ANTIBODY-DRUG CONJUGATE, AND INTERMEDIATE THEREOF, PREPARATION METHOD THEREFOR, AND APPLICATION THEREOF

Abstract

An antibody-drug conjugate, and an intermediate thereof, a preparation method therefor, and an application thereof. Provided is an antibody-drug conjugate as represented by formula I. The compound has good targetability, has a good inhibitory effect on HER3-positive tumor cells, and has good druggability and high safety. The antibody-drug conjugate has an inhibitory effect on HER3, has an inhibitory effect on SK-BR-3 and SW620 cells, and also has a good inhibitory effect on at least one of 22Rv1, LNCaP, NCI-H820, OVCAR-8, and HCC827 cells.

Claims

1. An antibody-drug conjugate, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of the pharmaceutically acceptable salt thereof; the antibody-drug conjugate has a structure shown in formula I; ##STR00088## wherein Ab is a HER3 antibody or a variant of the HER3 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 phenylalanine residue, alanine residue, glycine residue, glutamic acid residue, aspartic acid residue, cysteine 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 ##STR00089## 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 ##STR00090## 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 when Ab is the HER3 antibody, the HER3 antibody is HER3 antibody A, wherein the amino acid sequence of the light chain in HER3 antibody A is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 2; or, when Ab is the variant of the HER3 antibody, the variant of the HER3 antibody is a variant of the HER3 antibody A being at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the HER3 antibody A; the amino acid sequence of the light chain in HER3 antibody A is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 2; or, when D is the cytotoxic drug topoisomerase inhibitor, the cytotoxic drug topoisomerase inhibitor is ##STR00091## 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.

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 the amino acid sequence of the light chain in the variant of HER3 antibody A is shown in SEQ ID NO: 1; or, the amino acid sequence of the heavy chain in the variant of HER3 antibody A is the amino acid sequence shown in SEQ ID NO: 2 having one or more than one site mutation of E233P, L234V, L234F, L235A, L235E, or P331S; or, the b-terminal of L.sub.3 is connected to the sulfhydryl group on the antibody in the form of a thioether bond; or, 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 C.sub.1-C.sub.4 alkyl; 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 C.sub.1-C.sub.4 alkyl; 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 C.sub.1-C.sub.4 alkyl; or, when R.sup.1 is C.sub.1-C.sub.6 alkyl substituted by one or more than one NR 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 4 to 8; or, p is 2; or, n is 8 to 12; 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 C.sub.1-C.sub.4 alkyl.

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 3, wherein the amino acid sequence of the heavy chain in the variant of HER3 antibody A is the amino acid sequence shown in SEQ ID NO: 2 having site mutations of E233P, L234V, and L235A or having site mutations of L234F, L235E, and P331S; or, 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; 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; 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 ##STR00092## 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 ##STR00093## or, m is 7.49, 7.56, 7.59, 7.60, 7.63, 7.65, 7.67, 7.72, 7.78, 7.81, 7.82, or 7.83; or, (L.sub.1).sub.p is ##STR00094## 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; 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.

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 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, 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 ##STR00095##

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 2, wherein the antibody-drug conjugate is any one of the following schemes: scheme I: Ab is HER3 antibody A or the variant of HER3 antibody A, and the amino acid sequence of the light chain in the variant of HER3 antibody A is shown in SEQ ID NO: 1; the amino acid sequence of the heavy chain in the variant of HER3 antibody A is the amino acid sequence shown in SEQ ID NO: 2 having one or more than one site mutation of E233P, L234V, L234F, L235A, L235E, or P331S; D is ##STR00096## 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; 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; 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 the phenylalanine residue, alanine residue, glycine residue, isoleucine residue, leucine residue, proline residue, and valine residue; scheme II: Ab is HER3 antibody A or the variant of HER3 antibody A, and the amino acid sequence of the light chain in the variant of HER3 antibody A is shown in SEQ ID NO: 1; the amino acid sequence of the heavy chain in the variant of HER3 antibody A is the amino acid sequence shown in SEQ ID NO: 2 having one or more than one site mutation of E233P, L234V, L234F, L235A, L235E, or P331 S; D is ##STR00097## 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; scheme III: Ab is HER3 antibody A or the variant of HER3 antibody A, and the amino acid sequence of the light chain in the variant of HER3 antibody A is shown in SEQ ID NO: 1; the amino acid sequence of the heavy chain in the variant of HER3 antibody A is the amino acid sequence shown in SEQ ID NO: 2 having one or more than one site mutation of E233P, L234V, L234F, L235A, L235E, or P331S; D is ##STR00098## 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 7 to 8; 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 the valine residue and/or the alanine residue; scheme IV: Ab is HER3 antibody A or the variant of HER3 antibody A, and the amino acid sequence of the light chain in the variant of HER3 antibody A is shown in SEQ ID NO: 1; the amino acid sequence of the heavy chain in the variant of HER3 antibody A is the amino acid sequence shown in SEQ ID NO: 2 having one or more than one site mutation of E233P, L234V, L234F, L235A, L235E, or P331S; D is ##STR00099## 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; scheme V: Ab is HER3 antibody A or the variant of HER3 antibody A, and the amino acid sequence of the light chain in the variant of HER3 antibody A is shown in SEQ ID NO: 1; the amino acid sequence of the heavy chain in the variant of HER3 antibody A is the amino acid sequence shown in SEQ ID NO: 2 having one or more than one site mutation of E233P, L234V, L234F, L235A, L235E, or P331S; D is ##STR00100## m is 7 to 8; 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 the valine residue and/or the alanine residue.

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 2, wherein the antibody-drug conjugate is any one of the following compounds: ##STR00101## ##STR00102## ##STR00103## wherein Ab is HER3 antibody A or the variant of HER3 antibody A, and m is 7.49, 7.56, 7.59, 7.60, 7.63, 7.65, 7.67, 7.72, 7.78, 7.81, or 7.83.

8. The antibody-drug conjugate, the pharmaceutically acceptable salt thereof, the solvate thereof, or the solvate of the pharmaceutically acceptable salt thereof according to claim 7, wherein the antibody-drug conjugate is any one of the following schemes: ##STR00104## ##STR00105## ##STR00106## ##STR00107## ##STR00108## wherein Ab is HER3 antibody A or the variant of HER3 antibody A; the amino acid sequence of the light chain in the variant of HER3 antibody A is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain is selected from SEQ ID NO: 3 or SEQ ID NO: 4; ##STR00109## wherein Ab is HER3 antibody A or the variant of HER3 antibody A, and m is 7.56, 7.59, 7.63, 7.67, 7.72, 7.81, or 7.83; the amino acid sequence of the light chain in the variant of HER3 antibody A is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain is selected from SEQ ID NO: 3 or SEQ ID NO: 4; ##STR00110## ##STR00111## ##STR00112## wherein Ab is HER3 antibody A or the variant of HER3 antibody A; the amino acid sequence of the light chain in the variant of HER3 antibody A is shown in SEQ ID NO: 1, the amino acid sequence of the heavy chain is selected from SEQ ID NO: 3 or SEQ ID NO: 4.

9. 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 compounds: ##STR00113## Ab is HER3 antibody A, and m is 7.56; ##STR00114## Ab is the variant of HER3 antibody A; the amino acid sequence of the light chain in the variant of HER3 antibody A is shown in SEQ ID NO: 1, and the heavy chain is the amino acid sequence shown in SEQ ID NO: 3, and m is 7.67; ##STR00115## Ab is the variant of HER3 antibody A; the amino acid sequence of the light chain in the variant of HER3 antibody A is shown in SEQ ID NO: 1, and the heavy chain is the amino acid sequence shown in SEQ ID NO: 4, and m is 7.72; ##STR00116## Ab is HER3 antibody A, and m is 7.63; ##STR00117## Ab is the variant of HER3 antibody A; the amino acid sequence of the light chain in the variant of HER3 antibody A is shown in SEQ ID NO: 1, and the heavy chain is the amino acid sequence shown in SEQ ID NO: 3, and m is 7.59; ##STR00118## Ab is the variant of HER3 antibody A; the amino acid sequence of the light chain in the variant of HER3 antibody A is shown in SEQ ID NO: 1, and the heavy chain is the amino acid sequence shown in SEQ ID NO: 4, and m is 7.67; ##STR00119## Ab is HER3 antibody A, and m is 7.81; ##STR00120## Ab is the variant of HER3 antibody A; the amino acid sequence of the light chain in the variant of HER3 antibody A is shown in SEQ ID NO: 1, and the heavy chain is the amino acid sequence shown in SEQ ID NO: 3, and m is 7.63; ##STR00121## Ab is the variant of HER3 antibody A; the amino acid sequence of the light chain in the variant of HER3 antibody A is shown in SEQ ID NO: 1, and the heavy chain is the amino acid sequence shown in SEQ ID NO: 4, and m is 7.821 ##STR00122## Ab is HER3 antibody A, and m is 7.56; ##STR00123## Ab is HER3 antibody A, and m is 7.83; ##STR00124## Ab is HER3 antibody A, and m is 7.49; ##STR00125## Ab is HER3 antibody A, and m is 7.60; ##STR00126## Ab is HER3 antibody A, and m is 7.78; ##STR00127## Ab is HER3 antibody A, and m is 7.65; ##STR00128## Ab is HER3 antibody A, and m is 7.83; or ##STR00129## Ab is HER3 antibody A, and m is 7.82.

10. (canceled)

11. A method for preparing the antibody-drug conjugate according to claim 1, comprising the following step: carrying out a coupling reaction between a compound of formula II and Ab-hydrogen as shown below; ##STR00130##

12. 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.

13-15. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0134] FIG. 1 shows the construction of expression vectors for the light and heavy chains of an 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

[0135] The present disclosure will be further described below with reference to embodiments, but the present disclosure is not therefore limited to the scope of the embodiment. Experimental methods without specific conditions in the following embodiments are selected according to conventional methods and conditions, or according to the commercial specification.

DESCRIPTION OF ABBREVIATIONS

[0136] PCR polymerase chain reaction [0137] CHO Chinese hamster ovary cells [0138] HTRF homogeneous time-resolved fluorescence [0139] PB phosphate buffer [0140] EDTA ethylenediaminetetraacetic acid [0141] TECP tris(2-carboxyethyl)phosphine [0142] DMSO dimethyl sulfoxide [0143] DMF N,N-dimethylformamide [0144] HATU 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate [0145] v/v V/V, volume ratio [0146] UV ultraviolet visible light [0147] ELISA enzyme-linked immunosorbent assay [0148] BSA bovine serum albumin [0149] rpm revolutions per minute [0150] FBS fetal bovine serum

Example 1: Preparation of HER3 Antibody

[0151] In the present disclosure, the monoclonal antibody FDA026 with high affinity and specific targeting HER3 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 FDA026 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 FDA026-L/pV81 and the antibody heavy chain eukaryotic expression plasmid FDA026-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 sediment. The supernatant was collected and filtered through a 0.22 m filter membrane. The treated sample was purified using a Mab Select affinity chromatography column (purchased from GE) to obtain antibody FDA026.

[0152] Same preparation method as described above was used to prepare HER3 antibody FDA028 with Fc mutations (i.e., E233P, L234V, L235A) (the light chain amino acid sequence is shown in SEQ ID NO: 1 and the heavy chain sequence is shown in SEQ ID NO: 3) and HER3 antibody FDA029 with Fc mutations (i.e., L234F, L235E, P331S) (the amino acid sequence of the light chain is shown in SEQ ID NO: 1, and the heavy chain sequence is shown in SEQ ID NO: 4).

[0153] The light chain sequences of FDA026, FDA028, and FDA029 are shown below:

TABLE-US-00001 SEQIDNO:1: DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPP 50 KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYST 100 PRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA 150 KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC 200 EVTHQGLSSPVTKSFNRGEC 220

[0154] The heavy chain sequence of FDA026 is shown below:

TABLE-US-00002 SEQIDNO:2: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGE 50 INHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKW 100 TWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF 150 PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC 200 NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT 250 LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY 300 RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT 350 LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS 400 DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 447

[0155] The heavy chain sequence of FDA028 is shown below:

TABLE-US-00003 FDA028heavychain SEQIDNO:3 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIG EINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARD KWTWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

[0156] The heavy chain sequence of FDA029 is shown below:

TABLE-US-00004 FDA029heavychain SEQIDNO:4 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIG EINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARD KWTWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

Example 2: Synthesis of Linker-Drug Conjugates

Example 2-1: Synthesis of LE12

[0157] ##STR00060## ##STR00061##

[0158] Synthesis of Intermediate 2:

[0159] (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).

[0160] Synthesis of Intermediate 3:

[0161] 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).

[0162] Synthesis of Intermediate 5:

[0163] 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).

[0164] Synthesis of Intermediate 6:

[0165] 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 86%), ESI-MS m/z: 396 (M+H).

[0166] Synthesis of Intermediate 8:

[0167] 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.

[0168] The crude product obtained from the previous step was dissolved in 50 mL of anhydrous N,N-dimethylformamide, and then Fmoc-L-valine N-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).

[0169] Synthesis of Intermediate 9:

[0170] 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).

[0171] Synthesis of Compound LE12:

[0172] 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

[0173] ##STR00062## ##STR00063##

[0174] Synthesis of Intermediate 14

[0175] The 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 solution 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).

[0176] Synthesis of Intermediate 15

[0177] 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.

[0178] Synthesis of Intermediate 16

[0179] 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, yield of 83%), ESI-MS m/z: 840 (M+H).

[0180] Synthesis of Intermediate 17

[0181] 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).

[0182] Synthesis of Compound LE13

[0183] 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

[0184] ##STR00064## ##STR00065##

[0185] Synthesis of Intermediate 19

[0186] The commercially available intermediate 18 (300 mg, 0.8 mmol) was mixed with paraformaldehyde (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 mL, 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).

[0187] Synthesis of Intermediate 20

[0188] 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.

[0189] 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).

[0190] Synthesis of Intermediate 21

[0191] 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).

[0192] Synthesis of Compound LE14

[0193] 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

[0194] ##STR00066##

[0195] Intermediate VI could be prepared by replacing the methylamine hydrochloride in step 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 the obtained product was 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-00005 TABLE 1 Intermediates used for the synthesis of LE15 to LE20 Product R.sup.1 Amino Compound Maleimide Structure [00067] LE15 [00068] Methylsulfonyl ethylamine hydrochloride [00069] LE16 [00070] Methylsulfonyl ethylamine hydrochloride [00071] LE17 [00072] Dimethylethyl amine hydrochloride [00073] LE18 [00074] Methylsulfonyl ethylamine hydrochloride [00075] LE19 [00076] Methylsulfonyl ethylamine hydrochloride [00077] LE20 [00078] Methylsulfonyl ethylamine hydrochloride [00079] [00080][00081][00082]

Example 2-5: Synthesis of Compounds LE21 and LE22

[0196] ##STR00083## ##STR00084##

[0197] Synthesis of Compound DXD-1

[0198] 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).

[0199] Preparation of Intermediate V

[0200] 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.

[0201] Synthesis of LE21 to LE22

[0202] 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 N-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).

##STR00085##

Example 2-6: Synthesis of Compound LS13

[0203] Referring to the synthesis method of LEIS in Examples 2 to 4, SN-38 (7-ethyl-10-hydroxycamptothecin) was reacted with intermediate VII (10 is methyl sulfonyl 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, 10H).

##STR00086##

Example 2-7: Synthesis of Compound GGFG-Dxd

[0204] 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).

##STR00087##

Example 3: Preparation of Antibody-Drug Conjugates

[0205] The antibodies FDA026, FDA028, and FDA029 against HER3 were prepared according to the method of Example 1 and were respectively exchanged into 50 mM PB/1.0 mM EDTA buffer (pH 7.0) using a G25 desalting column. 12 equivalents of TECP were added thereto and the mixture was stirred at 37 C. for 2 hours to fully open the disulfide bonds between the antibody chains. Then, phosphoric acid was used to adjust the pH of the reduced antibody solution to 6.0 and the temperature of the water bath was lowered to 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 12 equivalents of linker-drug conjugate were added dropwise to the reduced antibody solution. Additional DMSO was added to a final concentration of 10% (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 50 mM PB/1.0 mM EDTA solution (pH 6.0). After purification, a final concentration of 6% sucrose was added and 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.

[0206] 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-00006 TABLE 2 Coupling conditions for preparing different antibody-drug conjugates (ADCs) Linker-Drug DAR Whether Aggregation Recovery ADC Number Antibody Conjugate Value Precipitation Ratio Rate FDA026-1402 FDA026 GGFG-Dxd 7.76 No 0.5% 90% FDA026-LE12 FDA026 LE12 7.56 No 0.6% 88% FDA026-LE13 FDA026 LE13 7.63 No 0.1% 90% FDA026-LE14 FDA026 LE14 7.81 No 0.1% 92% FDA026-LE15 FDA026 LE15 7.56 No 0.2% 90% FDA026-LE16 FDA026 LE16 7.83 No 0.2% 82% FDA026-LE17 FDA026 LE17 7.49 No 0.2% 82% FDA026-LE18 FDA026 LE18 7.60 No 0.3% 93% FDA026-LE19 FDA026 LE19 7.78 No 0.2% 88% FDA026-LE20 FDA026 LE20 7.65 No 0.2% 84% FDA026-LE21 FDA026 LE21 7.83 No 0.3% 91% FDA026-LE22 FDA026 LE22 7.72 No 0.4% 89% FDA026-LS13 FDA026 LS13 7.68 No 0.5% 92% FDA028-LE12 FDA028 LE12 7.67 No 0.6% 88% FDA028-LE13 FDA028 LE13 7.59 No 0.1% 90% FDA028-LE14 FDA028 LE14 7.63 No 0.1% 92% FDA029-LE12 FDA029 LE12 7.72 No 0.3% 89% FDA029-LE13 FDA029 LE13 7.67 No 0.2% 91% FDA029-LE14 FDA029 LE14 7.82 No 0.2% 90% / indicates that the recovery rate is not calculated

[0207] In practical research, it was found that the linker-drug conjugate GGFG-Dxd produces precipitation when coupled with other antibodies and has a high aggregation ratio, which is not universal. However, when the linker-drug conjugates of the present technical solution were attempted to be coupled with different antibodies, no precipitation was produced and the aggregation ratio was within the normal range, indicating that the linker-drug conjugates provided by the present disclosure have better physicochemical properties.

Effect Example 1: Evaluation of Binding Ability Between Antibody-Drug Conjugates and Antigens

[0208] The binding ability of FDA026 antibody to Her3 antigen before and after conjugation with linker-drug conjugate LE12-LE22 was evaluated by competitive ELISA method. The specific method is as follows: 100 ng/mL HER3 antigen (purchased from Sino Biological, product number: 10201-H8H) was coated on a hydrophilic ELISA plate strip at 100 L/well and blocked with 3% BSA at room temperature for 2 hours. 33 ng/mL biotin-labeled FDA026 antibody (DAR-2.82) was pre-mixed at a ratio of 1:1 with a series of final concentrations (100000, 10000, 1000, 400, 160, 64, 3.2, and 0.16 ng/mL) of FDA026 antibody and the antibody-drug conjugate prepared in Example 3, and then added to the ELISA plate coated with HER3 antigen. The plate was shaken horizontally at 200 rpm for 1 hour at room temperature. Pierce High Sensitivity Streptavidin-HRP, Pre-Diluted enzyme-linked antibody (Thermo, product number: 21134) (dilution ratio of 1:500) was added at 100 L/well and Sigma's TMB color developing solution (Sigma, product number: T0440) was used for color development for 25 minutes before being terminated with 1 mol/L H.sub.2SO.sub.4. The reference wavelength of the microplate reader was 650 nm, and the absorbance reading was measured at 450 nm. The results (as shown in Table 3) showed that there was no difference in the binding activity to the HER3 antigen before and after coupling of the FDA026 antibody with the linker-drug conjugate. Using the same method, the binding ability of the antibodies FDA028 and FDA029 to the HER3 antigen before and after coupling with the linker-drug conjugate was evaluated separately. The results (as shown in Table 3) indicated that there was no difference in the binding activity to the HER3 antigen before and after coupling of the antibodies FDA028 and FDA029 with the linker-drug conjugate.

TABLE-US-00007 TABLE 3 Data on the binding activity of HER3 antibody to HER3 before and after coupling with linker-drug conjugate Concentration ng/ml OD.sub.450-650 100000 10000 1000 400 160 64 3.2 0.32 Antibody 0.051 0.1 0.319 1.254 2.845 3.573 3.766 3.586 FDA026 FDA026-LE12 0.046 0.11 0.33 1.253 2.794 3.567 3.745 3.546 FDA026-LE13 0.054 0.09 0.32 1.247 2.764 3.613 3.756 3.596 FDA026-LE14 0.06 0.098 0.347 1.173 2.722 3.507 3.673 3.616 FDA026-LE15 0.052 0.11 0.33 1.25 2.85 3.58 3.766 3.58 FDA026-LE16 0.045 0.11 0.32 1.24 2.784 3.557 3.735 3.556 FDA026-LE17 0.055 0.09 0.33 1.253 2.767 3.623 3.766 3.58 FDA026-LE18 0.06 0.099 0.35 1.176 2.73 3.517 3.663 3.676 FDA026-LE19 0.053 0.011 0.035 1.182 2.83 3.521 3.665 3.681 FDA026-LE20 0.051 0.01 0.035 0.179 2.77 3.632 3.712 3.562 FDA026-LE21 0.056 0.089 0.365 1.182 2.783 3.567 3.723 3.602 FDA026-LE22 0.066 0.088 0.344 1.234 2.812 3.516 3.716 3.613 Antibody 0.053 0.11 0.33 1.25 2.86 3.56 3.78 3.53 FDA028 FDA028-LE12 0.051 0.13 0.31 1.26 2.79 3.61 3.77 3.54 FDA028-LE13 0.055 0.012 0.32 1.26 2.78 3.62 3.72 3.55 FDA028-LE14 0.06 0.099 0.34 1.25 2.77 3.63 3.75 3.62 Antibody 0.055 0.13 0.32 1.23 2.79 3.61 3.77 3.53 FDA029 FDA029-LE12 0.053 0.11 0.357 1.272 2.781 3.651 3.765 3.63 FDA029-LE13 0.062 0.09 0.361 1.278 2.763 3.631 3.771 3.64 FDA029-LE14 0.05 0.11 0.372 1.281 2.771 3.661 3.782 3.65

Effect Example 2: In Vitro Killing Activity Evaluation of Antibody-Drug Conjugates

[0209] SK-BR-3 (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, 0.08, 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 4.

[0210] Using the same method as above, the cytotoxic killing activity of each antibody-drug conjugate against multiple tumor cells purchased from ATCC including 22Rv1, LNCaP, SW620, NCI-H820, OVCAR-8, and HCC827 was tested. The results are shown in Table 4. From the results in Table 4, it can be seen that the antibody-drug conjugates provided by the present disclosure have excellent in vitro cytotoxic activity against SK-BR-3, 22Rv1, LNCaP, SW620, NCI-H820, OVCAR-8, and HCC827 cells, etc.

TABLE-US-00008 TABLE 4 In vitro killing activity of antibody-drug conjugates IC50 (nM) ADC SK-BR-3 22Rv1 LNCaP SW620 NCI-H820 OVCAR-8 HCC827 Number Cell Cell Cell Cell Cell Cell Cell FDA026- 37.8 52.32 97.64 56.33 38.67 Not Tested Not Tested LE12 FDA026- 42.4 64.36 87.77 67.54 Not Tested 54.33 Not Tested LE13 FDA026- Not Tested 37.5 85.6 36.5 38.4 50.23 62.38 LE14 FDA026- 53.12 Not Tested Not Tested 47.86 Not Tested Not Tested Not Tested LE15 FDA026- 46.18 Not Tested Not Tested 53.12 Not Tested Not Tested Not Tested LE16 FDA026- 40.36 Not Tested Not Tested 42.38 Not Tested Not Tested Not Tested LE17 FDA026- 45.32 Not Tested Not Tested 41.69 Not Tested Not Tested Not Tested LE18 FDA026- 52.38 Not Tested Not Tested 40.28 Not Tested Not Tested Not Tested LE19 FDA026- 46.15 Not Tested Not Tested 52.33 Not Tested Not Tested Not Tested LE20 FDA026- 46.3 98.26 Not Tested Not Tested 89.45 Not Tested Not Tested LE21 FDA026- 52.7 87.65 Not Tested Not Tested 78.44 89.34 96.75 LE22 FDA026- 43.2 36.9 93.26 40.20 41.64 55.46 70.75 1402 FDA026- >1 M >600 Not Tested >500 Not Tested Not Tested Not Tested LS13 FDA028- 38.2 55.3 96.5 56.45 38.76 Not Tested Not Tested LE12 FDA028- 42.4 65.2 88.23 67.82 Not Tested 54.45 Not Tested LE13 FDA028- Not Tested 37.2 93.28 41.2 43.12 55.75 70.13 LE14 FDA029- 38.45 55.34 96.8 56.52 38.68 Not Tested Not Tested LE12 FDA029- 42.35 66.1 88.34 67.75 Not Tested 54.56 Not Tested LE13 FDA029- Not Tested 37.6 93.32 41.6 43.23 55.87 70.25 LE14

Effect Example 3: Effects of Mutations at Fc-Terminal of Antibodies in Antibody-Drug Conjugates on Binding Activity and Cytotoxicity of Cells Expressing CD32a Receptors

Effect Example 3-1: Effects of Mutations at Fc-Terminal of Antibodies in Antibody Drug Conjugates on Binding Activity of Cells Expressing CD32a Receptors

[0211] Using the HTRF method to analyze the binding ability of the prepared ADC to cells expressing CD32a, HEK293 cells (ATCC) were quickly shaken in a 37 C. water bath to resuscitate the cells, centrifuged and the supernatant was removed, and 1.1 mL of 1 Tag-lite buffer was added to resuspend. 10 L of cells were seeded into a 384-well plate per well. Then, in the wells where cells were added, pre-prepared antibody-drug conjugate samples such as FDA026-LE14, FDA028-LE14, and FDA029-LE14 were added at a series dilution concentration (100000, 3333.33, 1111.11, 370.37, 123.46, 41.15, 13.72, 4.57, and 1.52 nM) at 5 L per well into a 384-well plate. Finally, 5 L of d2-labeled immunoglobulin IgG-d2 conjugate was added to each well of the above plate. This IgG-d2 conjugate could bind to CD32a and was incubated for 3 hours at room temperature in the dark to produce a FRET signal. After adding the above ADC samples to the plate, the ADC samples could competitively bind to CD32a with labeled IgG, reducing its FRET signal. Readings were taken using a SpectraMax Paradigm microplate reader with an excitation wavelength of 340 nm, a reference wavelength of 616 nm and a characteristic emission wavelength of 665 nm. The absorbance was measured and its IC50 value was calculated. The results are shown in Table 5. The binding ability of each antibody-drug conjugate to CD32 is shown in Table 5; compared with FDA026-LE14 that has not been modified, the binding ability of FDA028-LE14 and FDA029-LE14 that have been modified in Fc-terminal amino acids is weaker (about 10%); indicating that the binding of antibody-drug conjugates modified in Fc-terminal amino acids to CD32a is weaker.

TABLE-US-00009 TABLE 5 Binding ability of antibody-drug conjugates to CD32a before and after Fc-terminal amino acid modification IC50 value (nM) ADC Number 293 Cell FDA026-LE14 52.69 FDA028-LE14 523.81 FDA029-LE14 535.42

Effect Example 3-2: Effects of Mutations at Fc-Terminal of Antibodies in Antibody-Drug Conjugates on Cytotoxicity of Cells Expressing CD32a Receptors

[0212] K562 (ATCC) cells positively expressing FcRIIa (note that the cells did not express HER3 antigen) were selected as the experimental cell line for in vitro activity detection. 5000 cells per well were seeded in a 96-well cell culture plate and cultured for 16 to 20 hours. The antibody-drug conjugates FDA026-LE14, FDA028-LE14, and FDA029-LE14 prepared as above 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 for 10 minutes. 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 5. The results show that FDA026-LE14 conjugated drugs without Fc modification have certain killing effects on K562 cells, while the killing activities of ADC drugs FDA028-LE14 and FDA029-LE14 modified with Fc modification are significantly reduced.

[0213] Using the same experimental method as the above K562 cell line, HSC cells differentiated for 4 days (purchased from stem cell company, product number: 70008.1) were selected and treated with the drug for 6 days for cytotoxic activity detection. After 6 days of treatment with antibody-drug conjugates, flow analysis of HSC cells showed that their surface antigen abundance (i.e., binding ratio, CD32a 7.4; HER3 antigen 0.8; CD34 69.0) showed high expression of CD32 and almost no expression of HER3 antigen. The results of the activity measurement are shown in Table 6. The results show that FDA026-LE14 conjugated drugs without Fc modification have certain killing effects on HSC cells, while the killing activities of ADC drugs FDA028-LE14 and FDA029-LE14 with Fc modification are significantly reduced.

TABLE-US-00010 TABLE 6 Evaluation of the killing activity of antibody- drug conjugates before and after modification of Fc-terminal amino acid on K562 and HSC cells IC50 value (nM) ADC Number K562 Cell HSC Cell FDA026-LE14 16.65 31.6 FDA028-LE14 270.42 334 FDA029-LE14 323.35 363

Effect Example 4: In Vitro Plasma Stability Assay

[0214] 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 7.

TABLE-US-00011 TABLE 7 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 FDA026-1402 0.2% 0.5% 0.8% 1.3% 1.5% 2.2% FDA026-LE12 0.2% 0.6% 0.7% 1.3% 1.5% 2.2% FDA026-LE13 0.1% 0.5% 0.6% 1.2% 1.4% 2.3% FDA026-LE14 0.1% 0.2% 0.5% 1.1% 1.3% 2.0% FDA026-LS13 0.3% 0.7% 2.3% 3.8% 4.2% 5.2% FDA028-LE12 0.2% 0.5% 0.6% 1.2% 1.4% 2.1% FDA028-LE13 0.1% 0.7% 0.6% 1.3% 1.3% 2.2% FDA028-LE14 0.1% 0.3% 0.5% 1.1% 1.2% 2.0% FDA029-LE12 0.2% 0.5% 0.6% 1.1% 1.3% 2.2% FDA029-LE13 0.1% 0.7% 0.5% 1.2% 1.2% 2.2% FDA029-LE14 0.1% 0.2% 0.4% 1.0% 1.1% 2.1%

[0215] The plasma stability results show that the stability of the ADC obtained using the new technical solution is not inferior to FDA026-1402, 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 FDA026-1402.

Effect Example 5: In Vitro Enzyme Digestion Experiment of Linker-Drug Conjugates

[0216] The linker-drug conjugate (LE14 and GGFG-Dxd) was co-incubated with tissue proteinase 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 8) 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-00012 TABLE 8 In vitro enzyme digestion of LE14 and GGFG-Dxd at different pH Percentage of drug release in the sample % GGFG-Dxd LE14 Time (h) pH 5.0 pH 6.0 pH 7.0 pH 5.0 pH 6.0 pH 7.0 0 21.62 23.58 22.98 15 14.28 17.59 1 25 24.8 26.53 96.93 95.98 98.05 2 25.85 27.02 29.52 98.35 96.8 99.08 3 27.76 29.29 31.95 99.01 98.45 99.33 4 29.72 31.37 34.78 99.21 98.81 99.2 5 31.69 33.05 36.17 99.32 98.9 100 6 34.17 35.95 38.25 97.39 99 99.39

Effect Example 6: In Vitro Enzyme Digestion Experiment of FDA026-LS13

[0217] SK-BR-3 cell line was selected as the experimental cells. After the sample was incubated in tissue proteinase 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 on 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.

[0218] The above enzyme digestion samples obtained by incubating in a tissue proteinase 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%.

[0219] The experimental results (as shown in Table 9) show that after enzyme digestion treatment, the cytotoxic activity of FDA026-LS13 is almost the same as that of SN-38 at an equivalent dose, which also indicates that FDA026-LS13 has almost completely released SN-38 under the action of tissue proteinase B and played a role. However, FDA026-LS13 may have undergone unpredictable changes when it is endocytosed into lysosomes, resulting in SN-38 not being able to function effectively.

TABLE-US-00013 TABLE 9 Changes in killing activity of FDA026-LS13 on NCI-N87 cell line before and after enzyme digestion by tissue proteinase B system IC50 (based on SN-38 equivalent, nM) Before Enzyme After Enzyme Sample Digestion Digestion FDA026-LS13 >100 nM 6.56 nM SN-38 7.13 nM 7.32 nM

Effect Example 7: Testing Antitumor Activity of FDA026-LE14 in SW620 Human Colorectal Cancer Model

[0220] 6 to 8 week old female Balb/c nude mice were subcutaneously injected with 510.sup.6 human colorectal cancer cells (SW620) 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 8 groups according to tumor size and mouse weight, with 6 animals in each group. The groups were blank control group, Irinotecan hydrochloride group (100 mg/kg), and antibody-drug conjugate FDA026-1402, FDA028-LE14 and FDA026-LE14, respectively, with two dose groups of 5.0 mg/kg and 10.0 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 9, the average tumor volume of the Irinotecan hydrochloride (100 mg/kg) treatment group was 1120.09 mm.sup.3 on the 14th day after the end of treatment (i.e., the 46th day of observation, same below), while the average tumor volume of the blank control group was 2074.5 mm.sup.3 at the end of treatment. The average tumor volume of the FDA026-1402 treatment group at 5.0 mg/kg was 260.87 mm.sup.3 on the 14th day after the end of treatment, and the average tumor volume of the FDA026-1402 treatment group at 10 mg/kg was 0.00 mm.sup.3 on the 14th day after the end of treatment. The average tumor volume of the FDA026-LE14 treatment group at 5.0 mg/kg was 13.79 mm.sup.3 on the 14th day after the end of treatment, and the average tumor volume of the FDA026-LE14 treatment group at 10 mg/kg was 0.00 mm.sup.3 on the 14th day after the end of treatment. The average tumor volume of the FDA028-LE14 treatment group at 5.0 mg/kg was 9.86 mm.sup.3 on the 14th day after the end of treatment, and the average tumor volume of the FDA028-LE14 treatment group at 10 mg/kg was 0.00 mm.sup.3 on the 14th day after the end of treatment. The experimental results show that both FDA026-LE14 and FDA028-LE14 have good antitumor activity in vivo, and all experimental mice have no death or weight loss, indicating that FDA026-LE14 has good safety.

TABLE-US-00014 TABLE 10 Antitumor activity of FDA026-LE14 in SW620 human colorectal cancer model Observation Days Grouping 11 14 18 21 25 28 32 35 39 42 46 Average Tumor Volume/mm3 01 159.75 284.67 538.37 797.32 1458.10 1740.41 2074.54 / / / / 02 159.35 265.09 267.78 259.26 199.88 160.40 104.56 37.81 28.72 22.45 13.79 03 159.71 201.19 227.81 139.61 60.68 25.75 3.83 3.28 0.00 0.00 0.00 04 159.83 221.27 264.94 321.40 404.98 481.97 412.12 351.12 229.20 205.81 260.87 05 159.84 238.08 255.34 169.81 89.27 60.48 33.85 24.88 11.50 6.91 0.00 06 159.63 266.32 256.78 232.53 185.88 155.32 100.42 34.16 25.48 18.42 9.86 07 159.73 200.56 228.32 138.87 59.32 27.32 3.15 2.12 0.00 0.00 0.00 08 159.41 227.21 362.76 509.97 667.07 778.01 864.41 875.35 895.60 1017.45 1120.09 Standard Deviation 01 10.37 31.73 32.42 77.06 85.50 81.66 92.36 / / / / 02 10.81 30.80 21.55 22.67 31.96 34.21 20.45 10.13 8.38 6.24 6.25 03 11.03 23.15 33.03 29.12 13.67 7.39 3.83 3.28 0.00 0.00 0.00 04 10.74 27.49 32.75 53.43 65.56 87.31 85.57 90.69 63.81 69.14 86.58 05 10.93 18.63 20.43 21.58 12.07 7.35 9.19 6.84 5.87 4.37 0.00 06 10.82 25.36 26.54 25.36 23.46 26.57 19.23 9.47 8.53 5.47 5.32 07 11.31 18.34 22.01 21.32 12.01 7.12 5.32 5.93 0.00 0.00 0.00 08 10.40 31.79 63.03 98.30 119.71 162.30 206.93 204.40 218.88 253.08 273.36 Note: Group 01 is the blank control group; group 02 is the FDA026-LE14 group with a dose of 5 mg/kg; group 03 is the FDA026-LE14 group with a dose of 10 mg/kg; group 04 is the FDA026-1402 group with a dose of 5 mg/kg; group 05 is the FDA026-1402 group with a dose of 10 mg/kg; group 06 is the FDA028-LE14 group with a dose of 5 mg/kg; group 07 is the FDA028-LE14 group with a dose of 10 mg/kg; group 08 is the Irinotecan hydrochloride group with a dose of 100 mg/kg.