TROP2 TARGETING ANTIBODY-DRUG CONJUGATE, AND PREPARATION METHOD AND USE THEREFOR

Abstract

A TROP2 targeting antibody-drug conjugate, and a preparation method and use therefor. The antibody-drug conjugate is shown in formula I. Said type of compound has a good targeting property, a good inhibitory effect on tumor cells positively expressing TROP2, and good druggability and high safety. The present antibody drug conjugate has an inhibitory effect on TROP2, and also a good inhibitory effect on at least one of NCI-N87, MDA-MB-468 and BXPC3 cells.

Claims

1. An antibody-drug conjugate of formula I, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of the pharmaceutically acceptable salt thereof; ##STR00080## wherein Ab is a TROP2 antibody or a variant of the TROP2 antibody; the amino acid sequence of the light chain in the TROP2 antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 2; the variant of the TROP2 antibody is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the TROP2 antibody; m is 2 to 8; D is a cytotoxic drug topoisomerase inhibitor; R.sup.1 is C.sub.1-C.sub.6 alkyl substituted by one or more than one NR.sup.1-1R.sup.1-2, C.sub.1-C.sub.6 alkyl substituted by one or more than one R.sup.1-3S(O).sub.2, C.sub.1-C.sub.6 alkyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.6-C.sub.14 aryl, or 5- to 14-membered heteroaryl; the heteroatom in the 5- to 14-membered heteroaryl is selected from one or more than one of N, O, and S, and the number of heteroatoms is 1, 2, 3, or 4; the R.sup.1-1, R.sup.1-2 and R.sup.1-3 are each independently C.sub.1-C.sub.6 alkyl; L.sub.1 is independently one or more than one of a phenylalanine residue, alanine residue, glycine residue, aspartic acid residue, cysteine residue, glutamic acid residue, histidine residue, isoleucine residue, leucine residue, lysine residue, methionine residue, proline residue, serine residue, threonine residue, tryptophan residue, tyrosine residue, and valine residue; p is 2 to 4; L.sub.2 is ##STR00081## wherein n is independently 1 to 12, the c-terminal is connected to L.sub.1 through a carbonyl group, and the f-terminal is connected to the d-terminal of L.sub.3; L.sub.3 is ##STR00082## wherein the b-terminal is connected to the Ab, and the d-terminal is connected to the f-terminal of L.sub.2.

2. The antibody-drug conjugate, the pharmaceutically acceptable salt thereof, the solvate thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 1, wherein the b-terminal of L.sub.3 is connected to the sulfhydryl group on the antibody in the form of a thioether; or, when D is the cytotoxic drug topoisomerase inhibitor, the cytotoxic drug topoisomerase inhibitor is ##STR00083## R.sup.2 and R.sup.5 are each independently H, C.sub.1-C.sub.6 alkyl, or halogen; R.sup.3 and R.sup.6 are each independently H, C.sub.1-C.sub.6 alkyl, or halogen; R.sup.4 and R.sup.7 are each independently C.sub.1-C.sub.6 alkyl; or, when R.sup.1 is C.sub.1-C.sub.6 alkyl substituted by one or more than one NR.sup.1-1R.sup.1-2, the C.sub.1-C.sub.6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl; or, when R.sup.1 is C.sub.1-C.sub.6 alkyl substituted by more than one NR.sup.1-1R.sup.1-2, the more than one is two or three; or, when R.sup.1-1 and R.sup.1-2 are each independently C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl; or, when R.sup.1 is C.sub.1-C.sub.6 alkyl substituted by one or more than one R.sup.1-3S(O).sub.2, the C.sub.1-C.sub.6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl; or, when R.sup.1 is C.sub.1-C.sub.6 alkyl substituted by more than one R.sup.1-3S(O).sub.2, the more than one is two or three; or, when R.sup.1-3 is C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl; or, when R.sup.1 is C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl; or, m is an integer or non-integer of 3 to 5; or, n is 8 to 12; or, p is 2; or, when R.sup.1-1, R.sup.1-2, and R.sup.1-3 are independently C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl.

3. The antibody-drug conjugate, the pharmaceutically acceptable salt thereof, the solvate thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 2, wherein when R.sup.2 and R.sup.5 are each independently C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl; or, when R.sup.2 and R.sup.5 are each independently halogen, the halogen is fluorine, chlorine, bromine, or iodine; or, when R.sup.3 and R.sup.6 are each independently C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl; or, when R.sup.3 and R.sup.6 are each independently halogen, the halogen is fluorine, chlorine, bromine, or iodine; or, when R.sup.4 and R.sup.7 are each independently C.sub.1-C.sub.6 alkyl, the C.sub.1-C.sub.6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl; or, when R.sup.1 is C.sub.1-C.sub.6 alkyl substituted by one or more than one NR.sup.1-1R.sup.1-2, the NR.sup.1-1R.sup.1-2 is N(CH.sub.3).sub.2; or, when R.sup.1 is C.sub.1-C.sub.6 alkyl substituted by one NR.sup.1-1R.sup.1-2, the C.sub.1-C.sub.6 alkyl substituted by one NR.sup.1-1R.sup.1-2 is ##STR00084## or, when R.sup.1 is C.sub.1-C.sub.6 alkyl substituted by one R.sup.1-3S(O).sub.2, the C.sub.1-C.sub.6 alkyl substituted by one R.sup.1-3S(O).sub.2 is ##STR00085## or, m is 3.8 to 4.2; or, (L.sub.1).sub.p is ##STR00086## wherein the g-terminal is connected to the c-terminal of L.sub.2 through a carbonyl group; or, n is 8, 9, 10, 11, or 12.

4. The antibody-drug conjugate, the pharmaceutically acceptable salt thereof, the solvate thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 2, wherein R.sup.2 and R.sup.5 are each independently C.sub.1-C.sub.6 alkyl; or, R.sup.3 and R.sup.6 are each independently halogen; or, R.sup.4 and R.sup.7 are ethyl; or, L.sub.1 is one or more than one of the phenylalanine residue, alanine residue, glycine residue, isoleucine residue, leucine residue, proline residue, and valine residue; or, R.sup.1 is C.sub.1-C.sub.6 alkyl substituted by one or more than one NR.sup.1-1R.sup.1-2, C.sub.1-C.sub.6 alkyl substituted by one or more than one R.sup.1-3S(O).sub.2, C.sub.1-C.sub.6 alkyl, or C.sub.3-C.sub.10 cycloalkyl, D is ##STR00087##

5. The antibody-drug conjugate, the pharmaceutically acceptable salt thereof, the solvate thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 2, wherein the antibody-drug conjugate is any one of the following schemes: scheme I: Ab is the TROP2 antibody or the variant of the TROP2 antibody; D is ##STR00088## R.sup.2 and R.sup.5 are each independently H, C.sub.1-C.sub.6 alkyl, or halogen; R.sup.3 and R.sup.6 are each independently H, C.sub.1-C.sub.6 alkyl, or halogen; R.sup.4 and R.sup.7 are each independently C.sub.1-C.sub.6 alkyl; L.sub.1 is independently one or more than one of the phenylalanine residue, alanine residue, glycine residue, isoleucine residue, leucine residue, proline residue, and valine residue; scheme II: Ab is the TROP2 antibody or the variant of the TROP2 antibody; D is ##STR00089## R.sup.2 and R.sup.5 are each independently C.sub.1-C.sub.6 alkyl; R.sup.3 and R.sup.6 are each independently halogen; R.sup.1 is C.sub.1-C.sub.6 alkyl substituted by one or more than one NR.sup.1-1R.sup.1-2, C.sub.1-C.sub.6 alkyl substituted by one or more than one R.sup.1-3S(O).sub.2, or C.sub.1-C.sub.6 alkyl; L.sub.1 is independently one or more than one of the phenylalanine residue, alanine residue, glycine residue, and valine residue; scheme III: Ab is the TROP2 antibody; D is ##STR00090## R.sup.2 and R.sup.5 are each independently C.sub.1-C.sub.6 alkyl; R.sup.3 and R.sup.6 are each independently halogen; m is 3.5 to 4.5; R.sup.1 is C.sub.1-C.sub.6 alkyl substituted by one or more than one NR.sup.1-1R.sup.1-2, C.sub.1-C.sub.6 alkyl substituted by one or more than one R.sup.1-3S(O).sub.2, or C.sub.1-C.sub.6 alkyl; L.sub.1 is independently selected from the valine residue and the alanine residue; scheme IV: Ab is the TROP2 antibody; D is ##STR00091## R.sup.1 is C.sub.1-C.sub.6 alkyl substituted by one or more than one NR.sup.1-1R.sup.1-2, C.sub.1-C.sub.6 alkyl substituted by one or more than one R.sup.1-3S(O).sub.2, or C.sub.1-C.sub.6 alkyl; L.sub.1 is independently selected from the valine residue and the alanine residue; scheme V: Ab is the TROP2 antibody; D is ##STR00092## m is 3.5 to 4.5; R.sup.1 is C.sub.1-C.sub.6 alkyl substituted by one or more than one NR.sup.1-1R.sup.1-2, C.sub.1-C.sub.6 alkyl substituted by one or more than one R.sup.1-3S(O).sub.2, or C.sub.1-C.sub.6 alkyl; L.sub.1 is independently selected from the valine residue and the alanine residue.

6. The antibody-drug conjugate, the pharmaceutically acceptable salt thereof, the solvate thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 1, wherein the antibody-drug conjugate is any one of the following compounds: ##STR00093## ##STR00094## ##STR00095## wherein Ab is the TROP2 antibody or the variant of the TROP2 antibody, and m is 3.8 to 4.2.

7. The antibody-drug conjugate, the pharmaceutically acceptable salt thereof, the solvate thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 1, wherein the antibody-drug conjugate is any one of the following schemes: ##STR00096## ##STR00097## ##STR00098## wherein Ab is the TROP2 antibody or the variant of the TROP2 antibody; the amino acid sequence of the light chain in the variant of the TROP2 antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 2; ##STR00099## wherein Ab is the TROP2 antibody or the variant of the TROP2 antibody; m is 3.90, 4.00, or 4.10; the amino acid sequence of the light chain in the variant of the TROP2 antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 2; ##STR00100## wherein Ab is the TROP2 antibody or the variant of the TROP2 antibody; ##STR00101## ##STR00102## ##STR00103## ##STR00104##

8. A method for preparing the antibody-drug conjugate according to claim 1, comprising the following steps: carrying out a coupling reaction between a compound of formula II and Ab-hydrogen as shown below; ##STR00105##

9. A pharmaceutical composition, comprising substance X and a pharmaceutically acceptable excipient; the substance X is the antibody-drug conjugate, the pharmaceutically acceptable salt thereof, the solvate thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 1.

10-12. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0121] FIG. 1 shows the construction of expression vectors for the light and heavy chains of an FDA018 antibody; wherein Ab-L is the light chain of the antibody, and Ab-H is the heavy chain of the antibody.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0122] The present disclosure will be further described below with reference to examples, but the present disclosure is not therefore limited to the scope of the examples. Experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the commercial specification.

Description of Abbreviations

[0123] PCR polymerase chain reaction

[0124] CHO Chinese hamster ovary cells

[0125] HTRF homogeneous time-resolved fluorescence

[0126] PB phosphate buffer

[0127] EDTA ethylenediaminetetraacetic acid

[0128] TECP tris(2-carboxyethyl)phosphine

[0129] DMSO dimethyl sulfoxide

[0130] DMF N,N-dimethylformamide

[0131] His histidine

[0132] HATU 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate

[0133] v/v V/V, volume ratio

[0134] UV ultraviolet visible light

[0135] ELISA enzyme-linked immunosorbent assay

[0136] BSA bovine serum albumin

[0137] rpm revolutions per minute

[0138] FBS fetal bovine serum

Example 1: Preparation of TROP2 Antibody

[0139] In the present disclosure, the monoclonal antibody FDA018 with high affinity and specific targeting TROP2 was selected, the amino acid sequence of its light chain was shown in SEQ ID NO: 1, and the amino acid sequence of its heavy chain was shown in SEQ ID NO: 2. The light and heavy chain nucleotide sequences of FDA018 were obtained by whole gene synthesis (Suzhou Genewiz). They were separately constructed into the pV81 vector (as shown in FIG. 1) by double digestion with EcoR I and Hind III (purchased from TAKARA), and then transformed into Trans 1-T1 competent cells (purchased from Beijing TransGen Biotech, product number: CD501-03) by ligation, which were picked for cloning, PCR identification and sent for sequencing confirmation. Positive clones were cultured and expanded for plasmid extraction, thus obtaining the antibody light chain eukaryotic expression plasmid FDA018-L/pV81 and the antibody heavy chain eukaryotic expression plasmid FDA018-H/pV81. These two plasmids were linearized by digestion with XbaI (purchased from Takara, product number: 1093S). The light and heavy chain eukaryotic expression plasmids were transformed into CHO cells adapted to suspension growth (purchased from ATCC) at a ratio of 1.5/1 by electroporation. After electroporation, the cells were seeded at 2000 to 5000 cells/well in a 96-well plate. After 3 weeks of culture, the expression level was measured by HTRF method (homogeneous time-resolved fluorescence). The top ten cell pools in terms of expression level were selected for expansion and cryopreservation. A cell was revived into a 125 mL shake flask (culture volume of 30 mL) and cultured at 37? C., 5.0% CO.sub.2, and 130 rpm by vibration for 3 days, then expanded to a 1000 mL shake flask (culture volume of 300 mL) and cultured at 37? C., 5.0% CO.sub.2, and 130 rpm by vibration. Starting on the 4th day, 5 to 8% of the initial culture volume of replenishment culture medium was added every other day. The culture was ended on 10th to 12th day and the harvest liquid was centrifuged at 9500 rpm for 15 minutes to remove the cell precipitate. The supernatant was collected and filtered through a 0.22 m filter membrane. The treated sample was purified using a MabSelect affinity chromatography column (purchased from GE) to obtain antibody FDA018.

[0140] The amino acid sequence of the FDA018 light chain is shown below:

TABLE-US-00001 SEQIDNO:1: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYS50 ASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGA100 GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV150 DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG200 LSSPVTKSFNRGEC214

[0141] The amino acid sequence of FDA018 heavy chain is shown below: SEQ ID NO: 2:

TABLE-US-00002 SEQIDNO:2: QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGW50 INTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGG100 FGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV150 KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ200 TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK250 PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY300 NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP350 QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP400 VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG450 K451

Example 2: Synthesis of Linker-Drug Conjugates

Example 2-1: Synthesis of LE12

[0142] ##STR00050## ##STR00051##

[0143] Synthesis of Intermediate 2.

[0144] (S)-2-Azidopropionic acid (10 g, 86.9 mmol) and 4-aminobenzyl alcohol (21.40 g, 173.8 mmol) were dissolved in a mixed solvent of 300 mL of dichloromethane and methanol (in a volume ratio of 2:1), and then 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (21.49 g, 86.9 mmol) was added thereto. The reaction was carried out at room temperature for 5 hours. The solvent was then evaporated under reduced pressure and the obtained crude product was purified by silica gel column chromatography [dichloromethane:ethyl acetate=1:1 (v/v)] to obtain intermediate 2 (16.3 g, yield of 85%), ESI-MS m/z: 221 (M+H).

[0145] Synthesis of Intermediate 3:

[0146] Intermediate 2 (15 g, 68.2 mmol) was mixed with bis(p-nitrophenyl)carbonate (22.82 g, 75.02 mmol) and dissolved in 200 mL of anhydrous N,N-dimethylformamide, and then 25 mL of triethylamine was added thereto, and the reaction was carried out at room temperature for 2 hours. After the complete reaction of the raw materials was monitored by liquid chromatography-mass spectrometry, methylamine hydrochloride (6.91 g, 102.3 mmol) was added thereto, and the reaction was continued at room temperature for 1 hour. After the reaction was completed, most of the solvent was removed by distillation under reduced pressure, then 200 mL of water and 200 mL of ethyl acetate were added thereto. The organic phase was collected after the phases were separated, and the organic phase was dried and concentrated, and then the obtained crude product was purified by silica gel column chromatography [dichloromethane:ethyl acetate=10:1 (v/v)] to obtain intermediate 3 (18.9 g, yield of 100%), ESI-MS m/z: 278 (M+H).

[0147] Synthesis of Intermediate 5:

[0148] Intermediate 3 (10 g, 36.1 mmol) was mixed with polyformaldehyde (1.63 g, 54.2 mmol) and dissolved in 150 mL of anhydrous dichloromethane. Trimethylchlorosilane (6.28 g, 57.76 mmol) was slowly added thereto and the reaction was carried out at room temperature for 2 hours to obtain a crude solution of intermediate 4. The reaction was monitored by liquid chromatography-mass spectrometry after sampling and quenching with methanol. After the reaction was completed, the reaction mixture was filtered and then tert-butyl hydroxyacetate (9.54 g, 72.2 mmol) and triethylamine (10 mL, 72.2 mmol) were added to the filtrate and the reaction was continued at room temperature for 2 hours. After the reaction was completed, most of the solvent was removed by distillation under reduced pressure, and then the obtained crude product was purified by silica gel column chromatography [petroleum ether:ethyl acetate=3:1 (v/v)] to obtain intermediate 5 (11.2 g, yield of 74%), ESI-MS m/z: 422 (M+H).

[0149] Synthesis of Intermediate 6:

[0150] Intermediate 5 (10 g, 23.8 mmol) was dissolved in 80 mL of anhydrous tetrahydrofuran, and 80 mL of water was added thereto, and then tris(2-carboxyethylphosphine) hydrochloride (13.6 g, 47.6 mmol) was added thereto and the reaction was carried out for 4 hours at room temperature. After the reaction was completed, the tetrahydrofuran was removed by distillation under reduced pressure, and then the mixture was extracted with ethyl acetate. The obtained organic phase was dried and evaporated to remove the solvent under reduced pressure, and purified by silica gel column chromatography [dichloromethane:methanol=10:1 (v/v)] to obtain intermediate 6 (8.1 g, yield of 86%), ESI-MS m/z: 396 (M+H).

[0151] Synthesis of Intermediate 8:

[0152] Intermediate 6 (5 g, 12.7 mmol) was dissolved in 60 mL of a mixed solvent of dichloromethane and methanol (v/v=2:1), and 3 mL of trifluoroacetic acid was slowly added thereto, and the reaction was carried out at room temperature for 30 min. After the reaction was completed, an equal volume of water and ethyl acetate were added thereto, and the organic phase was dried and concentrated, and the obtained crude product was directly used in the next step.

[0153] The crude product obtained from the previous step was dissolved in 50 mL of anhydrous N,N-dimethylformamide, and then Fmoc-L-valine hydroxysuccinimide ester (8.3 g, 19.1 mmol) and triethylamine (5 mL) were added thereto, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, most of the solvent was removed by distillation under reduced pressure, and then the obtained crude product was purified by silica gel column chromatography [dichloromethane:methanol=10:1 (v/v)] to obtain intermediate 8 (5.4 g, yield of 64%), ESI-MS m/z: 661 (M+H).

[0154] Synthesis of Intermediate 9:

[0155] Intermediate 8 (1 g, 1.5 mmol) was mixed with Exatecan methanesulfonate (0.568 g, 1 mmol) in 30 mL of anhydrous N,N-dimethylformamide, and then 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (1.14 g, 3.0 mmol) and 2 mL of triethylamine were added thereto, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and then the obtained crude product was purified by silica gel column chromatography [chloroform:methanol=10:1 (v/v)] to obtain intermediate 9 (0.94 g, yield of 87%), ESI-MS m/z: 1078 (M+H).

[0156] Synthesis of Compound LE12:

[0157] Intermediate 9 (1 g, 0.929 mmol) was dissolved in 20 mL of anhydrous DMF, then 0.5 mL of 1,8-diazabicyclo[5.4.0]undec-7-ene was added thereto, and the reaction was carried out at room temperature for 1 hour. After the reaction of the raw materials was completed, N-succinimidyl 6-maleimidohexanoate (428.5 mg, 1.39 mmol) was added directly, and the reaction mixture was stirred at room temperature for 1 hour. The solvent was removed by distillation under reduced pressure, and then the obtained crude product was purified by silica gel column chromatography [chloroform:methanol=8:1 (v/v)] to obtain the title compound (0.7 g, yield of 73%), ESI-MS m/z: 1035 (M+H).

Example 2-2: Synthesis of Compound LE13

[0158] ##STR00052## ##STR00053##

[0159] Synthesis of Intermediate 14

[0160] Commercially available intermediate 12 (267 mg, 0.8 mmol) was mixed with paraformaldehyde (50 mg, 1.6 mmol) and dissolved in 20 mL of anhydrous dichloromethane. Then, trimethylchlorosilane (0.3 mL, 3.4 mmol) was added slowly. After the addition was completed, the reaction was carried out at room temperature for 2 hours. Then, the reaction was monitored by liquid chromatography-mass spectrometry after sampling and quenching with methanol. After the reaction was completed, the reaction mixture was filtered, and then tert-butyl 2-hydroxyacetate (211 mg, 1.6 mmol) and pempidine (0.5 mL) were added to the filtrate, and the reaction was continued at room temperature for about 2 hours. After the reaction was completed, most of the solvent was removed by distillation under reduced pressure, and the obtained crude product was purified by silica gel column chromatography [dichloromethane:methanol=20:1 (v/v)] to obtain intermediate 14 (260 mg, yield of 68%), ESI-MS m/z: 479 (M+H).

[0161] Synthesis of Intermediate 15

[0162] Intermediate 14 (238 mg, 0.50 mmol) was dissolved in 6 mL of a mixed solvent of dichloromethane and methanol (v/v=2:1), and 0.3 mL of trifluoroacetic acid was slowly added thereto, and the reaction was carried out at room temperature for 30 min. After the reaction was completed, an equal volume of water and ethyl acetate were added thereto, and the organic phase was dried and concentrated, and the obtained crude product was directly used in the next step.

[0163] Synthesis of Intermediate 16

[0164] The crude product obtained from the previous step was mixed with Exatecan methanesulfonate (170 mg, 0.30 mmol) in 5 mL of anhydrous N,N-dimethylformamide, and then 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (341 mg, 0.90 mmol) and 0.60 mL of triethylamine were added thereto, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and then the obtained crude product was purified by silica gel column chromatography [chloroform:methanol=10:1 (v/v)] to obtain intermediate 16 (210 mg, 83%), ESI-MS m/z: 840 (M+H).

[0165] Synthesis of Intermediate 17

[0166] Intermediate 16 (100 mg, 0.12 mmol) was dissolved in 15 mL of anhydrous tetrahydrofuran, and 3 mL of water was added thereto, then 0.3 mL of 1 mol/L triethylphosphine aqueous solution was added thereto, and the reaction was carried out at room temperature for 4 hours. After the reaction was monitored to be completed, the reaction mixture was distilled under reduced pressure to remove tetrahydrofuran. Sodium bicarbonate was added to the remaining aqueous solution to adjust the pH to neutral, and then dichloromethane was added for extraction. The obtained organic phase was dried and evaporated under reduced pressure to remove the solvent. The obtained crude product was purified by silica gel column chromatography [dichloromethane:methanol=10:1 (v/v)] to obtain intermediate 17 (69 mg, yield of 71%), ESI-MS m/z: 814 (M+H).

[0167] Synthesis of Compound LE13

[0168] Intermediate 17 (120 mg, 0.15 mmol) obtained according to the previous synthesis method was mixed with the commercially available raw material MC-V (102 mg, 0.33 mmol) in 40 mL of dichloromethane, and the condensation agent 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (82 mg, 0.33 mmol) was added to react overnight at room temperature. After the reaction was completed, the solvent was evaporated under reduced pressure and the obtained crude product was purified by silica gel column chromatography [dichloromethane:methanol=10:1 (v/v)] to obtain compound LE13 (116 mg, yield of 70%), ESI-MS m/z: 1106.5 (M+H).

Example 2-3: Synthesis of Compound LE14

[0169] ##STR00054##

[0170] Synthesis of Intermediate 19

[0171] Commercially available intermediate 18 (300 mg, 0.8 mmol) was mixed with polyformaldehyde (50 mg, 1.6 mmol) and dissolved in 20 mL anhydrous dichloromethane. Then, trimethylchlorosilane (0.3 mL, 3.4 mmol) was slowly added thereto, and the reaction was carried out at room temperature for 2 hours. The reaction was monitored by liquid chromatography-mass spectrometry after sampling and quenching with methanol. After the reaction was completed, the reaction mixture was filtered and then tert-butyl 2-hydroxyacetate (211 mg, 1.6 mmol) and triethylamine (0.22 m, 1.6 mmol) were added to the filtrate. The reaction was continued at room temperature for about 2 hours. After the reaction was completed, most of the solvent was removed by distillation under reduced pressure and the obtained crude product was purified by silica gel column chromatography [dichloromethane:methanol=20:1 (v/v)] to obtain intermediate 19 (349 mg, yield of 85%), ESI-MS m/z: 514 (M+H), .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.13 (s, 1H), 7.56 (d, J=7.5 Hz, 2H), 7.35 (s, 2H), 5.14 (s, 2H), 4.91 (s, 2H), 4.25 (q, J=7.1 Hz, 1H), 3.99 (d, J=42.5 Hz, 2H), 3.85 (t, J=6.2 Hz, 2H), 3.40 (dd, J=18.5, 7.6 Hz, 2H), 2.89 (d, J=48.6 Hz, 3H), 1.65 (d, J=6.8 Hz, 3H), 1.46 (s, 9H).

[0172] Synthesis of Intermediate 20

[0173] Intermediate 19 (257 mg, 0.50 mmol) was dissolved in 6 mL of a mixed solvent of dichloromethane and methanol (v/v=2:1), and 0.3 mL of trifluoroacetic acid was slowly added thereto, and the reaction was carried out at room temperature for 30 min. After the reaction was completed, an equal volume of water and ethyl acetate were added thereto, and the organic phase was dried and concentrated, and the obtained crude product was directly used in the next step.

[0174] The obtained crude product was mixed with Exatecan methanesulfonate (170 mg, 0.30 mmol) in 5 mL of anhydrous N,N-dimethylformamide, and then 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (341 mg, 0.90 mmol) and 0.60 mL of triethylamine were added thereto, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and then the obtained crude product was purified by silica gel column chromatography [dichloromethane:methanol=20:1 (v/v)] to obtain intermediate 20 (212 mg, yield of 81%), ESI-MS m/z: 875 (M+H). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 8.27 (d, J=34.7 Hz, 1H), 7.63-7.35 (m, 5H), 7.21-7.10 (m, 1H), 5.71-5.48 (m, 2H), 5.24-4.95 (m, 3H), 4.95-4.72 (m, 4H), 4.45 (s, 1H), 4.33-3.97 (m, 3H), 3.75 (s, 2H), 3.39-2.99 (m, 4H), 2.76 (d, J=15.3 Hz, 3H), 2.43-2.15 (m, 5H), 2.04 (s, 1H), 1.94-1.75 (m, 2H), 1.62 (d, J=6.6 Hz, 3H), 1.11-0.89 (m, 3H).

[0175] Synthesis of Intermediate 21

[0176] Intermediate 20 (77 mg, 0.09 mmol) was dissolved in 12 mL of anhydrous tetrahydrofuran, and 3 mL of water was added thereto, then 0.3 mL of 1 mol/L triethylphosphine aqueous solution was added thereto, and the reaction was carried out at room temperature for 4 hours. After the reaction was completed, the reaction mixture was distilled under reduced pressure to remove tetrahydrofuran. Sodium bicarbonate was added to the remaining aqueous solution to adjust the pH to neutral, and then dichloromethane was added for extraction. The obtained organic phase was dried and evaporated under reduced pressure to remove the solvent. The obtained crude product was purified by silica gel column chromatography [dichloromethane:methanol=10:1 (v/v)] to obtain intermediate 21 (53 mg, yield of 69%), ESI-MS m/z: 849 (M+H). .sup.1H NMR (400 MHz, DMSO) ? 8.52 (s, 1H), 7.79 (d, J=10.8 Hz, 1H), 7.67-7.55 (m, 2H), 7.47-7.21 (m, 3H), 6.51 (s, 1H), 5.60 (s, 1H), 5.52-5.32 (m, 2H), 5.30-5.11 (m, 2H), 5.11-4.94 (m, 2H), 4.94-4.74 (m, 2H), 4.02 (s, 2H), 3.81-3.66 (m, 2H), 3.60-3.35 (m, 4H), 3.24-3.08 (m, 2H), 2.94 (d, J=30.8 Hz, 3H), 2.39 (s, 3H), 2.28-2.04 (m, 2H), 2.00-1.73 (m, 2H), 1.22 (d, J=6.6 Hz, 3H), 0.96-0.70 (m, 3H).

[0177] Synthesis of Compound LE14

[0178] Intermediate 21 (134 mg, 0.16 mmol) was mixed with the commercially available raw material MC-V (102 mg, 0.33 mmol) in 40 mL of dichloromethane, and the condensation agent 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (82 mg, 0.33 mmol) was added to react overnight at room temperature. After the reaction was completed, the solvent was evaporated under reduced pressure and the obtained crude product was purified by silica gel column chromatography [dichloromethane:methanol=10:1 (v/v)] to obtain compound LE14 (137 mg, yield of 75%), ESI-MS m/z: 1141.4 (M+H). .sup.1H NMR (400 MHz, DMSO) ? 9.97 (s, 1H), 8.52 (s, 1H), 8.27-8.09 (m, 1H), 7.88-7.70 (m, 2H), 7.63-7.51 (m, 2H), 7.28 (s, 3H), 6.99 (s, 2H), 6.51 (s, 1H), 5.59 (s, 1H), 5.50-5.32 (m, 2H), 5.17 (s, 2H), 4.98 (s, 2H), 4.85 (d, J=17.3 Hz, 2H), 4.43-4.33 (m, 1H), 4.21-4.12 (m, 1H), 4.03 (s, 2H), 3.74-3.64 (m, 2H), 3.20-3.03 (m, 3H), 3.02-2.84 (m, 4H), 2.36 (s, 3H), 2.23-2.09 (m, 4H), 2.01-1.90 (m, 1H), 1.90 -1.78 (m, 2H), 1.55-1.39 (m, 4H), 1.30 (d, J=6.7 Hz, 3H), 1.23-1.11 (m, 2H), 0.93-0.77 (m, 9H).

Example 2-4: Synthesis of Compound LE15-LE20

[0179] ##STR00055##

[0180] Intermediate VI could be prepared by replacing the methylamine hydrochloride instep b with the corresponding commercially available amino compound, using Fmoc-L-valyl-L-alanine as the starting material and referring to steps a and b in the synthesis method of intermediate 3 in Example 2-1. The subsequent steps were carried out starting from intermediate VI, and with the same methods as those in steps c, d, f, and h of Example 2-1, and intermediate IX similar to intermediate 9 was obtained. Then, following the same steps i and j as Example 6, the amino protecting group was removed, and condensed with different commercially available maleimide compounds to obtain the final product. The structures of the amino compounds and maleimides used are shown in Table 1. Compound LE15: gray-white solid, ESI-MS m/z: 1121.2 (M+H); compound LE16: light yellow solid, ESI-MS m/z: 1167.1 (M+H); compound LE17: yellow solid, ESI-MS m/z: 1132.3 (M+H); compound LE18: light yellow solid, ESI-MS m/z: 1305.4 (M+H); compound LE19: light yellow solid, ESI-MS m/z: 1307.4 (M+H); compound LE20: light yellow solid, ESI-MS m/z: 1337.6 (M+H).

TABLE-US-00003 TABLE 1 Intermediates used for the synthesis of LE15 to LE20 Maleimide Structure Product R.sup.A Amino Compound [00056] LE15 [00057] Methylsulfonyl ethylamine hydrochloride [00058] LE16 [00059] Methylsulfonyl ethylamine hydrochloride [00060] LE17 [00061] Dimethyl- ethylamine hydrochloride [00062] LE18 [00063] Methylsulfonyl ethylamine hydrochloride [00064] LE19 [00065] Methylsulfonyl ethylamine hydrochloride [00066] LE20 [00067] Methylsulfonyl ethylamine hydrochloride [00068] [00069][00070][00071][00072][00073][00074]

Example 2-5: Synthesis of Compounds LE21 and LE22

[0181] ##STR00075## ##STR00076##

[0182] Synthesis of Compound DXD-1

[0183] Commercially available Exatecan methanesulfonate (0.568 g, 1 mmol) was mixed with commercially available 2-(tert-butyldimethylsilyloxy)acetic acid (CAS: 105459-05-0, 0.38 g, 2 mmol) in 20 mL anhydrous dichloromethane. The condensation agent HATU (0.76 g, 2 mmol) and 1 mL of pyridine were added thereto and stirred at room temperature for 2 hours. After the reaction was completed, the solvent was evaporated to dryness under reduced pressure, and the obtained crude product was purified by column chromatography [dichloromethane:methanol=50:1 (v/v)] to obtain title compound DXD-1 (0.55 g, yield of 90%), ESI-MS m/z: 608.1 (M+H). .sup.1H NMR (400 MHz, CDCl.sub.3) ? 7.73 (d, J=10.5 Hz, 1H), 7.64 (s, 1H), 7.05 (d, J=9.2 Hz, 1H), 5.80-5.62 (m, 2H), 5.41-5.14 (m, 4H), 4.29-4.15 (m, 2H), 4.08-4.03 (m, 1H), 3.27-3.07 (m, 2H), 2.45 (s, 3H), 2.38-2.28 (m, 2H), 1.96-1.81 (m, 2H), 1.04 (t, J=7.4 Hz, 3H), 0.80 (s, 9H), 0.11 (s, 3H), 0.03 (s, 3H).

[0184] Preparation of Intermediate V

[0185] Intermediate V could be prepared by replacing the methylamine hydrochloride in step b with the corresponding commercially available amino compound, referring to the preparation method of compound 4 in Example 2-1.

[0186] Synthesis of LE21 to LE22

[0187] Intermediate V was reacted with DXD-1, and then treated with 10% trifluoroacetic acid/dichloromethane solution to obtain intermediate X. Then, intermediate X was reacted according to the subsequent steps e, g, i, and j of compound 5 in Example 2-1: Intermediate X was reduced to obtain an amino compound, and the amino compound was then condensed with Fmoc-L-valine hydroxysuccinimide ester. Then the Fmoc protecting group of the amino group was removed from the obtained product, and the obtained amino product was then reacted with N-succinimidyl 6-maleimidohexanoate to obtain the final product. Compound LE21: yellow solid, ESI-MS m/z: 1141.2 (M+H); compound LE22: yellow solid, ESI-MS m/z: 1106.6 (M+H).

##STR00077##

Example 2-6: Synthesis of Compound LS13

[0188] Referring to the synthesis method of LE15 in Examples 2 to 4, SN-38 (7-ethyl-10-hydroxycamptothecin) was reacted with intermediate VII (R.sup.1 is methylsulfonyl ethyl) to obtain compound LS13 after deprotection, condensation and other steps: .sup.1H NMR (400 MHz, DMSO) ? 9.92 (d, J=22.4 Hz, 1H), 8.14 (s, 1H), 8.08 (d, J=9.1 Hz, 1H), 7.81 (d, J=8.0 Hz, 1H), 7.70-7.50 (m, 3H), 7.47 (d, J=7.2 Hz, 1H), 7.34 (d, J=7.2 Hz, 1H), 7.27 (s, 1H), 7.20 (s, 1H), 6.98 (s, 2H), 6.51 (s, 1H), 5.61 (s, 2H), 5.48-5.35 (m, 2H), 5.27 (s, 2H), 5.10 (d, J=20.6 Hz, 2H), 4.36 (s, 1H), 4.21-4.07 (m, 1H), 3.84 (s, 2H), 3.48 (s, 2H), 3.21-2.92 (m, 6H), 2.25-2.04 (m, 2H), 2.04-1.78 (m, 3H), 1.55-1.36 (m, 4H), 1.36-1.10 (m, 9H), 0.95-0.71 (m,

##STR00078##

Example 2-7: Synthesis of Compound GGFG-Dxd

[0189] Compound GGFG-Dxd was prepared according to the known synthesis method reported in WO2015146132A1. ESI-MS m/z: 1034.5 (M+H), .sup.1H-NMR (400 MHz, DMSO-d.sub.6) ? 8.61 (t, J=6.4 Hz, 1H), 8.50 (d, J=8.5 Hz, 1H), 8.28 (t, J=5.1 Hz, 1H), 8.11 (d, J=7.5 Hz, 1H), 8.05 (t, J=5.7 Hz, 1H), 7.99 (t, J=5.9 Hz, 1H), 7.77 (d, J=11.0 Hz, 1H), 7.31 (s, 1H), 7.25-7.16 (m, 5H), 6.98 (s, 2H), 6.51 (s, 1H), 5.59 (dt, J=7.4, 4.1 Hz, 1H), 5.41 (s, 2H), 5.20 (s, 2H), 4.64 (d, J=6.1 Hz, 2H), 4.53-4.40 (m, 1H), 4.02 (s, 2H), 3.74-3.37 (m, 8H), 3.18-3.00 (m, 2H), 3.04-2.97 (m, 1H), 2.77 (dd, J=13.5, 9.4 Hz, 1H), 2.38 (s, 3H), 2.19 (dd, J=14.9, 8.5 Hz, 2H), 2.11-2.05 (m, 2H), 1.86 (dd, J=14.0, 6.7 Hz, 2H), 1.45 (s, 4H), 1.20-1.14 (m, 2H), 0.87 (t, J=7.1 Hz, 3H).

##STR00079##

Example 3: Preparation of Antibody-Drug Conjugates

[0190] The antibody FDA018 against TROP2 was prepared according to the method of Example 1 and was replaced into 50 mM PB/1.0 mM EDTA buffer (pH 7.0) using a G25 desalting column. 5 equivalents of TECP were added thereto and the mixture was stirred at 25? C. for 1 hours to fully open the disulfide bonds between the antibody chains. Then, citric acid was used to adjust the pH of the reduced antibody solution to 5.0, and the sample was replaced with 20 mM citrate buffer and 1 mM EDTA buffer (pH 5.0) using a G25 desalting column. The temperature of the water bath was maintained at 25? C. for coupling reaction. The linker-drug conjugates LE12 to LE22, LS13, and GGFG-Dxd prepared according to the above Example 2 were dissolved in DMSO respectively and 4.5 equivalents of linker-drug conjugate were added dropwise to the reduced antibody solution. Additional DMSO was added to a final concentration of 5% (v/v) and the reaction was stirred at 25? C. for 0.5 hours. After the reaction was completed, the sample was filtered through a 0.22 ?m membrane. The tangential flow filtration system was used to purify and remove unconjugated small molecules. The buffer was a 25 mM His, 60 sucrose solution (pH 6.0). After purification, the sample was stored in a ?20? C. refrigerator. The absorbance values were measured at 280 nm and 370 nm by UV method, respectively, and the DAR value was calculated. The results are shown in Table 2 below.

[0191] The coupling reaction was carried out in the same manner as in this example and all samples were prepared according to the highest DAR (i.e., excessive coupling). The occurrence of precipitation during each coupling reaction was observed and the polymer ratio and recovery rate after each coupling reaction were calculated. The results are also shown in Table 2.

TABLE-US-00004 TABLE 2 Coupling conditions for preparing different antibody-drug conjugates (ADCs) Linker- Whether Drug DAR Precipi- Aggregation Recovery ADC Number Antibody Conjugate Value tation Ratio Rate FDA018-LE12 FDA018 LE12 3.90 No 0.5% 89% FDA018-LE13 FDA018 LE13 4.10 No 0.2% 98% FDA018-LE14 FDA018 LE14 4.00 No 0.1% 93% FDA018-LE15 FDA018 LE15 4.12 No 0.4% 91% FDA018-LE16 FDA018 LE16 4.03 No 0.3% 87% FDA018-LE17 FDA018 LE17 3.98 No 0.3% 88% FDA018-LE18 FDA018 LE18 3.96 No 0.5% 92% FDA018-LE19 FDA018 LE19 4.13 No 0.3% 91% FDA018-LE20 FDA018 LE20 3.88 No 0.1% 92% FDA018-LE21 FDA018 LE21 4.05 No 0.4% 91% FDA018-LE22 FDA018 LE22 4.00 No 0.1% 92% FDA018-LS13 FDA018 LS13 3.97 No 0.6% 89% FDA018-GGFG-Dxd FDA018 GGFG-Dxd 4.03 No 0.2% 92% / indicates that the recovery rate is not calculated

[0192] No precipitation was produced in the preparation procedure of the antibody-drug conjugate of the present disclosure, and the aggregation ratio was within the normal range, indicating that the linker-drug conjugates provided by the present disclosure have good physicochemical properties.

Effect Example 1: In Vitro Killing Activity Evaluation of Antibody-Drug Conjugates

[0193] NCI-N87 (ATCC) cells were selected as the cell line for in vitro activity detection. 2000 cells per well were seeded in a 96-well cell culture plate and cultured for 20 to 24 hours. The antibody-drug conjugates prepared according to the method of Example 3 were formulated into test solutions with 11 concentration gradients of 1000, 166.7, 55.6, 18.6, 6.17, 2.06, 0.69, 0.23, 0.08, 0.008, and 0 nM using L15 cell culture medium containing 10% FBS. The diluted test solutions were added to the culture plate containing the seeded cells at 100 ?L/well and incubated for 144 hours at 37? C. in a 5% CO.sub.2 incubator. CellTiter-Glo? Luminescent Cell Viability Assay Reagent (50 ?L/well) was added and the plate was shaken at 500 rpm at room temperature for 10 minutes to mix well. The data were read using a SpectraMaxL microplate reader (OD 570 nm, reading at 2 s intervals) and the IC50 results were calculated as shown in Table 3.

[0194] Using the same method as above, the cytotoxic activity of each antibody-drug conjugate against MDA-MB-468 and BXPC3 tumor cells purchased from ATCC was tested. The results are shown in Table 3. From the results in Table 3, it can be seen that the antibody-drug conjugates provided by the present disclosure have excellent in vitro killing activity against cells such as NCI-N87, MDA-MB-468, and BXPC3. However, the antibody-drug conjugate has no killing activity on Calu-6 negative cells, indicating that the prepared ADC has specific targeted killing activity.

TABLE-US-00005 TABLE 3 In vitro killing activity of antibody-drug conjugates IC50 (nM) MDA-MB-468 NCI-N87 BXPC3 Calu-6 ADC Number Cell Cell Cell Cell FDA018- 1.03 1.12 0.68 >1000 LE12 FDA018- 2.0 2.34 1.23 >1000 LE13 FDA018- 0.8 1.08 0.50 >1000 LE14 FDA018- 0.93 0.98 0.96 >1000 LE15 FDA018- 1.64 1.35 1.58 >1000 LE16 FDA018- 0.82 1.12 0.62 >1000 LE17 FDA018- 0.86 Not Tested Not Tested >1000 LE18 FDA018- 1.03 Not Tested Not Tested >1000 LE19 FDA018- Not Tested 1.15 Not Tested >1000 LE20 FDA018- Not Tested 1.23 1.23 >1000 LE21 FDA018- Not Tested 3.25 1.69 >1000 LE22 FDA018- 67.38 Not Tested 61.32 >1000 LS13 FDA018- 1.2 1.10 0.61 >1000 GGFG-DXD

Effect Example 2: In Vitro Plasma Stability Assay

[0195] This example evaluates the stability of the antibody-drug conjugate prepared according to the method of Example 3 in human plasma. Specifically, in this example, the antibody-drug conjugate of Example 3 was added to human plasma and placed in a 37? C. water bath for 1, 3, 7, 14, 21, and 28 days. An internal standard (Exatecan as an internal standard substance) was added and extracted and then detected by high-performance liquid chromatography to detect the release of free drugs. The results are shown in Table 4.

TABLE-US-00006 TABLE 4 Stability evaluation of different ADCs in human plasma Free Drug Ratio Sample Name Day 1 Day 3 Day 7 Day 14 Day 21 Day 28 FDA018- 0.1% 0.3% 0.6% 1.3% 1.4% 2.2% GGFG-DXD FDA018-LE12 0.2% 0.6% 0.8% 1.3% 1.6% 2.1% FDA018-LE13 0.1% 0.4% 0.7% 1.1% 1.3% 2.2% FDA018-LE14 0.1% 0.2% 0.4% 1.0% 1.4% 2.1% FDA018-LS13 0.2% 0.9% 2.2% 3.5% 4.3% 5.1%

[0196] The plasma stability results show that the stability of the ADC obtained using the new technical solution is not inferior to FDA018-GGFG-DXD, and some are even better. At the same time, the above activity test results also prove that some of the newly obtained ADCs have better activity than FDA018-GGFG-DXD.

Effect Example 3: In Vitro Enzyme Digestion Experiment of Linker-Drug Conjugates

[0197] The linker-drug conjugate (LE14 and GGFG-Dxd) was co-incubated with cathepsin B in three different pH (5.0, 6.0, 7.0) buffers. Samples were taken at different time points and entered into a high-performance liquid chromatography-mass spectrometry instrument. The external standard method (with DXD as the external standard) was used to determine the release percentage of the drug. The experimental results (as shown in Table 5) show that GGFG-Dxd has a slow speed of enzyme digestion within the pH range used, while LE14 of the present disclosure can be quickly enzymatically digested within the pH range of 5.0 to 7.0.

TABLE-US-00007 TABLE 5 In vitro enzyme digestion of LE14 and GGFG-Dxd at different pH Percentage of drug release in the sample % Time GGFG-Dxd LE14 (h) pH 5.0 pH 6.0 pH 7.0 pH 5.0 pH 6.0 pH 7.0 0 22.0 22.35 22.16 13.82 14.32 16.59 1 24.82 24.8 25.76 96.0 96.12 98.32 2 25.85 27.32 29.45 98.35 96.75 98.45 3 27.46 29.32 32.00 99.12 98.52 99.12 4 29.68 32.0 34.72 99.21 98.45 99.12 5 31.72 33.15 37.17 99.45 98.92 99.87 6 34.17 36.38 38.43 98.23 99.15 99.47

Effect Example 4: In Vitro Enzyme Digestion Experiment of FDA018-LS13

[0198] NCI-N87 cell line was selected as the experimental cell lines. After the sample was incubated in cathepsin B system (100 mM sodium acetate-acetic acid buffer, 4 mM dithiothreitol, pH 5.0) at 37? C. for 4 hours, the obtained sample was diluted with culture medium to different concentrations. 8 concentrations (1.5 to 10-fold dilution) were set from 70 nM to 0.003 nM of SN-38 concentration. The killing (inhibitory) ability of the cell line was observed for 144 hours. The IC50 value was calculated by reading the fluorescence data after chemical luminescent staining with CellTiter-Glo? Luminescent Cell Viability Assay.

[0199] The above enzyme digestion samples obtained by incubating in a cathepsin B system at 37? C. for 4 hours were precipitated with an appropriate amount of ethanol to remove protein and detected by high-performance liquid chromatography to release small molecule compounds. The 4-hour release rate was measured with an equal amount of SN-38 as a reference, and the results showed that the release rate reached 99%.

[0200] The experimental results (as shown in Table 6) show that after enzyme digestion treatment, the cytotoxic activity of FDA018-LS13 is almost the same as that of SN-38 at an equivalent dose, which also indicates that FDA018-LS13 has almost completely released SN-38 under the action of cathepsin B and played a role. However, FDA018-LS13 may have undergone unpredictable changes when it is endocytosed into lysosomes, resulting in SN-38 not being able to function effectively.

TABLE-US-00008 TABLE 6 Changes in killing activity of FDA018-LS13 on NCI-N87 cell line before and after enzyme digestion by cathepsin B system IC50 (based on SN-38 equivalent, nM) Before Enzyme Sample Digestion After Enzyme Digestion FDA018-LS13 >100 nM 6.48 nM SN-38 7.18 nM 7.43 nM

Effect Example 5: Testing Antitumor Activity of FDA018-LE14 in NCI-N87 Human Gastric Cancer Model

[0201] 6 to 8 week old female Balb/c nude mice were subcutaneously injected with 5?10.sup.6 human gastric cancer cells (NCI-N87) dissolved in 100 ?L of PBS solution on the right side of the neck and back. When the tumor grew to an average volume of 150 to 200 mm.sup.3, mice were randomly divided into 5 groups according to tumor size and mouse weight, with 6 animals in each group. The groups were a blank control group, and two dose groups of the antibody-drug conjugates FDA018-GGFG-DXD and FDA018-LE14, respectively. Specifically, group 01 was a blank control group; group 02 was FDA018-GGFG-DXD group at 4.0 mg/kg; group 03 was a FDA018-GGFG-DXD group at 2.0 mg/kg; group 04 was a FDA018-LE14 group at 4.0 mg/kg; group 05 was FDA018-LE14 group at 2.0 mg/kg; administered intraperitoneally once a week. The animal weight and tumor volume were measured twice a week, and the survival status of the experimental animals was observed during the experiment process. As shown in Table 7, the average tumor volume of the mice in the blank control group was 1388.47 mm.sup.3 at the end of treatment. The average tumor volume of the FDA018-GGFG-DXD treatment group at 2.0 mg/kg was 1235.21 mm.sup.3 on the 14th day after the end of treatment, and the average tumor volume of the FDA018-GGFG-DXD treatment group at 4.0 mg/kg was 721.09 mm.sup.3 on the 14th day after the end of treatment. The average tumor volume of the FDA018-LE14 treatment group at 2.0 mg/kg was 1342.31 mm.sup.3 on the 14th day after the end of treatment, and the average tumor volume of the FDA018-LE14 treatment group at 4.0 mg/kg was 435.36 mm.sup.3 on the 14th day after the end of treatment. The experimental results show that FDA018-LE14 has good in vivo antitumor activity, and all experimental mice have no death or weight loss, indicating that FDA018-LE14 has good safety.

TABLE-US-00009 TABLE 7 Antitumor activity of FDA018-LE14 in NCI-N87 human gastric cancer model Observation Days Group 5 7 11 14 18 21 Average Tumor Volume/mm.sup.3 01 205.31 ? 11.93 283.81 ? 26.98 395.50 ? 38.06 489.36 ? 34.44 621.74 ? 32.84 721.41 ? 19.24 02 205.35 ? 11.17 260.96 ? 23.58 235.65 ? 35.14 202.27 ? 36.43 250.53 ? 47.43 341.51 ? 59.92 03 205.54 ? 11.39 315.13 ? 29.11 332.46 ? 42.69 284.41 ? 50.34 419.84 ? 85.78 489.93 ? 88.24 04 205.32 ? 10.90 339.51 ? 32.35 268.58 ? 33.83 192.55 ? 36.69 191.16 ? 47.20 217.61 ? 72.84 05 205.32 ? 10.9 296.64 ? 32.35 320.03 ? 33.83 307.45 ? 34.59 386.87 ? 44.27 510.03 ? 43.55 Observation Days Group 25 28 32 35 39 42 Average Tumor Volume/mm.sup.3 01 783.85 ? 21.21 890.79 ? 35.51 994.80 ? 47.44 1176.92 ? 75.40 1348.83 ? 72.37 1388.47 ? 71.37 02 363.76 ? 56.95 412.44 ? 73.14 479.74 ? 88.12 527.30 ? 90.84 655.96 ? 135.74 721.09 ? 149.38 03 529.24 ? 100.84 628.75 ? 111.58 725.02 ? 131.43 892.29 ? 180.36 1065.85 ? 196.79 1235.21 ? 291.76 04 250.78 ? 83.71 273.13 ? 87.46 324.77 ? 101.84 368.20 ? 113.68 426.12 ? 142.41 435.36 ? 149.14 05 595.45 ? 68.98 699.04 ? 57.29 809.63 ? 87.10 922.72 ? 89.34 1192.74 ? 136.02 1342.31 ? 173.91