and data B shows the average body weight of the mouse at the time of measurement±SE.
(109)
(110)
(111) The disclosure is not limited to the scope of the specific embodiments disclosed in the embodiments, which are intended to illustrate several aspects of the disclosure, and any embodiments that are functionally equivalent are within the scope of the disclosure. In fact, various modifications of the disclosure are obvious to those skilled in the art and are in the scope of the appended claims.
(112) TABLE-US-00010 TABLE V amino acids Symbol or English Name Abbreviation Alanine A or Ala Arginine R or Arg Asparagine N or Asn Aspartic acid D or Asp Cysteine C or Cys Glutamine Q or Gln Glutamic acid E or Glu Glycine G or Gly Histidine H or His Isoleucine I or Ile Leucine L or Leu Lysine K or Lys Methionine M or Met Phenylalanine F or Phe Proline P or Pro Serine S or Ser Threonine T or Thr Tryptophan W or Trp Tyrosine Y or Tyr Valine V or Val
(113) TABLE-US-00011 heavy chain constant region (Fc) DNA sequence >IgG1-Fc SEQ ID NO: 1 GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTG GTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG AATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCT TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGAC CCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG GATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC TATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCGGGTAAA heavy chain constant region (Fc) amino acid sequence >IgG1-Fc SEQ ID NO: 2 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK light chain constant region (Kappa) DNA sequence >LC-Kappa SEQ ID NO: 3 ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG AAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA GAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC TGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC AGGGGAGAGTGTTAG light chain constant region (Kappa) amino acid sequence >LC-Kappa SEQ ID NO: 4 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC 2A1-LC-Cys205ins light chain constant region (Kappa) DNA sequence >LC-Cys205ins-Kappa SEQ ID NO: 5 ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG AAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA GAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC TGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCTGCGTCACAAAGAGCTTC AACAGGGGAGAGTGTTAG LC-Cys205ins light chain constant region (Kappa) amino acid sequence >LC-Cys205ins-Kappa SEQ ID NO: 6 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPCVTKSF NRGEC
wherein, the C in the GLSSPCVTKSF (SEQ ID NO:13) is the site-specific conjugation position. In one embodiment, the cysteine is conjugated with mc-vc-PAB-payload site-specifically.
(114) TABLE-US-00012 LC-Cys206ins light chain constant region (Kappa) DNA sequence >LC-Cys206ins-Kappa SEQ ID NO: 7 ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG GAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCAAATCTGGAA CTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGGGTAACTCC CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC TGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCTGCACAAAGAGCTTC AACAGGGGAGAGTGTTAG LC-Cys206ins light chain constant region (Kappa) amino acid sequence >LC-Cys206ins-Kappa SEQ ID NO: 8 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVCTKSF NRGEC
wherein, the C in the GLSSPVCTKSF (SEQ ID NO:14) is the site-specific conjugation position. In one embodiment, the cysteine is conjugated with mc-vc-PAB-payload site-specifically.
(115) TABLE-US-00013 IgG1-Fc-Cys439ins heavy chain constant region (Fc) DNA sequence >IgG1-Fc-Cys439ins SEQ ID NO: 9 GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTG GTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG AATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCT TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGAC CCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG GATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC TATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC CTCTCCTGCCTGTCTCCGGGTAAA IgG1-Fc-Cys439ins heavy chain constant region (Fc) amino acid sequence >IgG1-Fc-Cys439ins SEQ ID NO: 10 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLOSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSCLSPGK
Wherein the C in the TQKSLSCLSPGK (SEQ ID NO:15) sequence is the site-specific conjugation/coupling position, and undergoes site-specific conjugation with mc-vc-PAB-payload.
(116) TABLE-US-00014 LC-V205C light chain constant region (Kappa) DNA sequence >LC-V205C-Kappa SEQ ID NO: 11 ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG AAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA GAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC TGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCTGCACAAAGAGCTTCAAC AGGGGAGAGTGTTAG LC-V205C light chain constant region (Kappa) amino acid sequence >LC-V205C-Kappa SEQ ID NO: 12 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPCTKSFN RGEC
Wherein the C in the GLSSPCTKSF (SEQ ID NO:16) sequence is the site-specific conjugation/coupling position, and undergoes site-specific conjugation with mc-vc-PAB-payload.
(117) TABLE-US-00015 LC-V205C light chain constant region (Kappa) amino acid sequence SEQ ID NO: 13 GLSSPCVTKSF LC-V206C light chain constant region (Kappa) amino acid sequence SEQ ID NO: 14 GLSSPVCTKS Heavy chain amino acid sequence SEQ ID NO: 15 TQKSLSCLSPGK