DRUG CONJUGATE OF ERIBULIN DERIVATIVE, PREPARATION METHOD THEREFOR AND APPLICATION THEREOF IN MEDICINE

Abstract

The present disclosure relates to a drug conjugate of an Eribulin derivative, a preparation method therefor and an application thereof in medicine. Specifically, provided is an antibody-drug conjugate, which contains an Eribulin derivative drug portion. The present disclosure further relates to a method for treating cancer by administering the antibody-drug conjugate provided herein.

Claims

1. An antibody-drug conjugate having a structure of formula (I) or a pharmaceutically acceptable salt or solvate thereof:
Ab-(L-D).sub.k   (I) wherein, Ab is an antibody or an antigen-binding fragment thereof, L is a linker covalently linking Ab to D, and k is 1 to 20, -D is shown as in the formula below: ##STR00128## wherein R.sup.1a is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl and heteroaryl, and the alkyl, cycloalkyl, aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, preferably methyl; R.sup.1b is selected from the group consisting of hydrogen, alkyl, alkoxy, cycloalkyl, aryl and heteroaryl, and the alkyl, cycloalkyl, aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, preferably hydrogen; or R.sup.1a and R.sup.1b, together with carbon atoms connected thereto, form 5-8 membered heterocycloalkyl, and the heterocycloalkyl is optionally substituted with one or more substituents in alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, wherein R.sup.1a and R.sup.1b are not hydrogen at the same time.

2. (canceled)

3. The antibody-drug conjugate according to claim 1, wherein the linker comprises a cleavable peptide moiety.

4. The antibody-drug conjugate according to claim 3, wherein the cleavable peptide moiety is capable of being cleaved by an enzyme.

5. (canceled)

6. The antibody-drug conjugate according to claim 1, wherein the linker comprises a cleavable sulfonamide moiety or a disulfide moiety.

7. (canceled)

8. (canceled)

9. The antibody-drug conjugate according to claim 1, wherein the linker comprises a spacer unit linking to D.

10. The antibody-drug conjugate according to claim 9, wherein the spacer unit comprises p-aminobenzyloxycarbonyl (PAB).

11. (canceled)

12. The antibody-drug conjugate according to claim 9, wherein the spacer unit comprises the following moieties selected from the group consisting of: —(CR.sup.aR.sup.b).sub.m1—O(CR.sup.aR.sup.b).sub.m2—CR.sup.8R.sup.9—C(O)—, —(CR.sup.aR.sup.b).sub.m1NH—(CR.sup.aR.sup.b).sub.m2—CR.sup.8R.sup.9—C(O)—, —(CR.sup.aR.sup.b).sub.m1O—CR.sup.8R.sup.9(CR.sup.aR.sup.b).sub.m2—, —(CR.sup.aR.sup.b).sub.m1OCR.sup.8R.sup.9—C(O)—, —(CR.sup.aR.sup.b).sub.m1—O—(CR.sup.aR.sup.b).sub.m2C(O)— and —(CR.sup.aR.sup.b).sub.m1—S—(CR.sup.aR.sup.b).sub.m2—CR.sup.8R.sup.9—C(O)—, wherein R.sup.a and R.sup.b are identical or different and are each independently selected from the group consisting of hydrogen, deuterium, halogen and alkyl; R.sup.8 is selected from the group consisting of hydrogen, C.sub.3-6 cycloalkylalkyl and C.sub.3-6 cycloalkyl; R.sup.9 is selected from the group consisting of hydrogen, haloalkyl and C.sub.3-6 cycloalkyl, preferably hydrogen; or R.sup.8 and R.sup.9, together with carbon atoms connected thereto, form C.sub.3-6 cycloalkyl; m1 and m2 are each independently selected from the group consisting of 0, 1, 2 and 3.

13. (canceled)

14. The antibody-drug conjugate according claim 1, wherein L-D is a chemical moiety represented by formula below:
-Str-(Pep)-Sp-D Str is a stretching unit covalently linking to Ab, Sp is a spacer unit, Pep is selected from the group consisting of an amino acid unit, a disulfide moiety, a sulfonamide moiety and the following non-peptidic chemical moiety: ##STR00129## wherein W is —NH-heterocycloalkyl- or heterocycloalkyl; Y is heteroaryl, aryl, —C(O)C.sub.1-6 alkylene, C.sub.2-6 alkenylene, C.sub.1-6 alkylene or —C.sub.1-6 alkylene-NH—; each R.sup.2 is independently selected from the group consisting of C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.1-6 alkylene-NH.sub.2, —(C.sub.1-10 alkylene)NHC(NH)NH.sub.2 and —(C.sub.1-10 alkylene)NHC(O)NH.sub.2; R.sup.3 and R.sup.4 are each independently H, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, arylalkyl and heteroarylalkyl, or R.sup.3 and R.sup.4 together may form C.sub.3-7 cycloalkyl; R.sup.5 and R.sup.6 are each independently C.sub.1-10 alkyl, C.sub.2-10 alkenyl, arylalkyl, heteroarylalkyl and (C.sub.1-10 alkyl)OCH.sub.2—, or R.sup.5 and R.sup.6 together form a C.sub.3-7 cycloalkyl ring.

15. (canceled)

16. The antibody-drug conjugate according to claim 14, wherein Str is selected from a chemical moiety represented by the following formula: ##STR00130## wherein R.sup.7 is selected from the group consisting of -W1-C(O)—, —C(O)—W1-C(O)—, —(CH.sub.2CH.sub.2O).sub.p1C(O)—, —(CH.sub.2CH.sub.2O).sub.p1CH.sub.2C(O)— and —(CH.sub.2CH.sub.2O).sub.p1CH.sub.2CH.sub.2C(O)—, wherein W1 is selected from the group consisting of C.sub.1-8 alkylene, C.sub.1-8 alkylene-cycloalkyl and linear heteroalkyl of 1 to 8 atoms, and the heteroalkyl comprises 1 to 3 heteroatoms selected from the group consisting of N, O and S, wherein the C.sub.1-8 alkylene, cycloalkyl and linear heteroalkyl are each independently optionally further substituted with one or more substituents selected from the group consisting of halogen, deuterium, hydroxy, cyano, amino, alkyl, haloalkyl, deuterated alkyl, alkoxy and cycloalkyl; L.sup.1 is selected from the group consisting of —NR.sup.10(CH.sub.2CH.sub.2O).sub.p1CH.sub.2CH.sub.2C(O)—, —NR.sup.10(CH.sub.2CH.sub.2O).sub.p1CH.sub.2C(O)—, —S(CH.sub.2).sub.p1C(O)—, —(CH.sub.2).sub.p1C(O)— and a chemical bond, preferably a chemical bond; wherein, p1 is an integer from 1 to 20, and R.sup.10 is selected from the group consisting of hydrogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl.

17. (canceled)

18. (canceled)

19. The antibody-drug conjugate according to claim 16, wherein the linker L comprises: maleimide-(PEG).sub.2-Val-Cit, maleimide-(PEG).sub.6-Val-Cit, maleimide-(PEG).sub.8-Val-Cit, maleimide-(PEG).sub.4-CH.sub.2CH.sub.2C(O)-Val-lys, maleimide-(CH.sub.2).sub.5-Val-Cit, maleimide-(CH.sub.2).sub.5-Val-lys, maleimide-(CH.sub.2).sub.5-Gly-Gly-Phe-Gly, maleimide-(PEG).sub.2-Ala-Ala-Asn, maleimide-(PEG).sub.6-Ala-Ala-Asn, maleimide-(PEG).sub.8-Ala-Ala-Asn, maleimide-(PEG).sub.4-triazole-(PEG).sub.3-sulfonamide, maleimide-(PEG).sub.2-CH.sub.2CH.sub.2C(O)-Val-lys, maleimide-(PEG).sub.4-triazole-(PEG).sub.3-sulfonamide or Mal-(PEG).sub.4-triazole-(PEG).sub.3-disulfide.

20. (canceled)

21. (canceled)

22. The antibody-drug conjugate according to claim 14, wherein L-D is represented by a formula selected from the group consisting of: ##STR00131## wherein R.sup.2 is C.sub.1-6 alkyl, (C.sub.1-6 alkylene)NHC(NH)NH.sub.2 or (C.sub.1-6 alkylene)NHC(O)NH.sub.2; ##STR00132## wherein R.sup.2 is C.sub.1-6 alkyl, (C.sub.1-6 alkylene)NHC(NH)NH.sub.2 or (C.sub.1-6 alkylene)NHC(O)NH.sub.2; ##STR00133## wherein R.sup.2 is C.sub.1-6 alkyl, C.sub.2-6 alkenylene, (C.sub.1-6 alkylene)NHC(NH)NH.sub.2 or (C.sub.1-6 alkylene NHC(O)NH.sub.2; ##STR00134## wherein R.sup.2 is C.sub.1-6 alkyl, C.sub.2-6 alkenylene, (C.sub.1-6 alkylene)NHC(NH)NH.sub.2 or (C.sub.1-6 alkylene)NHC(O)NH.sub.2; ##STR00135## wherein R.sup.2 is C.sub.1-6 alkyl, (C.sub.1-6 alkylene)NHC(NH)NH.sub.2 or (C.sub.1-6 alkylene)NHC(O)NH.sub.2, and R.sup.5 and R.sup.6 together form a C.sub.3-7 cycloalkyl ring; ##STR00136## wherein R.sup.2 is C.sub.1-6 alkyl, (C.sub.1-6 alkylene)NHC(NH)NH.sub.2 or (C.sub.1-6 alkylene)N(CO)NH.sub.2, and R.sup.5 and R.sup.6 together form a C.sub.3-7 cycloalkyl ring; W and Str are as defined in claim 14, and D is as defined in claim 1.

23. The antibody-drug conjugate according to claim 22, wherein the antibody-drug conjugate is represented by the following formulas: ##STR00137## wherein R.sup.2 is selected from the group consisting of C.sub.1-6 alkylene-NH.sub.2, (C.sub.1-6 alkylene)NHC(NH)NH.sub.2 and (C.sub.1-6 alkylene)NHC(O)NH.sub.2, k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, p2 is selected from an integer from 2 to 6, and Y, R.sup.3 and R.sup.4 are defined as in claim 14; ##STR00138## wherein R.sup.2 is selected from the group consisting of C.sub.1-6 alkylene-NH.sub.2, (C.sub.1-6 alkylene)NHC(NH)NH.sub.2 and (C.sub.1-6 alkylene)NHC(O)NH.sub.2, k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, p2 is selected from an integer from 2 to 6, and Y, R.sup.3 and R.sup.4 are defined as in claim 14; ##STR00139## wherein R.sup.2 is C.sub.1-6 alkylene-NH.sub.2, (C.sub.1-6 alkylene)NHC(NH)NH.sub.2 or (C.sub.1-6 alkylene)NHC(O)NH.sub.2, and R.sup.5 and R.sup.6 form a C.sub.3-7 cycloalkyl ring; k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, and p2 is selected from an integer from 2 to 6; ##STR00140## wherein R.sup.2 is C.sub.1-6 alkylene-NH.sub.2, (C.sub.1-6 alkylene)NHC(NH)NH.sub.2 or (C.sub.1-6 alkylene)NHC(O)NH.sub.2, and R.sup.5 and R.sup.6 form a C.sub.3-7 cycloalkyl ring; k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, and p2 is selected from an integer from 2 to 6; Ab and D are as defined in claim 1.

24. The antibody drug conjugate according claim 12, wherein the antibody-drug conjugate is represented by the following formulas: ##STR00141## wherein R.sup.8 is selected from the group consisting of hydrogen, C.sub.3-6 cycloalkylalkyl and C.sub.3-6 cycloalkyl, preferably hydrogen; R.sup.9 is selected from the group consisting of hydrogen, haloalkyl and C.sub.3-6 cycloalkyl, preferably hydrogen, or R.sup.8 and R.sup.9, together with carbon atoms connected thereto, form C.sub.3-6 cycloalkyl, k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, and p2 is selected from an integer from 2 to 6; ##STR00142## wherein R.sup.8 is selected from the group consisting of hydrogen, C.sub.3-6 cycloalkylalkyl and C.sub.3-6 cycloalkyl, preferably hydrogen; R.sup.9 is selected from the group consisting of hydrogen, haloalkyl and C.sub.3-6 cycloalkyl, preferably hydrogen; or R.sup.8 and R.sup.9, together with carbon atoms connected thereto, form C.sub.3-6 cycloalkyl; k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, p1 is selected from the group consisting of 2, 4, 6 and 8, and p3 is selected from the group consisting of 0, 1 and 2; ##STR00143## k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, and p2 is selected from an integer from 2 to 6; ##STR00144## k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, and p2 is selected from an integer from 2 to 6; ##STR00145## k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, p1 is selected from the group consisting of 2, 4, 6 and 8, and p3 is selected from the group consisting of 0, 1 and 2; ##STR00146## k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, and p1 is selected from the group consisting of 2, 4, 6 and 8; p3 is selected from the group consisting of 0, 1 and 2; ##STR00147## k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, and p1 is selected from the group consisting of 2, 4, 6 and 8; ##STR00148## k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, and p1 is selected from the group consisting of 2, 4, 6 and 8; p3 is selected from the group consisting of 0, 1 and 2; ##STR00149## k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, and p1 is selected from the group consisting of 2, 4, 6 and 8; ##STR00150## k is selected from the group consisting o of 1 to 10 and may be an integer or a decimal, p1 is selected from the group consisting of 2, 4, 6 and 8, and p3 is selected from the group consisting of 0, 1 and 2; ##STR00151## k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, and p2 is selected from the group consisting of 2, 4, 6 and 8; ##STR00152## k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, and p2 is selected from the group consisting of 2, 4, 6 and 8: ##STR00153## k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, and p2 is selected from the group consisting of 2, 4, 6 and 8; ##STR00154## k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, and p2 is selected from the group consisting of 2, 4, 6 and 8; ##STR00155## k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, and p2 is selected from the group consisting of 2, 4, 6 and 8; ##STR00156## and may be an integer or a decimal, p1 is selected from the group consisting of 2, 4, 6 and 8, and p3 is selected from the group consisting of 0, 1 and 2; ##STR00157## k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, p1 is selected from the group consisting of 2, 4, 6 and 8, and p3 is selected from the group consisting of 0, 1 and 2; ##STR00158## wherein R.sup.8 is selected from the group consisting of hydrogen, haloalkyl and C.sub.3-6 cycloalkyl, preferably hydrogen; R.sup.9 is selected from the group consisting of hydrogen, haloalkyl and C.sub.3-6 cycloalkyl, preferably hydrogen, or R.sup.8 and R.sup.9, together with carbon atoms connected thereto, form C.sub.3-6 cycloalkyl, k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, and p2 is selected from an integer from 2 to 6; ##STR00159## wherein R.sup.8 is selected from the group consisting of hydrogen, haloalkyl and C.sub.3-6 cycloalkyl, preferably hydrogen; R.sup.9 is selected from the group consisting of hydrogen, haloalkyl and C.sub.3-6 cycloalkyl, preferably hydrogen, or R.sup.8 and R.sup.9, together with carbon atoms connected thereto, form C.sub.3-6 cycloalkyl, k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, and p2 is selected from an integer from 2 to 6; ##STR00160## wherein R.sup.8 is selected from the group consisting of hydrogen, haloalkyl and C.sub.3-6 cycloalkyl, preferably hydrogen; R.sup.9 is selected from the group consisting of hydrogen, haloalkyl and C.sub.3-6 cycloalkyl, preferably hydrogen, or R.sup.8 and R.sup.9, together with carbon atoms connected thereto, form C.sub.3-6 cycloalkyl, k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, p1 is selected from the group consisting of 2, 4, 6 and 8, and p3 is selected from the group consisting of 0, 1 and 2; ##STR00161## wherein R.sup.8 is selected from the group consisting of hydrogen, haloalkyl and C.sub.3-6 cycloalkyl, preferably hydrogen; R.sup.9 is selected from the group consisting of hydrogen, haloalkyl and C.sub.3-6 cycloalkyl, preferably hydrogen, or, R.sup.8 and R.sup.9, together with carbon atoms connected thereto, form C.sub.3-6 cycloalkyl, k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, p1 is selected from the group consisting of 2, 4, 6 and 8, and p3 is selected from the group consisting of 0, 1 and 2; Ab and D are as defined in claim 1.

25. The antibody-drug conjugate according to claim 1, wherein the antibody-drug conjugate is represented by the following formulas: ##STR00162## ##STR00163## ##STR00164## ##STR00165## ##STR00166## ##STR00167## wherein k is selected from the group consisting of 1 to 10 and may be an integer or a decimal; Ab and D are as defined in claim 1; further, R.sup.1a in D is preferably selected from methyl, and R.sup.1b in D is preferably selected from hydrogen.

26. The antibody-drug conjugate according to claim 1, wherein the antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

27. The antibody-drug conjugate according to claim 1, wherein the antibody or the antigen-binding fragment thereof is selected from the group consisting of an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD44 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUCl antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-Integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC44A4 antibody, an anti-CD79 antibody, an anti-TROP-2 antibody, an anti-CD79B antibody, an anti-Mesothelin antibody and an antigen-binding fragment thereof.

28. The antibody-drug conjugate according to claim 1, wherein the antibody or the antigen-binding fragment thereof is selected from the group consisting of Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, Glematumamab and an antigen-binding fragment thereof.

29. The antibody-drug conjugate according to claim 1, wherein the antibody is selected from an anti-CD79B antibody or an antigen-binding fragment thereof, and comprises a heavy chain variable region of the antibody and/or a light chain variable region of the antibody, wherein the heavy chain variable region of the antibody comprises: 1) a HCDR1, a HCDR2 and a HCDR3 set forth in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; or 2) a HCDR1, a HCDR2 and a HCDR3 set forth in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; and/or the light chain variable region of the antibody comprises: 1) a LCDR1, a LCDR2 and a LCDR3 set forth in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 respectively; or 2) a LCDR1, a LCDR2 and a LCDR3 set forth in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

30. (canceled)

31. (canceled)

32. (canceled)

33. The antibody-drug conjugate according to claim 1, wherein the antibody is an anti-TROP-2 antibody that comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a HCDR1, a HCDR2 and a HCDR3 having sequences identical to those of a HCDR1, a HCDR2 and a HCDR3 of a heavy chain variable region set forth in SEQ ID NO: 29, and the light chain variable region comprises a LCDR1, a LCDR2 and a LCDR3 having sequences identical to those of a LCDR1, a LCDR2 and a LCDR3 of a light chain variable region set forth in SEQ ID NO: 30.

34. (canceled)

35. (canceled)

36. (canceled)

37. (canceled)

38. (canceled)

39. The antibody-drug conjugate according to claim 1, wherein the antibody-drug conjugate is selected from the group consisting of the following structural formulas: ##STR00168## ##STR00169## ##STR00170## ##STR00171## ##STR00172## ##STR00173## ##STR00174## ##STR00175## ##STR00176## ##STR00177## ##STR00178## wherein: k is selected from the group consisting of 1 to 10 and may be an integer or a decimal, further, R.sup.1a in D is selected from methyl, and R.sup.1b in D is selected from hydrogen.

40. (canceled)

41. (canceled)

42. (canceled)

43. (canceled)

44. A pharmaceutical composition comprising a therapeutically effective amount of the antibody-drug conjugate according to claim 1, and a pharmaceutically acceptable carrier, diluent or excipient.

45. (canceled)

46. A method of treating or preventing a cancer in a subject in need thereof, the method comprising administering to the subject the antibody-drug conjugate according to claim 1, wherein the cancer is preferably breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urinary tract cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland carcinoma, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0173] FIG. 1 is a diagram showing the change trend of animal body weight in the blank vehicle group and different test administration groups in Test Example 7 (abscissa: days, ordinate: weight).

[0174] FIG. 2 is a diagram showing the change trend of animal tumor volume in vehicle and different test drug administration groups (abscissa: days, ordinate: tumor volume).

DETAILED DESCRIPTION

[0175] The following examples further illustrate the present disclosure, but the present disclosure is not limited thereto.

[0176] Experimental procedures without conditions specified in the examples of the present disclosure, are generally conducted according to conventional conditions, or according to conditions recommended by the manufacturer of the starting materials or commercial products, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Current Protocols in Molecular Biology, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. Reagents without specific origins indicated are commercially available conventional reagents.

[0177] The structure of the compound was determined by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectrometry (MS). NMR shift (6) is given in a unit of 10.sup.−6 (ppm). NMR spectra were measured using a Bruker AVANCE-400 nuclear magnetic resonance instrument, with deuterated dimethyl sulfoxide (DMSO-d.sub.6), deuterated chloroform (CDCl.sub.3) and deuterated methanol (CD.sub.3OD) as determination solvents, with tetramethylsilane (TMS) as internal standard.

[0178] Mass spectra were measured using Agilent 1200/1290 DAD-6110/6120 Quadrupole MS liquid chromatography-mass spectrometry system (manufacturer: Agilent; MS model: 6110/6120 Quadrupole MS), Waters ACQuity UPLC-QD/SQD (manufacturer: Waters, MS model: Waters ACQuity Qda Detector/waters SQ Detector) and THERMO Ultimate 3000-Q Exactive (manufacturer: THERMO, MS model: THERMO Q Exactive).

[0179] High performance liquid chromatography (HPLC) was performed using Agilent HPLC 1200DAD, Agilent HPLC 1200VWD or Waters HPLC e2695-2489 high pressure liquid chromatography.

[0180] Chiral HPLC was performed on Agilent 1260 DAD HPLC.

[0181] HPLC preparation was performed using Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP and Gilson GX-281 preparative chromatographs.

[0182] Chiral preparation was performed on a Shimadzu LC-20AP preparative chromatograph.

[0183] A CombiFlash Rf200 (TELEDYNE ISCO) system was used for rapid preparation.

[0184] Huanghai HSGF254 or Qingdao GF254 silica gel plates of specifications 0.15 mm to 0.2 mm were adopted for thin layer chromatography (TLC) analysis and 0.4 mm to 0.5 mm for TLC separation and purification.

[0185] The silica gel column chromatography generally used 200 to 300-mesh silica gel (Huanghai, Yantai) as the carrier.

[0186] Known starting materials described herein may be synthesized using or according to methods known in the art, or may be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., Chembee Chemicals, and other companies.

[0187] In the examples, the reactions can be performed in an argon atmosphere or a nitrogen atmosphere unless otherwise specified.

[0188] The argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a balloon containing about 1 L of argon or nitrogen.

[0189] A hydrogen atmosphere means that the reaction flask is connected to a balloon containing about 1 L of hydrogen.

[0190] Parr 3916EKX hydrogenator, Qinglan QL-500 hydrogenator or HC2-SS hydrogenator was used in the pressurized hydrogenation reactions.

[0191] The hydrogenation reactions usually involve 3 cycles of vacuumization and hydrogen purge.

[0192] A CEM Discover-S 908860 microwave reactor was used in the microwave reactions.

[0193] In the examples, a solution refers to an aqueous solution unless otherwise specified.

[0194] In the examples, the reaction temperature was room temperature, i.e., 20° C. to 30° C., unless otherwise specified.

[0195] The monitoring of the reaction progress in the examples was conducted by thin layer chromatography (TLC). The developing solvent for reactions, the eluent system for column chromatography purification, the developing solvent system for thin layer chromatography system and the volume ratio of the solvents were adjusted according to the polarity of the compound, or by adding a small amount of basic or acidic reagents such as triethylamine and acetic acid.

[0196] The antibody drug conjugate of the present disclosure is found in WO2020063676A, and the synthesis and tests of relevant compounds are incorporated herein by reference in their entirety. Non-limiting examples of synthesis are incorporated as follows:

1. Preparation of Antibodies

Example 1-1. Cloning and Expression of Protein Antigens

[0197] Antibodies (comprising light and heavy chains) and antigens were constructed by overlap extension PCR method known in the art, and DNA fragments obtained by overlap extension PCR were inserted into expression vector pEE6.4 (Lonza Biologics) using HindIII/BstBI enzymatic digestion site, and expressed in 293F cells (Invitrogen, Cat #R790-07) to obtain recombinant proteins. The obtained recombinant proteins were used for immunization or screening. The human CD79B gene sequence was derived from NCBI (NP_000617.1), the extracellular region (ECD) of which comprises 159 amino acids (Met1-Asp159).

[0198] The amino acid sequence of the fusion protein of a human CD79B extracellular domain (ECD) and a human Fc domain (human CD79B ECD-hFc) is shown in SEQ ID NO: 1:

TABLE-US-00013 SEQ ID NO: 1 ARSEDRYRNPKGSACSRIWQSPRFIARKRGFTVKMHCYMNSASGNVSWLW KQEMDENPQQLKLEKGRMEESQNESLATLTIQGIRFEDNGIYFCQQKCNN TSEVYQGCGTELRVMGFSTLAQLKQRNTLKDGIIMIQTLLIILFIIVPIF LLLDKDDSKAGMEEDHTYEGLDIDQTATYEDIVTLRTGEVKWSVGEHPGQ EEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0199] The amino acid sequence of a fusion protein of a human CD79B extracellular domain (ECD) and His tag (human CD79B ECD-His) is shown in SEQ ID NO: 2:

TABLE-US-00014 SEQ ID NO: 2 ARSEDRYRNPKGSACSRIWQSPRFIARKRGFTVKMHCYMNSASGNVSWLW KQEMDENPQQLKLEKGRMEESQNESLATLTIQGIRFEDNGIYFCQQKCNN TSEVYQGCGTELRVMGFSTLAQLKQRNTLKDGIIMIQTLLIILFIIVPIF LLLDKDDSKAGMEEDHTYEGLDIDQTATYEDIVTLRTGEVKWSVGEHPGQ EHHHHHH

Example 1-2. Preparation of Mouse Monoclonal Antibody

1. Mouse Immunization and Serum Titer Detection

[0200] The fusion protein of the human CD79B extracellular domain (ECD) and the human Fc domain (human CD79B ECD-hFc) and the fusion protein of human CD79B extracellular domain (ECD) and His tag (human CD79B ECD-His) were taken as immunogens, and Balb/c and SJL mice were immunized by an intraperitoneal injection method and stimulated to produce antibodies against the human CD79B extracellular domain (ECD). In addition, the fusion protein of cynomolgus monkey CD79B extracellular domain (ECD) and His tag (cyno CD79B ECD-His) was taken as immunogens, and SJL mice were immunized by an intraperitoneal injection method and stimulated to produce antibodies against the monkey CD79B extracellular domain (ECD).

Experimental Procedures:

[0201] 1) Intraperitoneal injection immunization: the antigen quantity required by the immunization according to the immunization program was calculated. Protein antigens were diluted to the corresponding concentrations with PBS as required, followed by emulsification of the antigens. The emulsified antigen and adjuvant mixture was transferred to a 2.0-mL sterile syringe and the air bubbles therein were evacuated. The tail of the mouse was grasped by the right hand, skin to the head and neck of the mouse was gently grasped by the thumb and the forefinger of the left hand, and the injection site on right abdomen of the mouse was wiped by a 75% alcohol cotton ball with abdominal cavity upward. The needle point of the syringe with the antigen medicine drawn in advance was inclined upwards and parallelly punctured into the skin with the mouse head downwards, the syringe was inserted into the abdominal cavity of the mouse at a 45-degree angle to the abdominal cavity, and the mixture of antigen and adjuvant was slowly injected. After the immunization was completed, observation was performed for at least 2 h.

[0202] 2) Mouse serum collection: the corresponding serum tube of each mouse was labeled, the mouse ear tag nail was checked, the mouse was grabbed by one hand, and about 100 μL of whole blood was taken through the submaxillary vein of the mouse face, left to stand at room temperature for about 2 h, and then centrifuged to collect upper serum of the centrifuge tube. The serum could be stored in a refrigerator at 4° C. within one week and used for related detections such as antibody titer and the like. If the serum was stored for a long time, the serum could be placed in a refrigerator at −80° C. to avoid repeated freezing and thawing.

[0203] 3) Immunized mouse ELISA serum titer determination: the 96-well plates were correspondingly labeled before experiment, and coated with 1 μg/mL antigen at 50 μL per well in a refrigerator at 4° C. overnight. The next day, the coated antigen plates from the previous day were removed and washed once with a plate washer (cleaning solution: 1×PBST). After washing, the plates were blocked with 1% BSA blocking solution prepared in 1×PBST at 37° C. for 1 h. After being washed with 1×PBST cleaning solution for 3 times, the plates were added with the serum to be detected with different dilution concentrations, and incubated in an incubator at 37° C. for 1 h. After being washed with 1×PBST cleaning solution for 3 times, the plates were added with 100 μL of goat anti-mouse secondary antibody diluted at 1:5000, and incubated in an incubator at 37° C. for 0.5 h. The plates were washed, the TMB color developing solution A solution and B solution in a ratio of 1:1 was taken for to color development. The color development reaction was terminated with 1 N hydrochloric acid for 15 min. Fluorescence value was read at 450 nm on a Spectra Max M5 multi-functional plate reader.

[0204] 4) Immunized mouse FACS serum titer assay: after centrifugation, the suspension of DoHH2 cells or monkey peripheral blood mononuclear cells was resuspended in PBS containing 0.1% BSA, counted, added with the test serum of each group of immunized mice and incubated at room temperature for 60 min. The cells were washed three times, then added with Anti-Mouse IgG (Fc specific)-FITC secondary antibody, and incubated at room temperature for 30 min in the dark. The cells were washed three times, gently resuspended in PBS containing 0.1% BSA, and then assayed using the machine.

[0205] Specific antibodies against CD79B were produced in immunized mice by the above assays, and the mice could be used for cell fusion to generate hybridoma cell lines capable of secreting antibodies specific for CD79B.

2. Hybridoma Preparation and Antibody Screening

[0206] The cell fusion is to promote the lymphocytes of the mice and the myeloma cell SP2/0 (ATCC, CCL-121™) to fuse into hybridoma cells under spontaneous or artificial induction, and the hybridoma cells have the antibody secretion function and can proliferate indefinitely. Lymphocytes and myeloma cells of immunized mice were fused by electrofusion and used for subsequent antibody screening.

[0207] 1) Electrofusion experiment: SP2/0 cells were expanded in 10% DMEM medium one week before fusion. The spleen and lymph nodes of the killed mice were picked up in a biosafety cabinet and washed and ground in a petri dish, and lymphocytes were collected. SP2/0 and lymphocytes were mixed in proportion, and the electrofusion apparatus was started and programmed to perform fusion. After fusion, the cells were plated in a 96-well plate and cultured overnight in an incubator at 37° C. with 5% CO.sub.2; the cell state was observed daily, and the cell fusion rate was counted 5 days after fusion. The fused hybridoma cells were screened 9-14 days after fusion, and the cells in positive wells were selected for amplification culturein a 24-well plate.

[0208] 2) Subcloning by limiting dilution method: the cell lines to be subcloned were resuspended in the 24-well culture wells and counted. The cell concentration of each cell strain was diluted to 5-10 cells/mL, the diluted cell suspension was added into a 15 cm disposable petri dish, and each well in a 96-well culture plate was added with 0.2 mL of suspension and contained 1-2 cells. The 96-well plate with the cells plated was cultured in an incubator at 37° C. with 5% CO.sub.2. After 7-10 days, the subclone plates were detected and screened according to the growth condition of the cells, and positive clones were selected to 24 wells for further positive confirmation.

[0209] 3) ELISA screening: the 96-well plates were correspondingly labeled before experiment, and coated with 1 μg/mL antigen at 50 μL per well in a refrigerator at 4° C. overnight. The next day, the coated antigen plates from the previous day were removed and washed once with a plate washer (cleaning solution: 1×PBST). After washing, the plates were blocked with 1% BSA blocking solution prepared in 1×PBST at 37° C. for 1 h. After being washed with 1×PBST cleaning solution for 3 times, the plates were added with 50 μL cell supernatant to be detected, and incubated in a 37° C. incubator for 1 h. After the plates were washed with 1×PBST cleaning solution for 3 times, added with 100 μL of goat anti-mouse secondary antibody diluted at 1:5000, and incubated in an incubator at 37° C. for 0.5 h. The plates were washed, the TMB color developing solution A solution and B solution in a ratio of 1:1 was taken for to color development. The color development reaction was terminated with 1 N hydrochloric acid for 15 min. Fluorescence value was read at 450 nm on a Spectra Max M5 multi-functional plate reader.

[0210] 4) FACS screening: after centrifugation, the cell suspension of DOHH2 was resuspended in PBS containing 0.1% BSA, counted, added with the test cell supernatant and incubated at room temperature for 60 min. The cells were washed three times, then added with Anti-Mouse IgG (Fc specific)-FITC secondary antibody, and incubated at room temperature for 30 min in the dark. The cells were washed three times, gently resuspended in PBS containing 0.1% BSA, and then assayed using the machine.

[0211] 5) Hybridoma positive clone identification: after fusion and subclone screening of mouse splenocytes, multiple specific antibodies against human CD79B antigen were obtained, and the antibodies from the 17 strains of hybridomas having the highest binding to ELISA and FACS were produced and purified. The ELISA assay results of the culture supernatant of the anti-human CD79B hybridoma positive clone cells are shown in Table 1. The FACS assay results of the culture supernatant of the anti-human CD79B hybridoma positive clone cells are shown in Table 2. Meanwhile, specific antibodies against monkey CD79B antigen were obtained, and the 4 strains of hybridomas having the highest binding to ELISA and FACS were taken for production and purification of antibodies. mIgG was used as negative control in the both assays.

TABLE-US-00015 TABLE 1 ELISA assay results of anti-human CD79B hybridoma positive clones Antibody No. Clone No. Results (OD450) Negative control mIgG 0.05 mAb015 83B2G2 3.41 mAb017 86F11F6 3.80

TABLE-US-00016 TABLE 2 FACS assay results of anti-human CD79B hybridoma positive clones Antibody No. Clone No. Mean fluorescence value Negative control mIgG 58 mAb015 83B2G2 10036 mAb017 86F11F6 8132

3. Production, Purification and Identification of Mouse Monoclonal Antibodies

[0212] 1) Production and purification of mouse monoclonal antibody: the hybridoma cells requiring antibody production were observed under a microscope to grow to ≥70% or more and to have a good cell status, and the cells were collected and counted by a Countstar IC1000-type cell counter. The cell concentration was adjusted to 1-5×10.sup.5 cells/mL using the prepared medium and transferred to a Roller Bottle. The Roller Bottle with the transferred cells were delivered into a roller bottle incubator for culturing at 37° C. for 10-15 days, the growth condition of the cells were observed every day, and the cells were taken out for purification when the culture solution turned orange and transparent. The cell supernatant was subjected to antibody purification using Protein A column according to a conventional method.

[0213] 2) ELISA assay on anti-human CD79B mouse monoclonal antibodies: the 96-well plates were correspondingly labeled before experiment, and coated with 1 μg/mL antigen at 50 μL per well in a refrigerator at 4° C. overnight. The next day, the coated antigen plates from the previous day were removed and washed once with a plate washer (cleaning solution: 1×PBST). After washing, the plates were blocked with 1% BSA blocking solution prepared in 1×PBST at 37° C. for 1 h. After being washed with 1×PBST cleaning solution for 3 times, the plates were added with 50 μL of diluted antibody at 1:10 at 100 nM, and incubated in a 37° C. incubator for 1 h. After the plates were washed with 1×PBST cleaning solution for 3 times, added with 100 μL of goat anti-mouse secondary antibody diluted at 1:5000, and incubated in an incubator at 37° C. for 0.5 h. The plates were washed, the TMB color developing solution A solution and B solution in a ratio of 1:1 was taken for to color development. The color development reaction was terminated with 1 N hydrochloric acid for 15 min. Fluorescence value was read at 450 nm on a Spectra Max M5 multi-functional plate reader. Among them, 4 anti-human CD79B mouse monoclonal antibodies had the highest ELISA binding capacity, including mAb015 and mAb017.

[0214] 3) FACS assay on anti-human CD79B mouse monoclonal antibodies: after centrifugation, the cell suspension of DOHH2 was resuspended in PBS containing 0.1% BSA, counted, added with 100 μL of antibody diluted 1:10 at 100 nM and incubated at room temperature for 1 h. The cells were washed three times, then added with Anti-Mouse IgG (Fc specific)-FITC secondary antibody, and incubated at room temperature for 30 min in the dark. The cells were washed three times, gently resuspended in PBS containing 0.1% BSA, and then assayed using the machine. Among them, 4 anti-human CD79B mouse monoclonal antibodies had the highest FACS binding capacity, including mAb015 and mAb017.

[0215] 4) SPR assay on anti-human CD79B mouse monoclonal antibodies: the affinity of the anti-human CD79B antibody for antigen human CD79B-His thereof was assayed by surface plasmon resonance (SPR) technology. The antigen human CD79B-His protein was immobilized to a CM5 chip. The coupling level was set at 100 RU. The running buffer was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). The diluted antibodies were flowed through the experimental and control channels at a flow rate of 30 μL/min for 3 min and dissociated for 5 min. Regeneration buffer (10 mM Glycine, pH 1.5) was then run at a flow rate of 30 μL/min for 30 s. Data were analyzed using Biacore 8K evaluation software.

Examples 1-3. Sequencing of Variable Region Amino Acid of Mouse Monoclonal Antibodies

[0216] The hybridoma monoclonal cell lines with high affinity obtained in Example 1-2 were subjected to variable region amino acid sequencing, followed by recombinant expression of human murine chimeric antibody (cAb), and further antibody identification. The heavy chain and light chain variable regions of the antibody gene were amplified by reverse transcription PCR, and connected to a vector for sequencing to obtain light and heavy chain sequences of the monoclonal antibody. The total cellular RNA of the well-activated single-cell strain in Example 2 was first extracted using an RNA purification kit (Qiagen, Cat #74134, steps referring to the manual). Then, a cDNA single strand, Oligo-dT primers cDNA reverse transcription, was prepared using the cDNA synthesis kit available from Invitrogen, Cat #18080-051. With the single strand as a template, light and heavy chain variable region sequences of the antibody were synthesized by adopting PCR method, and the PCR products were cloned to TA vector pMD-18T and then sent to sequencing. The obtained light and heavy chain sequences of the antibody were cloned into expression vectors (see Example 1-1), recombinant monoclonal antibodies were expressed, and after the activity was verified (see Example 1-2), humanization was performed.

[0217] The amino acid residues of the VH/VL CDRs of the anti-human CD79B antibody were identified using the Chothia numbering system and annotated.

[0218] Sequence of monoclonal antibody mAb015 of mouse hybridoma cell:

TABLE-US-00017 Heavy chain variable region: SEQ ID NO: 3 QVQLQQSGAELARPGASVKLSCKASGSSFTSYGINWVKQRTGQGLEWIGE IFPRSGNTYYNEKFEGKATLTADKSSSTAYMELRSLTSEDSAVYFCAKGD LGDFDYWGQGTTLTVSS Light chain variable region: SEQ ID NO: 4 DFLMTQTPLSLPVRLGDQASISCRSSQSIVHSDGNTYFEWYLQKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP WTFGGGTKLEIK

[0219] Sequence of monoclonal antibody mAb017 of mouse hybridoma cell:

TABLE-US-00018 Heavy chain variable region: SEQ ID NO: 5 QVQLQQSGAELARPGASVKLSCKASGYTFTTYGINWVKQRTGQGLEWIGE IYPRSGNIYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARGS DYDGDFAYWGQGTLVTVSA Light chain variable region: SEQ ID NO: 6 DVLMTQTPLSLPVSLGDQASISCRSSQSIVHHDGNTYLEWYLQKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP WTFGGGTQLEIK

[0220] The CDR sequences of murine are shown in Table 3:

TABLE-US-00019 TABLE 3 CDR sequences of murine anti-human CD79B antibodies Antibody CDR mAb015 mAb017 Heavy chain GSSFTSY (SEQ ID NO: 7) GYTFTTY (SEQ ID NO: 13) CDR1 Heavy chain FPRSGN (SEQ ID NO: 8) YPRSGN (SEQ ID NO: 14) CDR2 Heavy chain GDLGDFDY (SEQ ID NO: 9) GSDYDGDFAY (SEQ ID NO: 15) CDR3 Light chain RSSQSIVHSDGNTYFE (SEQ RSSQSIVHHDGNTYLE (SEQ CDR1 ID NO: 10) ID NO: 16) Light chain KVSNRFS (SEQ ID NO: 11) KVSNRFS (SEQ ID NO: 17) CDR2 Light chain FQGSHVPWT (SEQ ID NO:  FQGSHVPWT (SEQ ID NO: 18) CDR3 12)

Examples 1-4. Humanization of Anti-Human CD79B Antibodies

[0221] After homology comparison of the light and heavy chain sequences of the murine anti-CD79B monoclonal antibody obtained in Example 1-3 was performed in an antibody database, a humanized antibody model was established, and the optimal humanized anti-CD79B monoclonal antibody was screened as a preferred molecule according to model selection back mutation. The method started with searching a published crystal structure model database (such as a PDB database) of the mouse Fab, wherein the crystal structure had similar homology with the obtained murine candidate molecules, and the mouse Fab model was established by selecting the Fab crystal structure with high resolution (such as <2.5 Å). The light and heavy chain sequences of the murine antibody were compared with the sequences in the model, the sequences consistent with the sequences of the murine antibody in the model to obtain a murine antibody structural model, and inconsistent amino acids could be possible back mutation sites. The murine antibody structure model was run with Swiss-pdb viewer software to optimize energy (minimize). The different amino acid positions in the model except the CDR were back-mutated, and the resulting mutated antibodies (humanized) were compared with the antibodies before humanization for activity detection. The humanized antibody having good activity was reserved. Thereafter, the CDR regions were optimized, including avoiding glycosylation, deamidation, oxidation sites, and the like. The antibodies were cloned, expressed and purified by using a gene cloning and recombinant expression method, and the humanized antibodies hAb015-10 and hAb017-10 with the highest activity were finally selected by assays of ELISA, FACS, SPR and the like.

[0222] The sequences of humanized antibodies hAb015-10 and hAb017-10 are shown below.

TABLE-US-00020 hAb015-10 humanized antibody heavy chain: SEQ ID NO: 19 EVQLVQSGAEVKKPGSSVKVSCKASGSSFSSYGINWVKQAPGQGLEWIGE IFPRSGNTYYNEKFEGRATLTADKSTSTAYMELRSLRSEDTAVYYCAKGD LGDFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hAb015-10 humanized antibody light chain: SEQ ID NO: 20 DFVMTQTPLSLPVTPGEPASISCRSSQSIVHSDGNTYFEWYLQKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP WTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC hAb017-10 humanized antibody heavy chain: SEQ ID NO: 21 EVQLVQSGAEVKKPGASVKVSCKASGYTFTTYGINWVKQAPGQGLEWIGE IYPRSGNIYYNEKFKGKATLTADKSTSTAYMELRSLRSDDTAVYYCARGS YFPEPVTVSWNDYDGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hAb017-10 humanized antibody light chain: SEQ ID NO: 22 DVVMTQTPLSLPVTPGEPASISCRSSQSIVHHDGNTYLEWYLQKPGQSPQ LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVP WTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

Example 1-5. Construction of Cell Line with High TROP-2 Expression

[0223] pCDH-hTROP-2 lentiviral expression vector plasmids, pVSV-G and pCMV-dR8.91 lentiviral system packaging vectors were transfected into viral packaging cells 293T using Lipofectamine 3000 transfection reagent. The medium supernatant containing viruses was collected, filtered, and centrifuged at ultra-high speed. Chinese hamster ovary cells CHO-K1 was allowed to be infected with the concentrated virus, screened using puromycin for two to three weeks, and subjected to FACS single-cell sorting.

[0224] According to the TROP-2 expression level on the surface of the CHO-K1 cells infected by lentivirus determined by FACS, CHO-K1/hTROP-2 monoclonal cell strains with high TROP-2 expression were selected.

[0225] Amino acid sequence (Genbank: NP_002344.2) of TROP-2 is as follows:

TABLE-US-00021 SEQ ID NO: 35 MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPG GRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDN DGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVR THHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPT IQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEP LQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVVVVVALVAGMAVLVITNR RKSGKYKKVEIKELGELRKEPSL

[0226] Amino acid sequence of TROP-2-His:

TABLE-US-00022 SEQ ID NO: 36 MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPG GRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDN DGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVR THHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPT IQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEP LQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVVVVVALVAGMAVLVITNR RKSGKYKKVEIKELGELRKEPSLHHHHHH

Example 1-6. Preparation of Anti-Human TROP-2 Monoclonal Antibody

[0227] The anti-human TROP-2 monoclonal antibody in the present disclosure was prepared according to the method disclosed in WO03074566, and the site mutation modification design was carried out on CDR by using computer software and taking the antibody variable region gene of hRS7 as a template. The antibody variable region gene was inserted into a protein expression vector Phr-IgG (with signal peptide and constant region gene (CH1-Fc/CL) fragment) by molecular cloning and then expressed in HEK293 and Expi-CHO-S cells. Antibody purification was performed according to a conventional method. Activity verification was performed by using CHO-K1 cells and huTROP-2 protein (His27-Thr274 Accession #NP_002344.2) over-expressing huTROP-2 protein, and an antibody with better target binding activity was selected, wherein the variable region sequence of PD3 is as follows:

TABLE-US-00023 PD3 heavy chain variable region: SEQ ID NO: 29 EVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMG WINTYTGEPTYTQDFKGRFAFSLDTSVSTAYLQISSLKAEDTAVYYCAR GGFGSSYWYFDVWGQGTLVTVSS PD3 light chain variable region: SEQ ID NO: 30 DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIY SASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTF GAGTKVEIK

[0228] Note: the underlined portions are CDR regions determined according to Kabat numbering scheme.

TABLE-US-00024 TABLE 4 CDR regions of PD3 antibodies Antibodies PD3 Heavy chain CDR1 NYGMN (SEQ ID NO: 23) Heavy chain CDR2 WINTYTGEPTYTQDFKG (SEQ ID NO: 24) Heavy chain CDR3 GGFGSSYWYFDV (SEQ ID NO: 25) Light chain CDR1 KASQDVSIAVA (SEQ ID NO: 26) Light chain CDR2 SASYRYT (SEQ ID NO: 27) Light chain CDR3 QQHYITPLT (SEQ ID NO: 28)

[0229] The heavy chain constant region of the antibody may be selected from the group consisting of the constant regions of human IgG1, IgG2, IgG4 and variants thereof, and the light chain constant region of the antibody may be selected from the group consisting of the light chain constant regions of human κ and λ chains and variants thereof. Illustratively, the heavy chain constant region of the antibody is selected from the constant region of human IgG1 having a sequence set forth in SEQ ID NO: 11, and the light chain constant region of the antibody is selected from the constant region of human κ chain having a sequence set forth in SEQ ID NO: 12.

TABLE-US-00025 Human IgG1 Heavy chain constant region: SEQ ID NO: 31 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human κ light chain constant region: SEQ ID NO: 32 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC

[0230] Illustratively, the light/heavy chain constant regions described above are combined with the variable regions of the aforementioned PD3 antibody to form a complete antibody, the light/heavy chain sequences of which are as follows:

TABLE-US-00026 PD3 heavy chain: SEQ ID NO: 33 EVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMG WINTYTGEPTYTQDFKGRFAFSLDTSVSTAYLQISSLKAEDTAVYYCAR GGFGSSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK PD3 light chain: SEQ ID NO: 34 DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIY SASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTF GAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

2. Preparation of Compounds

Example 2-1: Synthesis of Compound L-1

[0231] ##STR00102## ##STR00103## ##STR00104##

Step 1: Preparation of Compound 2

[0232] Compound 1 (50 mg, 0.08 mmol, prepared by referring to the method disclosed in WO2017151979) was dissolved in 1.5 mL of N,N-dimethylformamide in an ice water bath, and the reaction system was added with DIPEA (N,N-diisopropylethylamine, 18 mg, 0.14 mmol) and then added with bis(p-nitrophenyl)carbonate (49 mg, 0.16 mmol). Then the mixture was stirred at room temperature, added with 20 mL of methyl tert-butyl ether, stirred for 20 min, filtered, and dried to obtain 36 mg of solid compound 2.

[0233] LC/MS (ESI): m/z 784.1 [M+H].sup.+.

Step 2: Preparation of Compound D-1b

[0234] Compound D-1a (eribulin, prepared as described in ZL201010236637.2) (72.91 mg, 0.1 mmol) was dissolved in 10 mL of tetrahydrofuran in an ice water bath, and the reaction system was added with Fmoc-OSu (fluorenylmethoxycarbonylsuccinimide, 41 mg, 0.12 mmol) and stirred at room temperature until the reaction was completed. The system was concentrated under reduced pressure, and used directly in the next step.

Step 3: Preparation of Compound D-1c

[0235] The crude compound D-1b obtained in the above step was dissolved in 10 mL of anhydrous ether; the reaction system was added with silver oxide (34.8 mg, 0.15 mmol), then added with methyl iodide (28.4 mg, 0.2 mmol), and stirred at room temperature until the reaction was completed. The reaction mixture was filtrated and concentrated under reduced pressure to obtain a crude product, which was directly used in the next step.

Step 4: Preparation of Compound D-1

[0236] The crude compound D-1c obtained in the above step was dissolved in 10 mL of tetrahydrofuran; the reaction system was added with 2 mL of diethylamine, and then stirred at room temperature until the reaction was completed. The reaction mixture was concentrated under reduced pressure to obtain crude product, and purified by silica gel column chromatography (eluent: dichloromethane/ethyl acetate/petroleum ether) to obtain 3 mg of the target compound D-1.

[0237] LC/MS (ESI): m/z 744.2 [M+H].sup.+.

Step 5: Preparation of Compound L-1

[0238] Compound D-1 (13.5 mg, 0.018 mmol) was dissolved in 1.5 mL of DMF; the reaction system was added with DIPEA (7 mg, 0.054 mmol) and added with compound 2 (18 mg, 1.3 mmol) in portions. The reaction mixture was stirred until the reaction was substantially completed and concentrated to obtain the crude product. The crude product was separated by prep-HPLC (column: Welch XTimate C18 (5.0 μm×30.0×150 mm), mobile phase: A-water (0.1% formic acid): B-acetonitrile, gradient elution=70:30-5:95 (16 min, flow rate: 30.0 mL/min)) to obtain 6.5 mg of compound L-1 (96.95% purity).

[0239] LC/MS (ESI): m/z 1388.3 [M+H].sup.+.

[0240] .sup.1HNMR (CDCl.sub.3, 400M) δ 0.85-0.90 (m, 3H), 0.93-1.00 (m, 3H), 1.08˜1.10 (m, 3H), 1.20˜1.50 (m, 15H), 1.75˜2.04 (m, 6H), 2.13˜2.55 (m, 16H), 2.70˜2.77 (m, 1H), 2.80˜2.96 (m, 2H), 3.16˜3.97 (m, 20H), 3.99˜4.39 (m, 8H), 4.60˜4.80 (m, 6H), 4.88˜5.10 (m, 5H), 5.24˜5.37 (m, 4H), 6.71 (s, 2H), 7.03 (d, J=6.8 Hz, 1H), 7.18˜7.30 (m, 3H), 7.63 (d, J=8.0 Hz, 2H), 8.92 (bs, 1H).

Example 2˜2: Synthesis of Compound D-2

[0241] ##STR00105##

[0242] (R)-2-cyclopropyl-2-hydroxyacetic acid (4.7 mg, 0.04 mmol, 1.5 eq) was added to a reaction flask, and THF was added thereto. The mixture was stirred for dissolving, and cooled in an ice water bath. The reaction system was added with EDCI HCl (8.0 mg, 0.04 mmol, 1.5 eq, 1-ethyl-3(3-dimethylpropylamine)carbodiimide hydrochloride) and HOBT (5.4 mg, 0.04 mmol, 1.50 eq, 1-hydroxybenzotriazole), then added with compound D-1a (20 mg, 0.027 mmol, 1.0 eq) and finally added with DIPEA (10.5 mg, 0.08 mmol, 3.0 eq). After the addition was completed, the reaction system was warmed to room temperature (20° C.) and stirred until the reaction was substantially completed, and added with 2 mL of water to quench the reaction. The reaction mixture was extracted with ethyl acetate (2×5 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product which was purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether) to obtain 6.0 mg of compound D-2 (98% purity).

[0243] MS: 827.8[M+H].sup.+.

[0244] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 6.82 (s, 1H), 5.08 (s, 1H), 4.93 (s, 1H), 4.89 (s, 1H), 4.81 (s, 1H), 4.70 (t, J=4.4 Hz, 1H), 4.61 (t, J=4.4 Hz, 1H), 4.42-4.25 (m, 3H), 4.23-4.16 (m, 1H), 4.12 (s, 1H), 4.03 (d, J=9.3 Hz, 3H), 4.02-3.87 (m, 3H), 3.82 (d, J=9.4 Hz, 1H), 3.78-3.70 (m, 3H), 3.58 (dd, J=50.7, 8.9 Hz, 4H), 3.43 (s, 3H), 3.32-3.23 (m, 2H), 2.88 (d, J=9.5 Hz, 2H), 2.72 (dd, J=16.0, 10.0 Hz, 1H), 2.46 (d, J=13.9 Hz, 4H), 2.33 (d, J=13.8 Hz, 3H), 2.19 (dd, J=21.4, 14.3 Hz, 4H), 2.08 (s, 1H), 1.97 (ddd, J=13.6, 9.4, 4.7 Hz, 5H), 1.44 (d, J=11.5 Hz, 3H), 1.27 (d, J=12.2 Hz, 4H), 1.10 (d, J=6.2 Hz, 3H), 0.68-0.45 (m, 4H).

Example 2-3: Synthesis of Compound D-3

[0245] ##STR00106##

[0246] Compound D-1 (22 mg, 0.03 mmol) and (R)-2-cyclopropyl-2-hydroxyacetic acid (7.0 mg, 0.06 mmol) were dissolved with 2 mL of DCM at room temperature in a nitrogen atmosphere; the reaction mixture was added with Et.sub.3N (21 μL, 0.15 mmol) and DMTMM (20.3 mg, 0.069 mmol, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride) in sequence and stirred overnight at room temperature. After the reaction was completed, H.sub.2O (2 mL) was added to quench the reaction, the reaction mixture was extracted with DCM (2 mL×3), and the organic phase was concentrated under reduced pressure (bath temperature 30° C.). The residue was purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether) to obtain compound D-3 (18 mg, 95% purity).

[0247] MS: 863.8 [M+Na].sup.+.

Example 2-4: Synthesis of Compound D-4

[0248] ##STR00107##

[0249] Compound D-4 was obtained by referring to Example 2-2 with replacing (R)-2-cyclopropyl-2-glycolic acid with glycolic acid.

[0250] MS: 787.82 [M+H].sup.+.

[0251] .sup.1HNMR (CDCl.sub.3, 400M):0.86˜0.90 (m, 1H), 1.04˜1.13 (m, 4H), 1.22˜1.41 (m, 4H), 1.71˜1.74 (m, 3H), 1.94˜2.00 (m, 5H), 2.15˜2.22 (m, 8H), 2.48 (S, 3H), 2.71˜2.75 (m, 2H), 2.87˜2.89 (m, 2H), 3.26˜3.31 (m, 2H), 3.43 (S, 3H), 3.53˜3.55 (m, 1H), 3.64˜3.70 (m, 2H), 3.74 (s, 1H), 3.80˜3.84 (m, 1H), 3.90˜4.05 (m, 4H), 4.12 (s, 3H), 4.18˜4.20 (m, 1H), 4.26˜4.39 (m, 3H), 4.61 (t, J=4.8 Hz, 1H), 4.619 (t, J=4.8 Hz, 1H), 4.82 (s, 1H), 4.82 (s, 1H), 4.88 (s, 1H), 4.93 (s, 1H), 5.08 (s, 1H), 6.89 (m, 1H).

Example 2-5: Preparation of Compound D-5

[0252] ##STR00108##

[0253] Compound D-5 was obtained by referring to Example 2-2 with replacing (R)-2-cyclopropyl-2-hydroxyacetic acid with 1-hydroxycyclopropane-1-carboxylic acid.

[0254] MS: 835.7 [M+Na].sup.+.

Example 2-6: Synthesis of Compound D-6

[0255] ##STR00109##

[0256] The compound of formula D-6 was prepared by referring to Example 2-2 using the starting materials (R)-2-cyclopropyl-2-hydroxyacetic acid and E1-30 (synthesized and obtained according to Bioorg. Med. Chem. Lett. 14 (2004) 5551-5554). MS: 850.64 [M+Na].sup.+.

Example 2-7: Synthesis of Compound D-7

[0257] ##STR00110##

[0258] The compound of formula D-7 was prepared by referring to Example 2-2 using the starting materials 1-hydroxycyclopropane-1-carboxylic acid and E1-30 (synthesized and obtained according to Bioorg. Med. Chem. Lett. 14 (2004) 5551-5554). MS: 836.73 [M+Na].sup.+.

Example 2-8: Synthesis of Compound D-8

[0259] ##STR00111##

[0260] The compound of formula D-8 was prepared by referring to the method in the Example 2-2 using the starting materials p-hydroxyethylbenzoic acid and E1-30. MS: 886.75 [M+Na].sup.+.

Test Example 1: In Vitro Cytotoxic Activity Screening

1.1. Principle and Method

[0261] The CTG is used for detecting the ATP content in the experiment, and the survival condition of the tumor cells is reflected. The final culture conditions were first determined by seeding cells at different densities and culturing the cells for 3 days and 5 days based on IC.sub.50 and the maximum inhibition rate. The killing effect of the toxin molecule was then assayed according to this condition. 1.2. Selection of cell lines

[0262] According to the purpose of the experiment, two disease models of breast cancer and NSCLC were selected, and SKBR3 tumor cells (HER2+, ATCC, Cat #HTB-30), MDA-MB-468 (HER2-, ATCC, Cat #HTB-132) and A549 (human non-small cell lung cancer cells, ATCC, Cat #CCL-185) were selected for screening.

1.3. Determination of Cell Culture Conditions

[0263] 1) Cell culture: A549, SK-BR-3 and MDA-MB-468 cells were cultured with Ham's F-12K (Kaighn's) medium (Gibco, 21127030) and McCoy's 5A medium (ThermoFisher, Cat #16600108) and Leibovitz's L-15 medium (ThermoFisher, Cat #11415-114) containing 10% FBS (Gibco, 10099-141), respectively.

[0264] 2) Cell plating: A549 cells were digested with trypsin, and the cells were terminated with the above medium, and 4.3×10.sup.5, 7.2×10.sup.5 and 11.5×10.sup.5 cells were added to the medium to give a final volume of 26 mL. 180 μL of cell suspension was added to each well in columns 2 to 11 of a 96-well plate (coming, Cat #3903) to give cell densities of 3K, 5K and 8K per well. Wells in column 12 were filled with 200 μL of culture medium and the remaining wells were filled with PBS. The above operations were repeated on the SKBR3 and MDA-MB-468 cells. The sample was duplicated.

[0265] 3) Drug preparation: stock solutions of positive control eribulin and the compound of the present disclosure were prepared in DMSO in a 96-well round bottom plate (coming, Cat #3788). 2 mM of stock solution (stock diluted 10-fold in DMSO) was prepared in column 1 of drug preparation plate 1, then 10-fold dilution in DMSO by gradient was performed in column 10, and the wells in column 11 were filled with DMSO. 95 μL of corresponding culture solution was added into each well from column 2 to column 11 of the drug preparation plate 2, 5 μL of solution was pipetted from column 2 to column 11 of drug preparation plate 1 to drug preparation plate 2, the solution was mixed well, 20 μL of solution was pipetted, added into the plated cells, and continuously cultured for 3 days and 5 days.

[0266] 4) CTG assay (Cell Titer-Glo™, luminescent cell viability assay, Promega): the cell plates were removed on day 3 and day 5, and balanced to room temperature. 90 μL of CTG was added into each well and reacted at room temperature for 10 min in the dark. The absorption value was read using a microplate reader and IC.sub.50 was calculated.

1.4. Data Results

[0267]

TABLE-US-00027 TABLE 5 SKBR3 MDA-MB-468 A549 Maximum Maximum Maximum IC.sub.50 inhibition IC.sub.50 inhibition IC.sub.50 inhibition Compound (nM) (%) (nM) (%) (nM) (%) Eribulin 0.7147 94.90 0.4819 87.47 0.6609 81.50 D-1 0.2052 96.87 0.1827 88.01 0.5151 81.34 D-2 0.8393 / 0.9088 / 3.156 / D-3 1.36 / 1.67 / 5.141 / D-4 1.301 / 1.776 / 5.614 / D-5 0.3834 / 05309 / 4.024 / D-6 0.2942 / 0.9963 / 1.849 / D-7 0.4811 / 0.9034 / 1.783 / D-8 0.3304 / 0.8428 / 0.8107 /

[0268] Conclusion: the compound D-1 had good killing effect in three tumor cell lines and was significantly superior to the positive drug eribulin.

Example 2-9: Synthesis of Compound D-9

[0269] ##STR00112## ##STR00113##

Step 1: Preparation of Compound D-9a

[0270] 0.3 mL of 1,4-dioxane and 0.3 mL of compound E-305 (31 mg, 0.042 mmol, synthesized and obtained according to Bioorg. Med. chem. Lett. 14 (2004) 5551-5554) were taken at room temperature, and then the mixture was added with fluorenylmethoxycarbonylsuccinimid (Fmoc-OSu) (17 mg, 0.050 mmol) and solid sodium carbonate (18 mg, 0.168 mmol) in sequence. The mixture was stirred overnight at room temperature until substantially complete conversion of the starting materials was detected. The reaction was quenched with water, extracted with ethyl acetate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether) to obtain 15 mg of the product. LC/MS (ESI): m/z 965.64 [M+H].sup.+.

Step 2: Preparation of Compound D-9b

[0271] Compound D-9a (7 mg, 0.007 mmol) was dissolved in dichloromethane (0.5 mL) at room temperature, followed by the addition of 4 A molecular sieves (10 mg), trimethyloxonium tetrafluoroborate (11 mg, 0.07 mmol) and proton sponge (16 mg, 0.07 mmol) in sequence, and the mixture was stirred at room temperature for 1 h. When complete conversion of the starting material was detected, the reaction system was quenched by addition of water, extracted with methyl tert-butyl ether, washed with 1 N dilute hydrochloric acid and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether=1:1) to give 7 mg of product.

[0272] LC/MS (ESI): m/z 979.68 [M+H].sup.+.

Step 3: Preparation of Compound D-9

[0273] 1 mL of tetrahydrofuran was taken to dissolve compound D-9b (10 mg, 0.01 mmol) in an ice water bath, the reaction mixture was added dropwise with DBU (6 μL, 0.04 mmol), and stirred until the reaction was completed. The reaction was quenched by addition of water, extracted with dichloromethane and concentrated under reduced pressure. The residue was separated by prep-HPLC (column: Welch Xtimate C18 (10×150 mm×5 m), mobile phase: A-water (20 mM NH.sub.4HCO.sub.3): B-acetonitrile, gradient elution=30% B to 95% B) to give 5 mg of compound D-9.

[0274] LC/MS (ESI): m/z 757.85 [M+H].sup.+.

Example 2-10: Synthesis of Compound D-10

[0275] ##STR00114## ##STR00115##

Step 1: Preparation of Compound D-10b

[0276] 2 mL of tetrahydrofuran was taken to dissolve compound D-10a (6 mg, 0.008 mmol, synthesized and obtained according to Bioorg. Med. Chem. Lett. 21 (2011) 1639-1643) in an ice water bath, and the mixture was added with lithium aluminum hydride solution (80 μL, 1 M in THF, 0.08 mmol) dropwise and stirred. The reaction temperature was slowly raised to 40° C. When substantially complete conversion of the starting material was detected by LCMS, the reaction was quenched by sodium sulfate decahydrate, stirred for half an hour in an ice water bath, and filtered. The obtained filtrate was concentrated under reduced pressure to obtain a crude product which was directly used in the next step.

[0277] LC/MS (ESI): m/z 758.4 [M+H].sup.+.

Step 2: Preparation of Compound D-10c

[0278] 0.5 mL of 1,4-dioxane and 0.5 mL of water were taken to dissolve the compound D-10b obtained in the previous steps at room temperature, the reaction system was added with fluorenylmethoxycarbonylsuccinimide (6.5 mg, 0.019 mmol) and sodium carbonate (6.8 mg, 0.064 mmol) in sequence and stirred overnight at room temperature until substantially complete conversion of the starting material was detected. The reaction was quenched with water, extracted with ethyl acetate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether=3:2) to obtain 14 mg of compound D-10c.

[0279] LC/MS (ESI): m/z 980.4 [M+H].sup.+.

Step 3: Preparation of Compound D-10d

[0280] 1 mL of dichloromethane was taken to dissolve the compound D-10c (14 mg, 0.014 mmol) obtained in the previous steps in an ice water bath, followed by the addition of Dess-Martin periodinane (18.2 mg, 0.042 mmol). The reaction system was stirred and allowed to slowly warm to room temperature, and then stirred until substantially complete conversion of the starting material was detected by LCMS. The reaction was quenched by addition of aqueous sodium bicarbonate solution, extracted with dichloromethane, and concentrated under reduced pressure; the residue was purified by silica gel column chromatography (eluent: ethyl acetate/petroleum ether=3:2) to give 8 mg of compound D-10d.

[0281] LC/MS (ESI): m/z 978.4 [M+H].sup.+.

Step 4: Preparation of Compound D-10

[0282] The compound D-10D (8 mg, 0.008 mmol) obtained in the previous steps was dissolved in 1 mL of tetrahydrofuran in an ice water bath, the reaction system was added with DBU (6 μL, 0.032 mmol) dropwise and stirred for 1 h until substantially complete conversion of the starting material was detected by LCMS. The reaction was quenched by addition of water, the reaction mixture was extracted with dichloromethane and concentrated under reduced pressure, and the residue was separated by prep-HPLC (column: Welch Boltimate C18 Core-Shell (4.6×50 mm×2.7 μm), mobile phase: A-water (20 mM NH.sub.4HCO.sub.3)) to obtain the target compound D-10 (1.3 mg).

[0283] LC/MS (ESI): m/z 755.93 [M+H].sup.+.

Test Example 2: In Vitro Cytotoxic Activity Screening

2.1. Principle and Method

[0284] The CTG is used for detecting the ATP content in the experiment, and the survival condition of the tumor cells is reflected.

2.2. Determination of Cell Culture Conditions

[0285] 1) Cell culture: A549, SK-BR-3 and MDA-MB-468 cells were cultured with Ham's F-12K (Kaighn's) medium (Gibco, 21127030) and McCoy's 5A medium (ThermoFisher, Cat #16600108) and Leibovitz's L-15 medium (ThermoFisher, Cat #11415-114) containing 10% FBS (Gibco, 10099-141), respectively.

[0286] A549, SKBR3 and MDA-MB-468 were digested with trypsin, and each was resuspended in culture medium to a cell density of 2.2×10.sup.4 cells/mL, and 135 μL of cell suspension was added to each well in columns 2 to 11 of a 96-well plate, and column 12 was set as blank control. The cells were incubated in an incubator for 24 h at 37° C. with 5% CO.sub.2.

[0287] 2) Drug Preparation:

[0288] a) Stock solution preparation: the test compound and positive control drug were dissolved in DMSO to give a stock solution at a concentration of 5 mM.

[0289] b) Drug preparation plate 1: stock solution in column 1 was initially diluted 40-fold, and 3-fold gradient dilutions were performed sequentially in column 2 to column 11. Column 12 was filled with DMSO.

[0290] c) Drug preparation plate 2: 196 μL of the corresponding culture medium was added into columns 2 to 11, and 4 μL of culture medium was pipetted from column 3 to column 12 of drug preparation plate 1 to column 2 to column 11 of drug preparation plate

2. The Culture Medium was Mixed Well.

2.3. Cell Treatment

[0291] 15 μL of culture medium was pipetted from the drug preparation plate 2 and added into the plated cells. The cells were continuously incubated in an incubator for 5 h at 37° C. with 5% CO.sub.2.

[0292] 2.4) CTG assay (Cell Titer-Glo™, luminescent cell viability assay): the cell plates were removed and balanced to room temperature. 75 μL of CTG was added into each well and reacted at room temperature for 10 min in the dark. The absorption value was read using a microplate reader and IC.sub.50 was calculated.

2.5. Data Results

[0293]

TABLE-US-00028 TABLE 6 SKBR3 MDA-MB-468 A549 Maximum Maximum Maximum IC.sub.50 inhibition IC.sub.50 inhibition IC.sub.50 inhibition Compound (nM) (%) (nM) (%) (nM) (%) D-3 1.176 96.36 1.983 90.94 2.171 63.03 D-4 1.086 96.85 1.881 89.43 3.076 62.09 E-305 0.571 96.42 1.168 88.53 2.102 61.27 E1-30 0.1659 96.15 0.3596 90.49 0.7813 62.46

[0294] The positive control drugs, compound E-305 and compound E1-30, had structural formula as shown below and were prepared as described in Bioorganic & Medicinal Chemistry Letters 14 (2004) 5551-5554:

##STR00116##

Example 2-11: Synthesis of Compound L-2

[0295] ##STR00117##

[0296] Compound D-1a (9 mg, 0.012 mmol) was dissolved in 0.3 mL of DMF in an ice water bath, and the mixture was added with DIPEA (3.5 mg, 0.028 mmol), followed by compound 2 (7.8 mg, 0.011 mmol) in portions, and stirred until the reaction was substantially completed. The reaction mixture was concentrated under reduced pressure to obtain the crude product, which was separated by prep-HPLC (column: Xbridge Prep C18 OBD 5 μm×19×250 mm; mobile phase: A-water (10 mmol NH.sub.4OAc); B-acetonitrile, gradient elution) to obtain 4.95 mg of compound L-2 (97% o purity).

[0297] LC/MS (ESI): m/z 1374.3 [M+H].sup.+.

Example 2-12: Synthesis of Compound L-3

[0298] ##STR00118## ##STR00119## ##STR00120##

Step 1: Preparation of Compound 4

[0299] Compound 4a (1.3 g, prepared by the method disclosed in WO2013106717) was dissolved in 50 mL of acetonitrile, followed by addition of potassium carbonate (6.2 g), benzyl bromide (1.35 mL) and tetrabutylammonium iodide (415 mg) in sequence. The reaction mixture was stirred at room temperature until the reaction was substantially completed, filtered, concentrated, and purified by silica gel column chromatography with petroleum ether/ethyl acetate as developing solvent to obtain compound 4b.

[0300] Compound 4b (121 mg) and 4c (180 mg) were added into a reaction flask, and the mixture was added with 4 mL of tetrahydrofuran. In a nitrogen atmosphere, the reaction system was reduced to about 0° C. in an ice water bath, added with potassium tert-butoxide (109 mg, 0.98 mmol), warmed to room temperature and stirred for 40 min. The reaction mixture was added with 10 mL of ice water and extracted with ethyl acetate (20 mL×2) and chloroform (10 mL×5); the organic phases were combined and concentrated. The resulting residue was dissolved in 4 mL of dioxane, and 2 mL of water, sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added. The mixture was stirred at room temperature for 2 h. 20 mL of water was added, followed by extraction with ethyl acetate (10 mL×3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The reaction mixture was purified by silica gel column chromatography with petroleum ether/ethyl acetate as developing solvent to obtain compound 4b, MS m/z (ESI): 515.0 [M+1].sup.+.

[0301] Compound 4b (20 mg, 0.038 mmol) was dissolved in 4.5 mL of a solvent mixture of tetrahydrofuran and ethyl acetate (V:V=2:1), and palladium on carbon (12 mg, 10% loading, dry basis) was added. The system was purged with hydrogen three times, and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was filtered through celite, and the filter cake was rinsed with ethyl acetate. The filtrate was concentrated to give the crude title compound 4 (13 mg), which was directly used in the next step without purification.

[0302] MS m/z (ESI): 424.9 [M+1].

Step 2: Preparation of Compound DZ-1a

[0303] Compound 4 (13.4 mg, 0.0316 mmol, 1.7 eq) and the mesylate of compound D-1a (15 mg, 0.0182 mmol, 1 eq) were weighed and dissolved in DMF (0.5 mL); the reaction mixture was added with triethylamine (10 mg, 0.0988 mmol, 5.4 eq) and DMTMM (9.8 mg, 0.0332 mmol, 1.8 eq) in an ice water bath, naturally warmed to room temperature and stirred until reaction was substantially completed. Water (2 mL) and ethyl acetate (3 mL) were added to dilute for separation, the aqueous phase was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by preparative thin layer chromatography (ethyl acetate/petroleum ether) to give 16 mg of compound DZ-1a with a yield of 86.7%.

[0304] LC/MS (ESI): m/z 1136.3 [M+H].sup.+.

Step 3: Preparation of Compound DZ-1b

[0305] Compound DZ-1a (16 mg, 0.0141 mmol, 1 eq) obtained in the above step was weighed and dissolved in THF (0.4 mL) in an ice water bath; the reaction mixture was added with triethylamine (4.2 mg, 0.057 mmol, 4 eq) and stirred under ice-bath until the reaction was substantially completed. The reaction mixture was diluted with dichloromethane (5 mL) and washed with water (2 mL×3); the organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product which was used directly in the next step.

[0306] LC/MS (ESI): m/z 914.3 [M+H].sup.+.

Step 4: Preparation of Compound L-3

[0307] The crude compound DZ-1b (16 mg, 0.0175 mmol, 1 eq) obtained in the above step and compound 6 (11.6 mg, 0.0246 mmol, 1.4 eq, prepared by the method described in EP2907824) were weighed and dissolved into DMF (0.5 mL); the reaction mixture was added with HATU (9.9 mg, 0.026 mmol, 1.5 eq) and N,N-Diisopropylethylamine (DIPEA) (5.5 mg, 0.0426 mmol, 2.4 eq) and stirred under ice bath until the reaction was substantially completed. Water (2 mL) and ethyl acetate (3 mL) were added to dilute for separation, the aqueous phase was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by prep-HPLC (column: XBridge Prep C18 OBD 5 μm×19×250 mm; mobile phase: A-water (10 mmol NH.sub.4OAc): B-acetonitrile, gradient elution) to give 10 mg of compound L-3.

[0308] LC/MS (ESI): m/z 1368.3 [M+H].sup.+.

Example 2-13: Synthesis of Compound L-4

[0309] ##STR00121## ##STR00122##

Step 1: Preparation of Compound DZ-2a

[0310] Compound 4 (11.6 mg, 0.0273 mmol, 1.5 eq) and compound D-1 (13.5 mg, 0.0181 mmol, 1 eq) were weighed and dissolved in N,N-dimethylformamide (0.5 mL); the reaction mixture was added with DMTMM (10.1 mg, 0.0343 mmol, 1.3 eq) in an ice water bath, and stirred until reaction was substantially completed. Water (2 mL) and ethyl acetate (3 mL) were added to terminate and dilute reaction, the reaction mixture was separated, the aqueous phase was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by preparative thin layer chromatography (ethyl acetate/petroleum ether) to give 10 mg of compound with a yield of 47.9%.

[0311] LC/MS (ESI): m/z 1150.2 [M+H].sup.+.

Step 2: Preparation of Compound DZ-2b

[0312] The product compound DZ-2a (10 mg, 0.0087 mmol, 1 eq) obtained from the previous step was weighed and dissolved into THF (1 mL); the reaction mixture was added with DBU (1,8-diazabicycloundec-7-ene) (5.2 mg, 0.034 mmol, 4 eq) and stirred in an ice water bath until the reaction was substantially completed. The reaction mixture was diluted with dichloromethane (5 mL) and washed with water (2 mL×3); the organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product which was used directly in the next step.

[0313] LC/MS (ESI): m/z 928.2 [M+H].sup.+.

Step 3: Preparation of Compound L-4

[0314] The product compound DZ-2b (16 mg, 0.0087 mmol, 1 eq) obtained in the above step and compound 6 (7.8 mg, 0.0165 mmol, 1.9 eq) were weighed and dissolved into DMF (0.5 mL); the reaction mixture was added with HATU (6.2 mg, 0.0163 mmol, 1.9 eq) and DIEA (5.7 mg, 0.0441 mmol, 5 eq) and stirred under ice bath until the reaction was substantially completed. Water (2 mL) and ethyl acetate (3 mL) were added to dilute for separation, the aqueous phase was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by prep-HPLC (column: XBridge Prep C18 OBD 5 μm×19×250 mm; mobile phase: A-water (10 mmol NH.sub.4OAc): B-acetonitrile, gradient elution) to give 3.5 mg of compound L-4 with a yield of 29.1% for two steps.

[0315] LC/MS (ESI): m/z 1382.2 [M+H].sup.+.

Examples 2-14: Preparation of antibody drug conjugate ADC-1

[0316] ##STR00123##

[0317] To an aqueous PBS buffer of antibody PD3 (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 0.9 mL, 60.6 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 15.2 μL, 152 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. with a water bath. Compound L-3 (0.83 mg, 606 nmol) was dissolved in 50 μL of DMSO, and the resulting solution was added to the above reaction mixture, which was then shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5, containing 0.001 M EDTA) to give the title product ADC-1 in PBS buffer (0.76 mg/mL, 10 mL), which was frozen and stored at 4° C.

[0318] The average value was calculated by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) ultraviolet assay: k=3.87.

Examples 2-15: Preparation of antibody drug conjugate ADC-2

[0319] ##STR00124##

[0320] To an aqueous PBS buffer of antibody PD3 (0.05 M aqueous PB buffer at pH 6.5; 10.0 mg/mL, 1.14 mL, 77.2 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 19.3 μL, 193 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. with a water bath.

[0321] Compound L-2 (0.83 mg, 772 nmol) was dissolved in 50 μL of DMSO, and the resulting solution was added to the above reaction mixture, which was then shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5, containing 0.001 M EDTA) to give the title product ADC-2 in PBS buffer (0.71 mg/mL, 12 mL), which was frozen and stored at 4° C. Mean was calculated by CE-SDS: k=3.88.

Examples 2-16: Preparation of Antibody Drug Conjugate ADC-3

[0322] ##STR00125##

[0323] To an aqueous PBS buffer of antibody PD3 (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 0.9 mL, 60.6 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 15.2 μL, 152 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. with a water bath.

[0324] Compound L-1 (0.84 mg, 605 nmol) was dissolved in 50 μL of DMSO, and the resulting solution was added to the above reaction mixture, which was then shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5, containing 0.001 M EDTA) to give the title product ADC-3 in PBS buffer (0.77 mg/mL, 10.2 mL), which was frozen and stored at 4° C. Mean was calculated by CE-SDS: k=3.81.

Examples 2-17: Preparation of Antibody Drug Conjugate ADC-4

[0325] ##STR00126##

[0326] To an aqueous PBS buffer of antibody PD3 (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 0.9 mL, 60.6 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 15.2 μL, 152 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. with a water bath.

[0327] Compound L-4 (0.84 mg, 608 nmol) was dissolved in 50 μL of DMSO, and the resulting solution was added to the above reaction mixture, which was then shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5, containing 0.001 M EDTA) to give the title product ADC-4 in PBS buffer (0.64 mg/mL, 13.5 mL), which was frozen and stored at 4° C. Mean was calculated by CE-SDS: k=3.88.

Examples 2-18: Preparation of Antibody Drug Conjugate ADC-5

[0328] ##STR00127##

[0329] To an aqueous PBS buffer of CD79B antibody hAb015-10 (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/ml, 1.0 mL, 67.3 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 16.8 μL, 168 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. with a water bath.

[0330] Compound L-1 (0.93 mg, 673 nmol) was dissolved in 50 μL of DMSO, and the resulting solution was added to the above reaction mixture, which was then shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5, containing 0.001 M EDTA) to give the title product ADC-5 in PBS buffer (0.68 mg/mL, 9.6 mL), which was frozen and stored at 4° C. Mean was calculated by CE-SDS: k=4.07.

Test Example 3: Evaluation and Comparison for Efficacy of ADC-5 and Polivy on Nude Mouse Subcutaneous Xenograft Tumor of Human Diffuse Large B-Cell Lymphoma WSU-DLCL2

3.1 Drug Information

[0331] Blank group: hIgG1;

ADC-5: colorless and clear liquid with a concentration of 0.68 mg/mL and a purity of 98.00%, shaded and sealed at 2-8° C.;

[0332] Polivy (polatuzumab): colorless and clear liquid with a concentration of 5.83 mg/mL and a purity of 97.69%, shaded and sealed at 2-8° C.

3.2 Drug Preparation

[0333] All solutions were diluted with normal saline to the desired concentration.

[0334] Normal saline with a specification of 10 mL:0.09 g was purchased from China Otsuka Pharmaceutical Co., Ltd.

3.3 Cells

[0335] Human diffuse large B-cell lymphoma WSU-DLCL2 cells were purchased from DSMZ. WSU-DLCL2 cells were cultured in a 10-cm petri dish with RPMI 1640 medium (Gibco) containing 10% fetal bovine serum, penicillin and streptomycin (GIBCO, Cat #15070-063) and incubated in an incubator at 37° C. with 5% CO.sub.2. Subculturing was carried out 2-3 times a week and cells were collected, counted and inoculated when the cells grew in long-term exponentially.

3.4 Experimental Animals

[0336] Female BALB/c-nu nude mice with a growth period of 28-35 days was purchased from Beijing Huafukang Biotechnology Co., Ltd. Production license No.: SCXK (Beijing) 2019-0008, animal certification No.: 1103221911012510. Housing environment: SPF grade.

3.5 Experimental Steps

[0337] Each nude mouse was subcutaneously inoculated with 2.0×10.sup.7 WSU-DLCL2 cells, and after the tumor volume grew to −100 mm.sup.3, the animals were grouped according to tumor volume (D0). The mice was administrated by intravenous injection (IV), and the administration volume was 10 mL/kg; specific dosages and schedules are shown in Table 3. The tumor volumes and body weights were measured twice a week and the results were recorded.

[0338] The use and welfare of the laboratory animals were carried out in compliance with the provisions of Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC). The health and death of the animals were monitored daily, and routine examinations included observation of the effects of the test substance and drug on the daily performance of the animals, such as behavioral activities, weight changes and appearance. 3.6 Experimental index

[0339] The experimental index is to study the influence of the drug on the tumor growth, and the specific index is T/C % or tumor growth inhibition TGI (%). Tumor diameters were measured twice weekly with a vernier caliper and tumor volume (V) was calculated according to the following formula:

V=½×a×b.sup.2

where a and b represent length and width, respectively. T/C (%)=(T−T0)/(C−C0)×100, where T and C were tumor volumes at the end of the experiment; T0 and C0 were tumor volumes at the beginning of the experiment.

Tumor inhibition rate (TGI) (%)=100−T/C(%).

Tumor growth inhibition (TGI) (%)=100−(T−T0)/T0×100 when tumor started to regress.

[0340] If the volume of tumor shrank compared with its initial volume, i.e., T<T0 or C<C0, it was defined as partial regression(PR) of tumor; if the tumor completely disappeared, it was defined as complete regression (CR) of tumor.

[0341] At the end of the experiment, at the experiment endpoint, or when the mean tumor volume in the solvent group reached 1500 mm.sup.3, the animals were sacrificed by CO.sub.2 anesthesia and dissected to give the tumors. The tumors were photographed.

3.7 Statistical Analysis

[0342] Unless otherwise indicated, comparison between tumor volumes of the two groups was made by two-tailed student's t-test, with P<0.05 defined as statistically significant difference.

3.8. Results

[0343] The tumor inhibition rates of ADC-5 and Polivy (IV; D0, 1 mg/kg) on nude mouse subcutaneous xenograft tumor of human diffuse large B-cell lymphoma WSU-DLCL2 were 53% and 37%, respectively; the tumor-bearing mice could well tolerate the above drugs, and symptoms such as significant weight loss and the like did not occur.

TABLE-US-00029 TABLE 7 Tumor Mean tumor volume inhibition P of (mean ± SEM, mm.sup.3) T/C % rate (%) value Group Route administration D0 D17 D17 D17 D17 Blank D0 IV 109.29 ± 1.16 810.03 ± 83.95 — — group D0 IV 111.07 ± 2.15 441.02 ± 96.42 47 53 0.014 ADC-5, 1 mg/kg Polivy D0 IV 108.07 ± 1.31 550.88 ± 52.37 63 37 0.044 1 mg/kg

[0344] Note: IV Intravenous Injection

Conclusion

[0345] The tumor inhibition rates of the antibody-drug conjugate ADC-5 and Polivy (positive control group) on nude mouse subcutaneous xenograft tumor of human diffuse large B-cell lymphoma WSU-DLCL2 were 53% and 37%, respectively; the antibody-drug conjugate ADC-5 and Polivy have significant anti-tumor activity; the tumor-bearing mice can well tolerate the above drug.

Test Example 4: Cell Killing Study

4.1. Objective

[0346] The objective of this study is to test the inhibitory activity of the anti-TROP-2 antibody (PD3)-drug conjugate of the present disclosure against the proliferation of different tumor cell lines: Miapaca2 tumor cells (human pancreatic cancer cells, Nanjing Kebai Biotechnology Co., Ltd., Cat #CBP60544), Fadu tumor cells (human squamous cell carcinoma, ATCC, Cat #HTB-43), SK-OV-3 (human ovarian cancer cells, ATCC, Cat #HTB-77), K562 (human chronic granulocytic leukemia cells, ATCC, Cat #CCL-243), HCC827 (human lung cancer cells, ATCC, Cat #CRL-2868) and BXPC3 (human pancreatic cancer cells, ATCC, Cat #CRL-1687). The cells were treated in vitro with the antibody drug conjugate at different concentrations. After 6 days of culture, the proliferation of cells was tested using CTG (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, Cat #G7573) reagents, and the in vitro activity of the antibody drug conjugate was evaluated according to IC.sub.50 value.

4.2. Method

[0347] 1) Cell culture: MiaPaCa2, Fadu, SK-OV-3, K562, HCC827 and BXPC3 cells were cultured in DMEM/high glucose medium (GE, SH30243.01), MEM medium (Gibco, 11095080), McCoy'S 5a medium (Gibco, 16600108), IMDM medium (ThermoFisher, 12440061), RPIM1640 medium (Gibco, 11875119) containing 10% FBS (Gibco, 10099-141).

[0348] 2) Cell plating: on the day of the study, after the cells were digested with Trypsin (0.25% Trypsin-EDTA (1×), Life Technologies, Cat #25200-072), MiaPaCa2, Fadu, SK-OV-3, K562, HCC827 and BXPC3 were suspended in cell suspensions using the corresponding media to a density of 3.7×10.sup.3 cells/mL, and 135 μL of suspension was added into each well of a 96-well plate (coming, Cat #3903) to culture 500 cells per well at 37° C. for 24 h.

[0349] 3) Drug preparation: the mother liquor of the test ADC was first adjusted to have a concentration of 4 μM and was added to the first column of a drug preparation plate (coming, Cat #3599). The plate was diluted 5-fold in gradient from column 2 to column 9, with PBS in column 10. 15 μL of the mother liquor in each well was added to the cell culture plate.

[0350] 4) CTG assay: after being cultured at 37° C. for 6 days, the cell culture plate was removed and balanced to room temperature. 75 μL of CTG was added into each well and reacted at room temperature for 10 min in the dark. The absorption value was read using a microplate reader (BMG labtech, PHERAstar FS).

4.3 Data Analysis

[0351] Data were processed and analyzed using Graphpad Prism 5. The results are shown in Table 8 below

TABLE-US-00030 TABLE 8 No. Miapaca K562 SKOV3 HCC827 BXPC3 Fadu ADC-1 64.05 73.21 54.14 0.30 0.16 0.96 ADC-2 24.59 27.95 0.27 0.11 0.05 0.11 ADC-3 31.22 44.10 10.42 0.01 0.09 0.60 ADC-4 29.95 39.44 18.73 1.37 1.38 4.04 E1-30 0.45 0.39 0.17 0.31 0.23 0.32

Test Example 5: Bystander Killing Study

5.1. Objective

[0352] When the antibody drug conjugate disclosed by the present disclosure is co-cultured in TROP-2 positive cells BXPC3 (human in-situ pancreatic adenocarcinoma cell, Procell) and TROP-2 negative cells MiaPaCa2 (human pancreatic adenocarcinoma cell, Procell), a concentration of 5 nM at which the antibody drug conjugate has killing effect on the TROP-2 positive cells BXPC3 and does not have killing effect on the TROP-2 negative cells MiaPaCa2 was selected for the study, and whether the antibody drug conjugate has bystander killing effect on the TROP-2 negative cells Miapaca2 in a co-culture system of the two is examined.

5.2. Method

[0353] 1) Cell culture: MiaPaCa2 and BXPC3 cells were cultured in DMEM/high glucose medium (GE, SH30243.01) and RPIM1640 medium (Gibco, 11875119) containing 10% FBS (Gibco, 10099-141).

[0354] 2) Cell plating: on the day of the study, the cells were digested with Trypsin (0.25% Trypsin-EDTA (1×), Life Technologies, Cat #25200-072), neutralized with fresh RPIM1640 medium (containing 10% FBS), and centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and the cells were resuspended in RPMI1640+10% FBS. After the cells were counted, the cell density of BXPC3 was adjusted to 6×10.sup.4 cells/mL and the cell density of MiaPaCa2 was adjusted to 1.5×10.sup.4 cells/mL. 500 μL of BXPC3 cells and 500 μL of MiaPaCa2 cells were added into each well in plate 1 in a 12-well plate. 500 μL of MiaPaCa2 cells and 500 μL of culture medium containing RPMI1640+10% FBS were added into plate 2 in the 12-well plate. The plate was cultured at 37° C. in 5% carbon dioxide for 24 h.

[0355] 3) Antibody drug conjugate preparation: antibody drug conjugates ADC-1, ADC-2, ADC-3 and ADC-4 were each diluted to a concentration of 600 nM with RPMI1640, and 50 μL of antibody drug conjugate was taken and diluted to a concentration of 200 nM with 100 μL of culture medium (40×, a final concentration of 5 nM). 25 μL of the antibody drug conjugate was added into the cell culture plate. A PBS solvent control group was additionally set and culture was continued for 6 days.

[0356] 4) Flow analysis: the cells in the 12-well plate (plate 1 and plate 2) were digested with trypsin, neutralized with fresh medium, and centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and the cells were resuspended in 1 mL of FACS buffer (PBS+2.5% FBS). 20 μL of the cells were taken, stained with 20 μL of trypan blue (Sigma, T8154-100ML) and counted. The cells in the plate 1 were centrifuged at 1000 rpm for 3 min, the supernatant was discarded, the cells were resuspended in 100 μL of FACS Buffer, 2 μL of TROP-2 (EGP-1) monoclonal antibody (MR54) (ThermoFisher, Cat #12-6024-42) was added, and the cells were incubated on ice for 30 min. The cells were then centrifuged at 2000 rpm for 1 min at 4° C., 150 μL of FACS buffer was added to resuspend the cells, and the above procedure was repeated twice. Detection was performed by flow cytometry (BD, FACSVerse).

5.3 Data Analysis

[0357] The data were processed and analyzed using Flowjo 10.0.

[0358] Conclusion: all antibody conjugates showed a bystander effect in the study.

Test Example 6. Pharmacokinetic Study

1. Overview

[0359] Non-naïve beagles were taken as test animals, the drug concentrations in plasma at various times after intravenous administration of beagles with compound D-1 and eribulin were measured by using LC/MS. The pharmacokinetic performance in beagles of the compounds of the present disclosure was studied and the pharmacokinetic profile thereof was evaluated.

2. Methodology

2.1. Test Compounds

Compound D-1 and Eribulin

2.2. Test Animals

[0360] Male 6 beagles were divided into 2 groups of 3, purchased from Shanghai Medicilon Inc., and subjected to administration test.

2.3. Drug Preparation

[0361] Compound D-1 was weighed, dissolved by adding 5% volume of DMSO, 20% PG and 20% PEG400, and then prepared into a colorless and clear solution of 0.25 mg/mL by adding 55% of normal saline.

Eribulin was weighed, dissolved by adding 5% volume of DMSO, 20% PG and 20% PEG400, and then prepared into a colorless and clear solution of 0.25 mg/mL by adding 55% of normal saline.

2.4. Administration

[0362] A group of beagles were intravenously injected with compound D-1 at a dose of 0.5 mg/kg and at a volume of 2 mL/kg.

Another group of beagles were intravenously injected with eribulin at a dose of 0.5 mg/kg and at a volume of 2 mL/kg.

3. Procedures

[0363] The beagles were injected with compound D-1, 1 mL of blood samples were collected before administration and at 5 min, 0.25 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h, 8.0 h, 12.0 h and 24.0 h after administration, the collected blood samples were placed in EDTA-K2 anticoagulant blood collection tubes, the collected whole blood samples were placed on ice, and plasma was centrifuged (centrifugal force: 2200 g, centrifugal time: 10 min, 2-8° C.) in 1 h. Plasma samples were stored in a refrigerator at −80° C. prior to testing. The beagles were injected with compound eribulin, 1 mL of blood samples were collected before administration and at 5 min, 0.25 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h, 8.0 h, 12.0 h and 24.0 h after administration, the collected blood samples were placed in EDTA-K2 anticoagulant blood collection tubes, the collected whole blood samples were placed on ice, and plasma was centrifuged (centrifugal force: 2200 g, centrifugal time: 10 min, 2-8° C.) in 1 h. Plasma samples were stored in a refrigerator at −80° C. prior to testing. The plasma concentration of the test compounds in beagles after drug injection was determined: after administration, 25 μL of beagle plasma at various times after administration was taken, and added with 50 μL (100 ng/mL) of internal standard solution camptothecin (National Institutes for Drug Control) and 200 μL of acetonitrile. The mixture was vortexed for 5 min and centrifuged for 10 min (3700 rpm/min). 3 to 4 μL of the supernatant of the plasma sample was subjected to LC/MS assay (API4000 triple quadrupole tandem mass spectrometer (No. 2), Applied Biosystems, USA; Shimadzu, LC-30AD ultra high performance liquid chromatography system, Shimadzu, Japan.) The analysis was performed.

4. Pharmacokinetic Parameters

[0364] Pharmacokinetic parameters for the compound of the present disclosure are shown in Table 9 below.

TABLE-US-00031 TABLE 9 Pharmaceutical study for beagles Area Apparent Plasma under Half Retention volume of concentration curve life Time Clearance distribution C5 min AUC0-t T½ MRT CL Vz No. (ng/mL) (ng/mL*h) (h) (h) (ml/min/kg) (ml/kg) Compound 82.1 433 29.3 41.1 8.9 22039 D-1 Eribulin 217 106 3.5 3.7 78 15556

Test Example 7: Efficacy of ADC-2 and ADC-3 on BALB/c Nude Mouse Subcutaneous Xenograft Tumor of Human Pharyngeal Squamous Cell Carcinoma FaDu Cell Line

(1) Cell Culture

[0365] The human pharyngeal squamous cell carcinoma Fadu cell line (ScienCell Laboratory, ml-cs-0374) used in this study was cultured in MEM medium (supplemented with 10% (v/v) fetal bovine serum (FBS) (GIBCO, 10099-141) and 0.1% phosphate buffer) in an incubator at 37° C. with 5% CO.sub.2. Mice were anesthetized with 3-4% isoflurane before inoculation. Before the cells were continuously cultured for ten passages, 100 μL of Fadu cell culture medium at a density of 5×10.sup.6 and an equal volume of Matrigel (solarbio) were mixed well and inoculated subcutaneously to the right side of the back of the mice near the axilla.

(2) Animal Grouping and Administration Regimen

[0366] When tumors grew to an average of about 100-150 mm.sup.3, mice were randomly grouped of 8 by tumor volume and body weight. The day of grouping and administration was defined as day 0. The grouping and administration regimen is shown in Table 10 below:

TABLE-US-00032 TABLE 10 Grouping and administration regimen Number Test of animals Dose Administration Groups compounds per group (mg/kg) regimen G1 Blank vehicle 8 N/A Administration group route: I.P G2 ADC-3 8 3 Administered G3 ADC-3 8 1.5 twice G4 ADC-2 8 3 at day 0 and G5 ADC-2 8 1.5 day 14

[0367] Administration volume: the administration volume was adjusted at 10 μL/g according to the body weight of mice.

[0368] Tumor volume: tumor volumes were measured twice a week for 4 consecutive weeks after the grouping. Tumor volume (V) was calculated as follows: v=(length×width.sup.2)/2. The relative tumor volume (RTV) per mouse was calculated as: RTV=Vt/V0, where Vt is the measured volume for each time and V0 is the volume at the start of the treatment.

[0369] Animal body weight: mice body weights were measured and recorded twice a week after the grouping.

[0370] Animal state observation: no abnormalities were observed in animals given vehicle or test drugs in this study. At the end of the experiment, some animals developed ulcers.

(3) Drug Withdrawal and Administration Criteria Recovery in Study

[0371] In the study process, when the body weight of the mice was reduced by ≥15%, the administration was interrupted; and the interruption period should be long enough to recover the body weight of the mice. The administration to only one mouse was interrupted, while the administration to other mice was normally performed; the study was continuously performed when the body weight of mice recovered at drug withdrawal with reference to the following criteria: the body weight of the mice was reduced by <10%.

(4) Endpoint

[0372] At the end of the in vivo experiment, all animals were asphyxiated by CO.sub.2 and then sacrificed by cervical dislocation. Tumors were collected, weighed and photographed. Dead tumor-bearing animals would not be sampled before the in vivo experiment was completed.

(5) Statistical Analysis

[0373] Results would be presented as mean±S.E.M. Comparisons between the two groups would be tested with Dunnett's multi-comparison test. Statistically significant differences were considered if p<0.05, which was recorded as *, p<0.01 recorded as **, and p<0.001 recorded as ***.

(6) Results

[0374] Weight: the trend of body weight change of animals in vehicle group and administration groups with different test drugs is shown in FIG. 1. Animal body weight increased normally with the progress of the experiment.

Tumor Volume:

[0375] In the experimental period, after animals in the blank vehicle group (G1) were inoculated, grouped and administered, the tumor slowly grew; tumor volume increased rapidly until day 14. Mean value of tumor volume for G1 at day 0 of the experiment was: 125.05±3.66 mm.sup.3; mean value of tumor volume at day 25 was: 1854.48±99.50 mm.sup.3. The experimental data of the tumor volume and the relative tumor volume showed that the human pharyngeal squamous cell carcinoma Fadu cell line was successfully established in BALB/c mouse subcutaneous xenograft tumor model.

[0376] Mean value of tumor volume of animals in ADC-3 high-dose (3 mg/kg) group (G2) at day 0 of the experiment was: 121.40±3.18 mm.sup.3; mean value of tumor volume at day 25 was: 721.56±169.15 mm.sup.3. During the experimental period, the ADC-3 high-dose group could significantly inhibit the growth of the tumor compared with the blank vehicle group.

Relative Tumor Proliferation Rate and Tumor Weight Inhibition Rate:

[0377] Relative tumor proliferation rate (T/C %) was used for evaluating the effect of the anti-tumor activity of the drug, the relative tumor proliferation rate T/C (%)=mean RTV of treatment group (T)/mean RTV of negative control group (C)×100%. The relative tumor proliferation rate of each group at each time point are shown in Table 11, and the trend diagram is shown in FIG. 2.

TABLE-US-00033 TABLE 11 Relative tumor proliferation rate of each group at each time point Days of Blank treat- vehicle ADC-3 ADC-3 ADC-2 ADC-2 ment group 3 mg/kg 1.5 mg/kg 3 mg/kg 1.5 mg/kg 0 N/A 97.08% 100.09% 97.59% 98.47% 4 N/A 79.18% 99.71% 99.84% 97.38% 7 N/A 57.93% 69.61% 56.73% 70.41% 11 N/A 34.24% 68.23% 44.34% 73.83% 14 N/A 34.27% 74.62% 48.35% 83.36% 18 N/A 32.07% 63.41% 50.87% 73.08% 21 N/A 30.56% 64.07% 50.08% 76.87% 25 N/A 38.91% 76.25% 60.07% 84.03%

Conclusion:

[0378] After the ADC-3 group was administrated, the tumor volume in the high-dose group was significantly reduced compared with that in the model group, and the tumor volume in the low-dose group was reduced compared with that in the model group, while the two groups had no statistical difference. The drug showed an effect in inhibiting dose-dependent tumor growth. Meanwhile, the in-vivo efficacy of the ADC-3 group was better than that of the ADC-2 group in day 25 after the administration of 3 mg/kg dose, and the two groups had significant difference.

[0379] Although specific embodiments of the present disclosure have been described above, it will be appreciated by those skilled in the art that these embodiments are merely illustrative and that many changes or modifications can be made to these embodiments without departing from the principles and spirit of the present disclosure. The scope of protection of the present disclosure is therefore defined by the appended claims.