RU2830167C1 - Conjugate of trop-2 antibody and exatecan analogue and medical use thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a conjugate of an anti-TROP-2 antibody and an exatecan analogue and its medical use. Presented is a conjugate of an anti-TROP-2 antibody and an exatecan analogue of general formula (Pc-L-Y-D), where Pc is an anti-TROP-2 antibody or an antigen-binding fragment thereof.

EFFECT: greater tumor efficacy.

35 cl, 5 dwg, 9 tbl, 3 ex

Description

This application claims priority from Chinese patent application (Application No. CN202010073438.8) filed on January 22, 2020.

AREA OF TECHNOLOGY

The present invention relates to an antibody to TROP-2 and a conjugate of an antibody to TROP-2 and an analogue of exatecan, a method for producing them, pharmaceutical compositions containing them, and their use for producing a medicinal product for treating a disease or condition mediated by TROP-2, in particular their use for producing an anticancer medicinal product.

PRIOR ART

The information set forth herein is intended to provide only general information relating to the present invention and may not necessarily constitute prior art.

TROP-2, human trophoblast surface glycoprotein antigen 2, also known as tumor-associated calcium signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1), gastrointestinal tumor-associated antigen (GA733-1), and surface marker 1 (M1S1), is a cell surface glycoprotein encoded and expressed by the Tacstd2 gene in the 1p32 region of chromosome 1p32. TROP-2 is a member of the GA733 protein family and has a higher structural sequence similarity to epithelial cell adhesion molecule (EpCAM, also known as Trop1 and TACSTD1) with 49% homology.

The primary structure of TROP-2 protein is a polypeptide of about 36 kDa consisting of about 323 amino acids, and the primary structure is modified post-translationally by N-terminal glycosylation to form a type I cell membrane glycoprotein different from EpCAM, i.e., TROP-2 protein. TROP-2 protein spans the cell membrane and has an N-terminal extracellular domain (Trop2EC), and the extracellular domain is immobilized on the cell membrane via a unidirectional transmembrane helix (TM) connected to a short intracellular tail (Trop2IC) of a hydrophobic polypeptide consisting of 26 amino acid residues.

It has now been established that TROP-2 is important in the processes of embryonic development and tumor cell proliferation and metastasis. TROP-2 is initially found in the trophoblast cell and serves as its surface marker, the trophoblast cell is derived from extraembryonic trophoblast, and TROP-2 promotes embryo implantation and placental tissue formation and plays an important role in maintaining the proliferative characteristics of embryonic stem cells and organ formation and development. In addition, TROP-2 is also an important factor associated with tumor development, and is highly expressed in various tumors such as pancreatic cancer, breast cancer, colon cancer, gastric cancer, oral squamous cell cancer and ovarian cancer, and can promote the processes of tumor cell proliferation, invasion, metastasis, diffusion and the like, and is closely associated with shortened survival and poor prognosis of tumor subjects, so it is of great significance to the research of antitumor drug targeting TROP-2.

Antibody-drug conjugate (ADC) binds monoclonal antibody or antibody fragment to biologically active cytotoxin through a linker compound, fully utilizing the binding specificity of antibody to surface antigens of normal cells and tumor cells and the high efficiency of cytotoxic substance, and avoiding the defects of poor therapeutic effect of antibody and serious toxic side effects of toxic substance. This means that antibody-drug conjugate can more accurately kill tumor cells and have reduced effects on normal cells compared with conventional chemotherapeutic drugs in the past.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to an antibody to TROP-2, an ADC thereof and a use thereof, and provides a medicament ADC in which the antibody to TROP-2 or an antigen-binding fragment is conjugated to an exatecan analogue, a cytotoxic substance.

The present invention relates to a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof,

Where:

Y is selected from the group consisting of -O-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-, -O-CR ¹ R ² -(CR ^a R ^b ) _m -, -O-CR ¹ R ² -, -NH-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)- and -S-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-;

R ^a and R ^b are identical or different and are each independently selected from the group consisting of hydrogen, deuterium, halogen, alkyl, haloalkyl, deuterated alkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl and heterocyclyl; or R ^a and R ^b , together with the carbon atoms to which they are attached, form cycloalkyl or heterocyclyl;

R ¹ is selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; R ² is selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; or R ¹ and R ^2, together with the carbon atoms to which they are attached, form cycloalkyl or heterocyclyl;

or R ^a and R ² , together with the carbon atoms to which they are attached, form cycloalkyl or heterocyclyl;

m is an integer from 0 to 4; non-limiting examples of m are, for example, m selected from the group consisting of 0, 1, 2, 3, and 4;

n is a decimal or integer number between 1 and 10;

L is a linker moiety;

Pc is an antibody to TROP-2 or its antigen-binding fragment.

In some embodiments of the invention, in the ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof as disclosed above, the antibody to TROP-2 or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having sequences identical to the sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region presented in SEQ ID NO: 3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having sequences identical to the sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region presented in SEQ ID NO: 4.

In some embodiments of the invention, in the ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above-mentioned embodiments of the invention, the antibody to TROP-2 or an antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 presented in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 presented in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

In some embodiments of the invention, in the ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above-mentioned embodiments of the invention, the antibody to TROP-2 is a mouse antibody, a chimeric antibody, a humanized antibody, or a human antibody.

In some embodiments of the invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments of the invention, the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence presented in SEQ ID NO: 3 or has at least 90-100% identity thereto, including, but not limited to, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%, and the light chain variable region has the amino acid sequence presented in SEQ ID NO: 4 or has at least 90-100% identical thereto, including, but not limited to, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

In some embodiments of the invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above-mentioned embodiments of the invention, the antibody to TROP-2 or an antigen-binding fragment thereof comprises a heavy chain variable region presented in SEQ ID NO: 3 and a light chain variable region presented in SEQ ID NO: 4.

In some embodiments of the invention, in the ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above-mentioned embodiments of the invention, the antibody to TROP-2 or an antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 constant regions, and the light chain constant region is selected from the group consisting of human κ and λ chain constant regions; and more preferably, said antibody comprises a heavy chain constant region having the sequence presented in SEQ ID NO: 11 and a light chain constant region having the sequence presented in SEQ ID NO: 12.

In some embodiments of the invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above-mentioned embodiments of the invention, the antibody to TROP-2 comprises a heavy chain as presented in SEQ ID NO: 13 and a light chain as presented in SEQ ID NO: 14.

In some embodiments of the invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof, as described above, the antibody to TROP-2 comprises a heavy chain having the sequence of SEQ ID NO: 15 and a light chain having the sequence of SEQ ID NO: 16, or comprises a heavy chain having the sequence of SEQ ID NO: 17 and a light chain having the sequence of SEQ ID NO: 18.

In some embodiments of the invention, in the ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, n is a decimal or integer from 2 to 10, preferably from 4 to 8. In some embodiments, n is an average of 0 to 10, preferably from 1 to 10, more preferably from 1 to 8 or from 2 to 8 or from 2 to 7 or from 2 to 4 or from 3 to 8 or from 3 to 7 or from 3 to 6 or from 4 to 7 or from 4 to 6 or from 4 to 5; in some embodiments, n is an average of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

In some embodiments, the present invention relates to a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments of the invention, wherein:

Y represents -O-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-;

R ^a and R ^b are identical or different and each is independently selected from the group consisting of hydrogen, deuterium, halogen and alkyl;

_R ¹ is haloalkyl or C _3-6 cycloalkyl;

R ² is selected from _{the group consisting of hydrogen, haloalkyl and C 3-6 cycloalkyl} ;

or _R ¹ and R ² together with the carbon atoms to which they are attached form C _3-6 cycloalkyl;

m represents 0 or 1.

In some embodiments, in the ligand-drug conjugate of general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, Y is selected from the group consisting of

where the O-terminus of Y is attached to the linker unit L.

In some embodiments of the present invention, in a ligand-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any of the above embodiments of the invention, the linker unit -L- is -L ¹ -L ² -L ³ -L ⁴ -, wherein

L ¹ is selected from the group consisting of -(succinimidyl-3-yl-N)-WC(O)-, -CH ₂ -C(O)-NR ³ -WC(O)- and -C(O) _{-WC(O)-, wherein W is selected from the group consisting of C 1-8 alkyl} , C _1-8 alkylcycloalkyl and linear heteroalkyl having from 1 to 8 atoms, wherein said heteroalkyl contains from 1 to 3 heteroatoms selected from the group consisting of N, O and S, wherein _{independently} each _{of said C 1-8 alkyl} , cycloalkyl or linear heteroalkyl is optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

L ² is selected from the group consisting of -NR ⁴ (CH ₂ CH ₂ O) pCH ₂ CH ₂ C(O)-, -NR ⁴ (CH ₂ CH ₂ O) pCH ₂ C(O)-, -S(CH ₂ ) pC(O)- and a chemical bond, where p is an integer from 1 to 20;

L ³ is a peptide residue consisting of 2-7 amino acids, wherein the amino acids are selected from the group consisting of amino acid residues formed from the amino acids phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

L ⁴ is selected from the group consisting of -NR ⁵ (CR ⁶ R ⁷ ) _t -, -C(O)NR ⁵ , -C(O)NR ⁵ (CH ₂ ) _t - and a chemical bond, where t is an integer from 1 to 6;

R ³ , R ⁴ and R ⁵ are the same or different and are each independently selected from the group consisting of hydrogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

R ⁶ and R ⁷ are the same or different and are each independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl, and hydroxyalkyl.

In some embodiments, in a ligand-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, the linker unit -L- is -L ¹ -L ² -L ³ -L ⁴ -, wherein

L ¹ is selected from the group consisting of -(succinimidyl-3-yl-N)-WC(O)-, -CH ₂ -C(O)-NR ³ -WC ₍ O)- and -C(O) _{-WC(O)-, wherein W is selected from the group consisting of C 1-8 alkyl} , C _1-8 alkylcycloalkyl and linear heteroalkyl having from 1 to 8 chain atoms, wherein said heteroalkyl contains from 1 to 3 heteroatoms selected from the group consisting of N, O and S, wherein independently each of said C _1-8 alkyl, cycloalkyl or linear heteroalkyl is optionally further substituted _with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

L ² is selected from the group consisting of -NR ⁴ (CH ₂ CH ₂ O) pCH ₂ CH ₂ C(O)-, -NR ⁴ (CH ₂ CH ₂ O) pCH ₂ C(O)-, -S(CH ₂ ) pC(O)- and a chemical bond, where p is an integer from 1 to 20;

L ³ is a peptide residue consisting of 2-7 amino acid residues, wherein the amino acid residues are selected from the group consisting of amino acid residues formed from the amino acids phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

L ⁴ is selected from the group consisting of -NR ⁵ (CR ⁶ R ⁷ ) _t -, -C(O)NR ⁵ -, -C(O)NR ⁵ (CH ₂ ) _t - and a chemical bond, where t is an integer from 1 to 6, and non-limiting examples of t are 1, 2, 3, 4, 5 and 6;

R ³ , R ⁴ and R ⁵ are the same or different and are each independently selected from the group consisting of hydrogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

R ⁶ and R ⁷ are the same or different and are each independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl, and hydroxyalkyl.

In some embodiments, in a ligand-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, the linker unit -L- is -L ¹ -L ² -L ³ -L ⁴ -, wherein

L ¹ is s ¹ is an integer from 2 to 8, and non-limiting examples of s ¹ are 2, 3, 4, 5, 6, 7, and 8;

L ² is a chemical bond;

L ³ is a tetrapeptide residue, preferably a GGFG tetrapeptide residue;

L ⁴ is -NR ⁵ (CR ⁶ R ⁷ )t-, where R ⁵ , R ⁶ and R ⁷ are the same or different and each independently represents hydrogen or alkyl, and t is 1 or 2;

where the L ¹ end is attached to Pc and the L ⁴ end is attached to Y.

In some embodiments, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments of the invention, -L- is

.

In some embodiments, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, -L-Y- is selected from the group consisting of:

In some embodiments, a ligand-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any of the above-mentioned embodiments of the invention is a ligand-drug conjugate of the general formula (Pc-L _a -YD) or a pharmaceutically acceptable salt thereof,

,

Where:

W, L², L³, R⁵, R⁶ And R⁷are as defined in the above-mentioned linker unit -L-;

Pc, n, R ¹ , R ² and m are as defined in the general formula (Pc-LYD).

In some embodiments, a ligand-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any of the above embodiments of the invention is a ligand-drug conjugate of the general formula (Pc-L _b -YD) or a pharmaceutically acceptable salt thereof,

,

Where:

s ¹ is an integer between 2 and 8;

Pc, R ¹ , R ² , R ⁵ , R ⁶ , R ⁷ , m and n are as defined in the general formula (Pc-L _a -YD).

In some embodiments, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, the ligand-drug conjugate is selected from the group consisting of:

,

And

,

where Pc and n are as defined in the general formula (Pc-L-Y-D).

In some embodiments of the present invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any of the above embodiments, the ligand-drug conjugate is selected from the group consisting of:

, Where:

n is a decimal or integer number between 4 and 8;

Pc is an anti-TROP-2 antibody comprising the heavy chain shown in SEQ ID NO: 13 and the light chain shown in SEQ ID NO: 14.

In some embodiments of the invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof, as described above, the ligand-drug conjugate is:

,

Where:

n is a decimal or integer number from 1 to 8, preferably from 2 to 4 or from 4 to 8, and more preferably from 4 to 6;

Pc is an anti-TROP-2 antibody comprising the heavy chain shown in SEQ ID NO: 13 and the light chain shown in SEQ ID NO: 14;

or comprising a heavy chain as set forth in SEQ ID NO: 15 and a light chain as set forth in SEQ ID NO: 16;

or comprising a heavy chain as set forth in SEQ ID NO: 17 and a light chain as set forth in SEQ ID NO: 18.

In some embodiments of the invention, in a ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof, as described above, the ligand-drug conjugate is:

,

And

,

Where:

n is a decimal or integer number from 1 to 8, preferably from 2 to 4 or from 4 to 8, and more preferably from 4 to 6;

PD3 is an anti-TROP-2 antibody comprising the heavy chain shown in SEQ ID NO: 13 and the light chain shown in SEQ ID NO: 14;

hRS7 is an anti-TROP-2 antibody comprising the heavy chain shown in SEQ ID NO: 15 and the light chain shown in SEQ ID NO: 16;

TINA is an anti-TROP-2 antibody comprising the heavy chain shown in SEQ ID NO: 17 and the light chain shown in SEQ ID NO: 18.

The present invention also relates to an antibody to TROP-2 or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having sequences identical to the sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region presented in SEQ ID NO: 3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having sequences identical to the sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region presented in SEQ ID NO: 4.

In some embodiments of the invention, in an anti-TROP-2 antibody or antigen-binding fragment thereof as described above, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

In some embodiments of the invention, in an anti-TROP-2 antibody or antigen-binding fragment thereof according to any of the above embodiments of the invention, the anti-TROP-2 antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

In some embodiments, in an anti-TROP-2 antibody or antigen-binding fragment thereof according to any of the above embodiments, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region has an amino acid sequence as set forth in SEQ ID NO: 3 or has at least 90-100% identity thereto, including but not limited to at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%, and the light chain variable region has an amino acid sequence as set forth in SEQ ID NO: 4 or has at least 90-100% identity thereto, including but not limited to at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100%.

In some embodiments of the invention, in an anti-TROP-2 antibody or antigen-binding fragment thereof according to any of the above-mentioned embodiments of the invention, the anti-TROP-2 antibody or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region of an antibody; preferably, the heavy chain constant region is selected from the group consisting of constant regions of human IgG1, IgG2, IgG3 and IgG4 and normal variants thereof, and the light chain constant region is selected from the group consisting of constant regions of human κ and λ chain antibodies and normal variants thereof; and more preferably, the antibody comprises the heavy chain constant region presented in SEQ ID NO: 11 and the light chain constant region presented in SEQ ID NO: 12.

In some embodiments of the invention, in an anti-TROP-2 antibody or antigen-binding fragment thereof according to any of the above-mentioned embodiments of the invention, the anti-TROP-2 antibody comprises a heavy chain as presented in SEQ ID NO: 13 and a light chain as presented in SEQ ID NO: 14.

In another aspect, the present invention relates to a nucleic acid molecule encoding an antibody to TROP-2 as described above. In another aspect, the present invention relates to a nucleic acid molecule encoding an antibody to TROP-2 or an antigen-binding fragment thereof as described above.

In another aspect, the present invention relates to a host cell comprising a nucleic acid molecule as described above.

The present invention further provides a method for producing a ligand-drug conjugate of the general formula (Pc-L _a -YD) or a pharmaceutically acceptable salt thereof, comprising the following steps:

,

subjecting the reduced Pc and the compound of general formula (L _a -YD) to a coupling reaction to obtain a compound of general formula (Pc-L _a -YD);

Where:

Pc is an antibody to TROP-2 or its antigen-binding fragment;

n, m, W, L ² , L ³ , R ¹ , R ² , R ⁵ , R ⁶ and R ⁷ are as defined in the above general formula (Pc-L _a -YD).

The present invention further provides a method for producing an antibody-drug conjugate of the general formula (Pc-L'-D), wherein the method comprises the following step:

,

subjecting the reduced Pc and the compound of general formula (L'-D) to a coupling reaction to obtain a compound of general formula (Pc-L'-D); where:

Pc is an antibody to TROP-2 or an antigen-binding fragment thereof as described above;

n is as defined in the general formula (Pc-L-Y-D).

In another aspect, the present invention relates to a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any of the above-mentioned embodiments of the invention, or an anti-TROP-2 antibody or antigen-binding fragment thereof according to any of the above-mentioned embodiments of the invention, and one or more pharmaceutically acceptable excipients, diluents or carriers. In some embodiments, a single dose of the pharmaceutical composition comprises 0.1-3000 mg or 1-1000 mg of an anti-TROP-2 antibody as described above or an antibody-drug conjugate as described above.

In another aspect, the present invention relates to the use of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any of the above-mentioned embodiments of the invention, or an antibody to TROP-2 or an antigen-binding fragment thereof according to any of the above-mentioned embodiments of the invention, or a pharmaceutical composition containing the same, as a medicine.

In another aspect, the present invention relates to a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any of the above-mentioned embodiments of the invention, an antibody to TROP-2 or an antigen-binding fragment thereof according to any of the above-mentioned embodiments of the invention, or a pharmaceutical composition comprising the same, in the preparation of a medicament for the treatment of a disease or condition or a tumor mediated by TROP-2, wherein the disease or condition mediated by TROP-2 is a cancer with a high level of TROP-2 expression, a moderate level of TROP-2 expression, or a low level of TROP-2 expression.

In another aspect, the present invention relates to the use of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any of the above-mentioned embodiments of the invention, an anti-TROP-2 antibody or an antigen-binding fragment thereof according to any of the above-mentioned embodiments of the invention, or a pharmaceutical composition comprising the same, for the preparation of a medicament for the treatment and/or prevention of tumors and cancers, wherein the tumors and cancers are preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, neuroglioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, throat cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, bowel cancer, colon cancer, rectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Krukenberg tumor, myeloproliferative tumor, squamous cell carcinoma, Ewing sarcoma, urothelial carcinoma, or Merkel cell carcinoma; more preferably, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small cell leukocyte lymphoma, T-cell/histiocyte-rich large B-cell lymphoma and lymphoplasmacytic lymphoma, the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer, and the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myeloid cell leukemia.

In another aspect, the present invention also relates to a method of treating and/or preventing a tumor, comprising administering to a patient in need thereof a therapeutically effective dose of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any of the above-mentioned embodiments of the invention, an antibody to TROP-2 or an antigen-binding fragment thereof according to any of the above-mentioned embodiments of the invention, or a pharmaceutical composition comprising the same; wherein the tumor is a cancer with a high level of TROP-2 expression, a moderate level of TROP-2 expression, or a low level of TROP-2 expression.

In another aspect, the present invention also relates to a method for treating or preventing tumors or cancers, comprising administering to a subject in need thereof a therapeutically effective dose of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any of the above embodiments of the invention, or an anti-TROP-2 antibody or antigen-binding fragment thereof according to any of the above embodiments of the invention, or a pharmaceutical composition comprising said, wherein the tumors and cancers are preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, neuroglioma, glioblastoma multiforme, neuroblastoma, central nervous system carcinoma, neuroendocrine tumor, throat cancer, pharyngeal squamous cell carcinoma, oral squamous cell carcinoma, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, bowel cancer, colon cancer, rectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Krukenberg tumor, myeloproliferative tumor, squamous cell carcinoma, Ewing sarcoma, urothelial carcinoma, or Merkel cell carcinoma; more preferably, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small cell leukocyte lymphoma, T-cell/histiocyte-rich large B-cell lymphoma and lymphoplasmacytic lymphoma, the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer, and the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myeloid cell leukemia.

In another aspect, the present invention further relates to the above-mentioned anti-TROP-2 antibody or antibody-drug conjugate thereof for use as a medicine, preferably as a medicine for treating cancers or tumors, and more preferably as a medicine for treating TROP-2-mediated cancer.

The active compound (e.g., a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to the present invention) can be formulated in a form suitable for administration by any suitable route, preferably in a unit dose form or in a single dose form that can be self-administered by a subject. A unit dose of the active compound or composition described herein can be in the form of a tablet, capsule, wafer, vial, powder, granule, lozenge, suppository, powder for reconstitution, or liquid formulation.

The dosage of administration of the active compound or composition used in the method of treatment according to the present invention usually varies depending on the severity of the disease, the weight of the subject and the potency of the active compound. As a rule, a suitable unit dose can be from 0.1 to 1000 mg.

The pharmaceutical composition of the present invention may contain, in addition to the active compound, one or more excipients selected from the group consisting of: a filler, a diluent, a binder, a wetting agent, a disintegrant, an excipient, etc. Depending on the route of administration, the composition may contain from 0.1 to 99% by weight of the active compound.

The anti-TROP-2 antibody and the antibody-drug conjugate provided in the present invention have good affinity for cell surface antigens, good endocytosis efficiency and high tumor inhibition efficiency, as well as wider application windows of the drug, and are suitable for clinical use of the drug.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Figure 1 shows the results of experiments on binding of antibodies to TROP-2 to cells expressing TROP-2.

Figure 2 shows the results of secondary cell killing activity of ADC on BxPC3 cells and mixed MiaPaCa2 cells.

Figure 3 shows the inhibitory activity of various ADCs against FaDu xenograft tumor in mice.

Figure 4 shows the inhibitory activity of different doses of ADC against SKOV3 xenograft tumor in mice.

Figure 5 shows the inhibitory activity of different doses of ADC against Colo205 xenograft tumor in mice.

DETAILED DESCRIPTION OF THE INVENTION

1. Terminology

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used to practice or test the present invention, the preferred methods and materials are described herein. In the description and claims of the present invention, the following terms are used in accordance with the definitions given below.

When a trade name is used in this description, it is intended to include the composition of the commercial product under the said trade name and the medicinal product and the active component of the medicinal product of the commercial product under the said trade name.

Unless otherwise specified, the terms used in the description and claims have the meanings given below.

The term "drug" refers to a cytotoxic drug, which may have a chemical molecule in a tumor cell that is strong enough to disrupt its normal growth. A cytotoxic drug can kill cells themselves at a high enough concentration; however, due to a lack of specificity, a cytotoxic drug can cause apoptosis of normal cells while killing tumor cells, leading to serious side effects. The term "cytotoxic drug" includes toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g., At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and radioactive isotopes of Lu), chemotherapeutic drugs, antibiotics and nucleolytic enzymes.

The term "linker unit", "linker" or "linker moiety" refers to a chemical structural fragment or linkage that is linked to a ligand at one end and linked to a drug at the other end, and may also be linked to other linkers and then linked to a drug.

The linker may comprise one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), para-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("SMCC", also referred to herein as "MCC"), and N-succinimidyl (4-iodoacetyl)aminobenzoate ("SIAB"). The linker may be selected from the following, or a combination thereof: an extender, a spacer, and an amino acid unit. The linker may be synthesized using methods known in the art, such as those described in US2005-0238649A1. The linker may be a "cleavable linker" that facilitates the release of drugs in cells. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers may be used (Chari et al., Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020).

Linker elements include, but are not limited to:

MC - 6-maleimidocaproyl, with the structure:

,

Val-Cit or "vc" - valine-citrulline (an example of a dipeptide in a protease-cleavable linker),

citrulline - 2-amino-5-ureidopentanoic acid,

PAB - para-aminobenzyloxycarbonyl (an example of "self-cleaving" linker components),

Me-Val-Cit - N-methyl-valine-citrulline (where the linker peptide bond has been modified to prevent its cleavage by cathepsin B),

MC(PEG)6-OH - maleimidocaproyl polyethylene glycol (attached to antibody cysteine),

SPP - N-succinimidyl-4-(2-pyridylthio)valerate,

SPDP - N-succinimidyl 3-(2-pyridyldithio)propionate,

SMCC - succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate,

IT - iminothiolane.

The term "ligand drug conjugate" means that the ligand is linked to a biologically active drug via a linker unit, and the "ligand drug conjugate" is preferably an "antibody drug conjugate". As used herein, "antibody drug conjugate" (ADC) means that a monoclonal antibody or antibody fragment is linked to a biologically active toxic drug via a linker unit. The antibody can be conjugated to the drug directly or via a linker. n is the average number of drug units conjugated to each antibody, can be an integer or a decimal, and can range from, for example, about 0 to about 20 drug units; in some embodiments, from 1 to about 10 drug units; and in some embodiments, from 1 to about 8 drug modules, such as 2, 3, 4, 5, 6, 7, or 8 drug modules. In the antibody-drug conjugate mixture of the present invention, the average drug load of each antibody is from about 1 to about 10, including, but not limited to, from about 3 to about 7, from about 3 to about 6, from about 3 to about 5, from about 1 to about 9, about 7, or about 4.

The three-letter and one-letter codes for amino acids used in this specification are disclosed in J. biol. chem, 243, p3558 (1968).

The term "antibody" refers to an immunoglobulin, which has a tetrapeptide chain structure formed by linking two heavy chains and two light chains by interchain disulfide bonds. Based on differences in the amino acid composition and the order of the constant regions of the heavy chain, immunoglobulins can be divided into five classes, otherwise called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ chain, δ chain, γ chain, α chain, and ε chain, respectively. Ig of one class can be divided into different subclasses based on differences in the amino acid composition of the hinge regions and the number and position of the disulfide bonds of the heavy chains; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as κ or λ chains based on differences in the constant regions. Each of the five Ig classes can have either a κ chain or a λ chain.

In the heavy and light chains of full-length antibodies, the sequences of about 110 amino acids near the N-terminus vary considerably and are thus called variable regions (Fv regions); the remaining amino acid sequences near the C-terminus are relatively stable and are thus called constant regions. The variable region includes 3 hypervariable regions (HVRs) and 4 framework regions (FRs) with relatively conserved sequences. The three hypervariable regions determine the specificity of the antibody and are also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) or heavy chain variable region (HCVR) consists of 3 CDRs and 4 FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three light chain CDR regions are designated LCDR1, LCDR2, and LCDR3, and the three heavy chain CDR regions are designated HCDR1, HCDR2, and HCDR3.

The terms "fully humanized antibody", "fully human antibody", "human antibody" or "absolutely human antibody", also known as "humanized variable region and constant region, for fully humanized monoclonal antibody", have both humanized immunogenicity and toxic side effects. The main suitable technologies for producing fully human antibodies include: human hybridoma technology, EBV-transformed B-lymphocyte technology, phage display technology, transgenic mouse antibody technology, single B-cell antibody technology, and the like.

The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. A fragment of a full-length antibody can be used to perform the antigen-binding function of the antibody. The binding fragment included in the "antigen-binding fragment" is selected from the group consisting of Fab, Fab', F(ab') ₂ , single-chain antibody (scFv), dimerized V region (diabody), disulfide-stabilized V region (dsFv), and antigen-binding fragments of peptides containing CDRs; examples include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab') ₂ fragments, divalent fragments comprising two Fab fragments linked by disulfide bridges at the hinge regions; (iii) Fd fragments consisting of the VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of one arm of an antibody; (v) single domains or dAb fragments (Ward et al., (1989) Nature 341:544-546) consisting of VH domains; and (vi) isolated complementarity determining regions (CDRs) or (vii) combinations of two or more isolated CDRs, which may optionally be linked by synthetic linkers. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker by recombination, allowing it to produce a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single-chain antibodies are also intended to be included within the term "antigen-binding fragment" of an antibody. Such antibody fragments are prepared using conventional techniques known to those skilled in the art and are screened for suitability in the same manner as intact antibodies. Antigen-binding portions may be produced using recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be of various isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

In general, Fab is an antibody fragment having a molecular weight of about 50,000 and possessing antigen-binding activity, among fragments obtained by treating an IgG antibody molecule with papain protease (e.g., cleavage of the amino acid residue at position 224 of the H chain), in which a portion at the N-terminus of the H chain is linked to the L chain by a disulfide bond.

In general, F(ab') ₂ is an antibody fragment produced by cleavage of the portion below the disulfide bond in the hinge region of IgG by the enzyme pepsin. It has a molecular weight of about 100,000, has antigen-binding activity, and contains two Fab regions linked at the hinge position.

In general, Fab' is an antibody fragment having a molecular weight of about 50,000 and possessing antigen-binding activity, which is obtained by cleavage of the disulfide bond in the hinge region of F(ab') ₂ as described above.

In addition, Fab' can be produced by inserting DNA encoding a Fab' fragment into a prokaryotic or eukaryotic expression vector and introducing the vector into the prokaryotic or eukaryotic organism to express Fab'.

The term "single chain antibody", "single chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or VH) and an antibody light chain variable domain (or VL) linked by a linker. Such scFv molecules have the general structure: NH2 _- VL-linker-VH-COOH or _NH2 -VH-linker-VL-COOH. Suitable linkers in the prior art consist of repeating amino acid sequences GGGGS or variants thereof, for example, 1 to 4 repeating variants (Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al. (1995) Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

The term "CDR" refers to one of six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. In general, there are three CDRs (HCDR1, HCDR2, and HCDR3) in each variable region of the heavy chain and three CDRs (LCDR1, LCDR2, and LCDR3) in each variable region of the light chain. The amino acid sequence boundaries of the CDRs may be defined using any of a number of well-known schemes, including the "Kabat" numbering scheme (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5 ^th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chotia" numbering scheme (see Al-Lazikani et al. (1997) JMB 273: 927-948), and the ImMunoGenTics (IMGT) numbering scheme (Lefranc MP, Immunologist, 7, 132-136(1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003), etc.). For example, for the classical format, according to the Kabat scheme, the amino acid residues of the CDRs in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the amino acid residues of the CDRs in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). According to the Chothia scheme, the amino acid residues of the CDRs in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). According to the CDR definitions by combining the Kabat scheme and the Chothia scheme, the CDR consists of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. According to the IMGT scheme, the amino acid residues of the CDRs in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the amino acid residues of the CDRs in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). According to the IMGT scheme, the CDRs of an antibody can be determined using the IMGT/DomainGap Align program.

The term "framework region" refers to the portion of a VL or VH variable domain that serves as a framework for the CDRs of the variable domain. This is essentially a variable domain without the CDRs.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody binds. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, volume 66, G.E.Morris, Ed. (1996).

The terms "specific binding", "selective binding", "selective binding to" and "specific binding to" refer to the binding of an antibody to an epitope on a given antigen. Typically, the antibody binds with an affinity (KD) of less than about 10 ^-7 M, such as less than about 10 ^-8 , 10 ^-9 , or 10 ^-10 M or less.

The term "KD" refers to the equilibrium dissociation constant for an antibody-antigen interaction. In general, an antibody or antigen-binding fragment of the present invention binds to TROP-2 or an epitope thereof with an equilibrium dissociation constant (KD) of less than about ^10-7 M, such as less than about ^10-8 M or ^10-9 M; for example, the KD value is determined using a FACS method for the affinity of an antibody of the present invention for a cell surface antigen.

The term "nucleic acid molecule" refers to a DNA molecule or an RNA molecule. A nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

"Identity" of an amino acid sequence refers to the percentage of amino acid residues shared by a first sequence and a second sequence, wherein amino acid sequence alignments introduce gaps as necessary to achieve the maximum percent sequence identity, and any conservative substitution is not considered part of the sequence identity. For the purpose of determining the percent amino acid sequence identity, alignments can be achieved in a variety of ways that are within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine parameters suitable for measuring alignment, including any algorithms needed to achieve maximum alignment of the entire length of the aligned sequences.

The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded loop of DNA into which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. The vectors disclosed herein are capable of autonomously replicating in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors), or are capable of integrating into the host cell genome after introduction into the host cell and thereby replicating with the host genome (e.g., non-episomal mammalian vectors).

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, for example, as described in Chapters 5-8 and 15 of Antibodies: A Laboratory Manual, Cold Spring Harbor Press. The antibody or antigen-binding fragment described in the present invention is genetically engineered to contain one or more additional human FRs in a CDR of non-human origin. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website or from the immunoglobulin journal, Lefranc, G., The Immunoglobulin FactsBook, Academic Press, 2001ISBN012441351, by comparison with the IMGT human antibody germline variable region gene database using the MOE software.

The term "host cell" refers to the cell into which the expression vector has been introduced. Host cells may include microbial (e.g., bacterial), plant, or animal cells. Transformable bacteria include members of the Enterobacteriaceae family, such as Escherichia coli or Salmonella strains; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NS0 cells.

The engineered antibody or antigen-binding fragment of the present invention can be produced and purified by conventional techniques. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector. The recombinant immunoglobulin expression vectors can be stably transfected into host cells. As more recommended in the prior art, mammalian expression systems will result in glycosylation of the antibody, in particular at the N-terminal site of the Fc region. Positive clones are expanded in the medium in an antibody bioreactor. The secreted antibody culture can be purified using conventional techniques, for example, using an A or G Sepharose FF column. Non-specifically bound fractions are washed away. Bound antibody is eluted by a pH gradient method, and antibody fragments are detected by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and collected. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods such as molecular sieves and ion exchange. The resulting product can be immediately frozen, for example at -70°C, or lyophilized.

The term "peptide" refers to a molecule formed by joining two or more amino acid molecules by peptide bonds and is a structural and functional fragment of a protein.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a linear or branched group containing from 1 to 20 carbon atoms, preferably alkyl containing from 1 to 12 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) carbon atoms, more preferably alkyl containing from 1 to 10 carbon atoms, and most preferably alkyl containing from 1 to 6 carbon atoms (containing 1, 2, 3, 4, 5 or 6 carbon atoms). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl and various side chain isomers thereof, etc. More preferably, the lower alkyl has from 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl and the like. Alkyl may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available site on the compound, wherein the substituent preferably represents one or more of the following groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

The term "heteroalkyl" refers to an alkyl containing one or more heteroatoms selected from the group consisting of N, O and S, wherein alkyl is as defined above.

The term "alkylene" refers to a saturated linear or branched aliphatic hydrocarbon group having 2 residues derived from a parent alkane by the removal of two hydrogen atoms from the same or two different carbon atoms. It is a linear or branched group containing from 1 to 20 carbon atoms, preferably alkylene containing from 1 to 12 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) carbon atoms, more preferably alkylene containing from 1 to 6 carbon atoms (containing 1, 2, 3, 4, 5 or 6 carbon atoms). Non-limiting examples of alkylene include, but are not limited to, methylene( _-CH2- ), 1,1-ethylidene(-CH _{( CH3} )-), 1,2-ethylidene( _-CH2CH2 )-, 1,1-propylidene(-CH( _CH2CH3 )-), _1,2 -propylidene(-CH2CH _{( CH3} )-), 1,3-propylidene ₍ -CH2CH2CH2-) _{, 1,4} - _butylidene (-CH2CH2CH2CH2-), _1,5 -butylidene( _{-CH2CH2CH2CH2CH2-} ) _and the _{like .} The alkylene _may be substituted or _{unsubstituted .} When substituting, the substituent may be substituted at any available site on the compound with one or more substituents, preferably independently optionally selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

The term "alkoxy" refers to the group -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is as defined above. Non-limiting examples of alkoxy include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy and cyclohexyloxy. Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocycloalkylthio.

The term "haloalkyl" refers to an alkyl group in which the hydrogen is replaced by one or more halogens, wherein alkyl is as defined above.

The term "deuterated alkyl" refers to an alkyl group in which the hydrogen is replaced by one or more deuterium atoms, wherein alkyl is as defined above.

The term "hydroxyalkyl" refers to an alkyl group in which the hydrogen is replaced by one or more hydroxy groups, wherein alkyl is as defined above.

The term "hydroxy" refers to the -OH group.

The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

The term "amino" refers to -NH ₂ .

The term "nitro" refers to -NO ₂ .

The term "cyano" refers to -CN.

The present invention also comprises various deuterated forms of the compound of formula (Pc-L-Y-D). Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of the compound of general formula (Pc-L-Y-D) according to the relevant literature. Commercially available deuterated starting materials can be used to prepare deuterated forms of the compound of general formula (I), or they can be synthesized using conventional methods with deuterated reagents, including, but not limited to, deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

The term "optional" or "optionally" means that the event or circumstance subsequently described may, but need not, occur, and that the description includes instances where the event or circumstance does or does not occur. For example, "a heterocyclyl group optionally substituted with alkyl" means that alkyl may, but need not, be present, and that the description includes instances where the heterocyclyl group is or is not substituted with alkyl.

"Substituted" means that one or more, preferably up to 5 and more preferably 1, 2 or 3 hydrogen atoms in the group are independently replaced by a substituent. The substituent is only in its possible chemical position, and those skilled in the art will be able to determine (experimentally or theoretically) possible or impossible substitutions without much effort. For example, it may be unstable when an amino or hydroxy having a free hydrogen is bonded to a carbon atom having an unsaturated (e.g., olefinic) bond.

The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration into the body, which facilitates absorption of the active ingredient, thereby implementing biological activity.

The term "pharmaceutically acceptable salt" refers to a salt of the antibody drug conjugates of the present invention. Such salts are safe and effective when used in subjects and have the desired biological activity. The ligand drug conjugate of the present invention contains at least one amino group and thus can form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodate, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, mesylate, ethanesulfonate, benzenesulfonate and p-toluenesulfonate.

The term "average drug loading" or "drug loading" refers to the average amount of cytotoxic drug loaded per ligand in antibody-drug conjugate molecules, and may also be expressed in terms of the drug to antibody ratio. The drug loading may be from 0 to 12, preferably from 1 to 10 cytotoxic drugs per ligand (Pc). In embodiments of the present invention, the drug loading is represented in n, which can also be referred to as the DAR (drug-antibody ratio) value, and exemplary values can be average values of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The average amount of drug per ADC molecule after coupling reactions can be characterized by conventional methods such as UV (ultraviolet)/visible spectroscopy, mass spectrometry, ELISA (enzyme-linked immunosorbent assay) and HPLC (high performance liquid chromatography).

In one embodiment of the present invention, the cytotoxic drug is conjugated to the mercapto group of the antibody via a linker moiety.

The loading of the ligand-drug conjugate can be controlled by the following non-limiting methods, including:

(1) control of the molar ratio of linker reagent to monoclonal antibody,

(2) control of reaction time and temperature, and

(3) selection of different reagents.

For obtaining conventional pharmaceutical compositions, reference is made to the Chinese Pharmacopoeia.

The term "carrier" for a drug according to the present invention refers to a system that can change the way a drug enters a human body and the distribution of a drug in a human body, control the release rate of a drug, and deliver a drug to a target organ. The system of targeting and releasing a drug from a carrier can reduce the degradation and loss of a drug, reduce side effects, and improve bioavailability. For example, polymeric surfactants that can be used as carriers can self-assemble due to their unique amphiphilic structures to form various forms of aggregates, such as micelles, microemulsions, gels, liquid crystals, and vesicles, as preferred examples. The aggregates have the ability to encapsulate drug molecules and have good membrane permeability, and therefore can be used as excellent drug carriers.

The term "excipient" refers to an additive other than the main drug to a pharmaceutical composition. It may also be referred to as an adjuvant. For example, binders, fillers, disintegrants, lubricants in tablets; a main part in semi-solid ointment and cream preparations; preservatives, antioxidants, flavors, flavors, co-solvents, emulsifiers, solubilizers, tonicity adjusting agents, coloring agents and the like in liquid formulations may be referred to as excipients.

The term "diluent", also called filler, is primarily used to increase the weight and volume of a tablet. The addition of a diluent not only ensures a certain volume, but also reduces the dose deviation of the main ingredients, and improves the compressibility of the drug, etc. When a drug in the form of a tablet contains oily components, it is necessary to add an absorbent to absorb the oily components so as to maintain a "dry" state and thus facilitate the preparation of a tablet. Examples include starch, lactose, inorganic calcium salts, microcrystalline cellulose, etc.

The pharmaceutical composition may be in the form of a sterile aqueous injection solution. Available and acceptable carriers or solvents include water, Ringer's solution and isotonic sodium chloride solution. The sterile injection preparation may be a sterile oil-in-water microemulsion for injection in which the active ingredient is dissolved in the oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerol and processed to form a microemulsion. The injection or microemulsion can be administered locally into the bloodstream of a subject in large quantities. Alternatively, it may be advisable to administer solutions and microemulsions in a manner that maintains a constant circulating concentration of the compound of the present invention. To maintain such a constant concentration, a continuous intravenous delivery device can be used. An example of such a device is the Deltec CADD-PLUS intravenous injection pump. TM. 5400.

The pharmaceutical composition can be in the form of a sterile aqueous or oily injection suspension for intramuscular and subcutaneous administration. The suspension can be prepared according to the prior art using suitable dispersants or wetting agents and suspending agents mentioned above. The sterile injection composition can also be a sterile injection or suspension prepared in a parenterally acceptable non-toxic diluent or solvent, for example, a solution prepared in 1,3-butanediol. In addition, a sterile fixed oil can usually be used as a solvent or suspending medium. For this purpose, any mixture of fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of the injection.

2. Method of synthesis

For the purposes of synthesis, the following technical synthesis schemes are adopted.

The method for obtaining a compound of the general formula (Pc-L _a -YD) includes the following stages:

subjecting the reduced Pc to a coupling reaction with the general formula (L _a -YD) to obtain a compound of the general formula (Pc-L _a -YD), wherein the reducing agent is preferably TCEP (tris(2-carboxyethyl)phosphine); in particular, the disulfide bonds in the antibody are preferably reduced;

Where:

Pc, W, L ² , L ³ , R ¹ , R ² , R ⁵ , R ⁶ , R ⁷ , m and n are as defined in the above general formula (Pc-L _a -YD).

One or more embodiments of the present invention are described in detail in the description above. Although any methods and materials similar or identical to those described herein can be used to make or test the present invention, the preferred methods and materials are described below. Other features, purposes, and advantages of the invention will be apparent from the description and claims. In the description and claims, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. All patents and publications cited in the description are incorporated by reference. The following examples are provided to more fully illustrate the preferred embodiments of the present invention. These examples should not be construed in any way as limiting the scope of the present invention, which is defined by the claims.

Examples

Experimental procedures without specific conditions mentioned in the following examples and test examples are generally carried out under routine conditions or under conditions recommended by the manufacturers 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 indication of origin are commercially available routine reagents.

1. Obtaining antibodies

Example 1-1. Construction of a cell strain with high expression of TROP-2

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

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

The amino acid sequence of TROP-2 (Genbank: NP_002344.2) is as follows:

MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHR PTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVVVVVALVAGMAVLVITNRRKSGKYKKVEIKELGELRKEPSL

SEQ ID NO:1;

Trop2-His amino acid sequence:

MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIIDLRHRPTA GAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVVVVVALVAGMAVLVITNRRKSGKYKKVEIKELGELRKEPSLHHHHHH

SEQ ID NO: 2.

Example 1-2. Production of monoclonal antibody to human TROP-2

The monoclonal antibody against human TROP-2 in the present application was prepared according to the method disclosed in WO03074566, and the design of mutation site modification was performed on CDR using computer software and adopting the variable region gene of hRS7 antibody as a template. The variable region gene of the antibody was inserted into the expression vector of Phr-IgG protein (with a signal peptide and a constant region gene fragment (CH1-Fc/CL)) by molecular cloning, and then expressed in HEK293 and Expi-CHO-S cells. The purification of the antibody was performed according to a conventional method. Activity testing was performed using CHO-K1 cells and huTROP-2 protein (His27-Thr274 accession number NP_002344.2) overexpressing huTROP-2 protein, and the antibody with the best target binding activity was selected, where the sequence of the PD3 variable region is as follows:

PD3 heavy chain variable region:

EVQLVQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMG WINTYTGEPTYTQDFKG RFAFSLDTSVSTAYLQISSLKAEDTAVYYCAR GGFGSSYWYFDV WGQGTLVTVSS

SEQ ID NO:3;

PD3 light chain variable region:

DIQLTQSPSSLSSASVGDRVSITC KASQDVSIAVA WYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLT FGAGTKVEIK

SEQ ID NO:4;

Note: The underlined parts are CDRs defined according to the Kabat numbering scheme.

Table 1. CDR of antibodies to PD3 Antibodies PD3 Heavy chain CDR1 NYGMN (SEQ ID NO: 5) Heavy chain CDR2 WINTYTGEPTYTQDFKG (SEQ ID NO: 6) Heavy chain CDR3 GGFGSSYWYFDV (SEQ ID NO: 7) Light chain CDR1 KASQDVSIAVA (SEQ ID NO: 8) Light chain CDR2 SASYRYT (SEQ ID NO: 9) Light chain CDR3 QQHYITPLT (SEQ ID NO: 10)

The constant region of the heavy chain of the antibody may be selected from the constant regions of human IgG1, IgG2, IgG4 and variants thereof, and the constant region of the light chain of the antibody may be selected from the constant regions of the light chain of human κ and λ chains and variants thereof. As an example, the constant region of the heavy chain of the antibody is selected from the constant region of human IgG1 having the sequence presented in SEQ ID NO: 11, and the constant region of the light chain of the antibody is selected from the constant region of the human κ chain having the sequence presented in SEQ ID NO: 12.

Human IgG1 heavy chain constant region:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 11;

Humanized κ light chain constant region:

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 12;

As an example, the light/heavy chain constant regions described above are combined with the variable regions of the above-mentioned PD3 antibody to form a complete antibody whose light/heavy chain sequences are as follows:

Heavy Chain PD3:

EVQLVQSGSELKKPGASVKVSCKASGYTFT NYGMN WVKQAPGQGLKWMG WINTYTGEPTYTQDFKG RFAFSLDTSVSTAYLQISSLKAEDTAVYYCAR GGFGSSYWYFDV WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 13;

Light chain PD3:

DIQLTQSPSSLSSASVGDRVSITC KASQDVSIAVA WYQQKPGKAPKLLIY SASYRYT GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC QQHYITPLT FGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:14;

The hRS7 control molecule used in the present invention is designed with reference to patent WO03074566, and the anti-TINA antibody is designed with reference to patent WO2015098099a1, the sequences of which are as follows:

Heavy duty chain hRS7:

QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWG QGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:15;

hRS7 Light Chain:

DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:16;

TINA Heavy Chain:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:17;

TINA Light Chain:

DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 18.

2. Obtaining connections

The experimental procedures without conditions indicated in the examples of the present invention were generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer of the starting materials or commercial products. Reagents without indication of a specific origin are commercially available common reagents.

The structure of the compounds was identified using nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR spectra were measured using a Bruker AVANCE-400 NMR instrument, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform ( _CDCl3 ) and deuterated methanol ( _CD3OD ) as solvents and tetramethylsilane (TMS) as an internal standard. Chemical shifts are reported in units of 10 ^-6 (ppm).

MS analysis was performed using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

UPLC (ultra-performance liquid chromatography) analysis was performed using a Waters Acquity UPLC SQD liquid chromatography-mass spectrometry system.

UPLC analysis was performed using an Agilent 1200DAD high-pressure liquid chromatograph (Sunfire C18 150×4.6 mm chromatography column) and a Waters 2695-2996 high-pressure liquid chromatograph (Gimini C18 150×4.6 mm chromatography column).

UV-HPLC (high performance liquid chromatography using a UV (ultraviolet) detector) analysis was performed using a Thermo nanodrop2000 ultraviolet spectrophotometer.

The degree of proliferation inhibition and IC ₅₀ (half-maximal inhibitory concentration) values were measured using a PHERA starFS microplate reader (BMG, Germany).

Huanghai HSGF254 or Qingdao GF254 silica gel plates with specifications from 0.15 mm to 0.2 mm were used for thin layer chromatography (TLC) and 0.4 mm to 0.5 mm for TLC separation and purification.

Yantai Yellow Sea 200-300 mesh silica gel was commonly used as a carrier in column chromatography.

The known starting materials of the present invention can be synthesized using or according to methods known in the art, or can be purchased from ABCR GmbH & Co.KG, Acros Organnics, Aldrich Chemical Company, Accela ChemBio Inc, Chembee Chemicals, etc.

In the examples, all reactions were carried out under argon or nitrogen atmosphere unless otherwise stated.

Argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a cylinder containing about 1 liter of argon or nitrogen.

Hydrogen atmosphere means that the reaction flask is connected to a cylinder containing about 1 liter of hydrogen.

Parr 3916EKX hydrogenator, Qinglan QL-500 hydrogenator or HC2-SS hydrogenator were used in pressure hydrogenation reactions.

The hydrogenation reaction typically involves 3 cycles of vacuum and hydrogen purge.

A CEM Discover-S 908860 microwave reactor was used for the microwave reaction.

In the examples, the solution in the reaction refers to an aqueous solution unless otherwise stated.

In the examples, the reaction temperature is room temperature unless otherwise stated.

Room temperature is the optimum reaction temperature, which ranges from 20 to 30°C.

Preparation of PBS (phosphate buffered saline) buffer at pH 6.5 in the examples: 8.5 g _{KH2PO4 ,} 8.56 g _{K2HPO4 3H2O} , 5.85 g NaCl and 1.5 g EDTA (ethylenediaminetetraacetic acid) were added to the flask and the volume was adjusted to 2 L. All additions were dissolved _by ultrasound and the solution was mixed well by shaking to obtain the required buffer.

The elution system for column chromatography and the developing solvent system for thin layer chromatography used for the purification of the compound included: A: dichloromethanol and isopropanol system, B: dichloromethanol and methanol system, and C: petroleum ether and ethyl acetate system. The volume ratio of the solvents was adjusted according to the polarity of the compound, or a small amount of triethylamine and an acidic or basic reagent was added.

Some of the compounds of the present invention were characterized by Q-TOF LC/MS (liquid chromatography with mass spectrometry) analysis. For Q-TOF LC/MS analysis, an Agilent 6530 accurate mass quadrupole time-of-flight mass spectrometer and an Agilent 1290-Infinity ultra-high performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 μm, 2.1×75 mm chromatography column) were used.

The drug portion of the Y-D antibody-drug conjugates of the present invention is disclosed in PCT/CN2019/107873, and the synthesis and tests of the corresponding compounds are incorporated herein by reference. Non-limiting examples of the synthesis are incorporated by reference as follows:

Example 2-1

N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropane-1-carboxamide 1

To exatecan mesylate 1b (2.0 mg, 3.76 μmol, prepared as described in patent application "EP0737686A1") was added 1 ml of N,N-dimethylformamide. The mixture was cooled to 0-5 °C in an ice water bath and triethylamine was added dropwise. The reaction mixture was stirred until it became clear. To the reaction mixture were successively added 1-hydroxycyclopropylcarboxylic acid 1a (1.4 mg, 3.7 μmol, prepared by the known method "Tetrahedron Letters, 25(12), 1269-72; 1984") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (3.8 mg, 13.7 μmol). After addition, the reaction mixture was stirred at 0-5°C for 2 h, quenched with 5 mL of water and extracted with ethyl acetate (8 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 mL × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography using developing solvent system B to give the title product 1 (1.6 mg, yield 82.1%).

MS m/z (ESI): 520.2 [M+1].

^1H NMR (400 MHz, CDCl ₃ ): δ 7.90-7.84 (m, 1H), 7.80-7.68 (m, 1H), 5.80-5.70 (m, 1H) , 5.62-5.54 (m, 2H), 5.44-5.32 (m, 2H), 5.28-5.10 (m, 2H), 3.40-3.15 (m, 3H), 2.44 (s, 3H), 2.23(t, 1H), 2.06-1, 75 (m, 2H), 1.68-1.56 (m, 1H), 1.22-1.18 (m, 2H), 1.04-0.98 (m, 2H), 0.89 (t, 3H).

Example 2-2

(S)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15 -G exahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide 2-A

(R)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15 -G exahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide 2-B

To 1b (4 mg, 7.53 μmol) were added 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide. The system was purged with argon three times and the mixture was cooled to 0-5 °C in an ice water bath, followed by dropwise addition of 0.3 mL of N-methylmorpholine. The reaction mixture was stirred until it became clear. To the reaction mixture were successively added 2-cyclopropyl-2-hydroxyacetic acid 2a (2.3 mg, 19.8 μmol, prepared as described in patent application "WO2013106717"), 1-hydroxybenzotriazole (3 mg, 22.4 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol). After the addition, the reaction mixture was stirred at 0-5 °C for 1 h. The ice water bath was removed, and the reaction mixture was warmed to 30 °C, stirred for 2 h, and concentrated under reduced pressure. The obtained crude compound 2 was purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19 × 250 mm; mobile phase: A - water (10 mmol NH ₄ OAc), B - acetonitrile, gradient elution, flow rate: 18 mL/min), and the appropriate fractions were collected and concentrated under reduced pressure to give the title product (2-A: 1.5 mg, 2-B: 1.5 mg).

MS m/z (ESI (electrospray ionization): 534.0 [M+1].

Compound with single 2-B configuration (lower retention time)

UPLC analysis: retention time: 1.06 min; purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm; mobile phase: A - water (5 mmol NH ₄ OAc), B - acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.37 (d, 1H), 7.76 (d, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.58-5.56 (m, 1H), 5.48 (d, 1H), 5.41 (s, 2H), 5.32-5.29 (m, 2H), 3.60 (t, 1H), 3.19-3.13 (m, 1H), 2.38 (s, 3H), 2.20-2, 14 (m, 1H), 1.98 (q, 2H), 1.87-1.83 (m, 1H), 1.50-1.40 (m, 1H), 1.34-1.28 (m, 1H), 0.86 (t, 3H), 0.50-0.39 (m, 4H).

Compound with single 2-A configuration (longer retention time)

UPLC analysis: retention time: 1.10 min; purity: 86% (chromatographic column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm; mobile phase: A - water (5 mmol NH ₄ OAc), B - acetonitrile).

^1H NMR(400 MHz, DMSO- _d6 ): δ 8.35 (d, 1H), 7.78 (d, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.58-5.53 (m, 1H), 5.42 (s, 2H), 5.37 (d, 1H), 5.32 (t, 1H), 3.62 (t, 1H), 3.20-3.15 (m, 2H), 2.40 (s, 3H), 2.25-2.16 (m, 1H), 1.98 (q, 2H), 1.87-1.82 (m, 1H), 1.50-1.40 (m, 1H), 1.21-1.14 (m, 1H), 0.87 (t, 3H), 0.47-0.35 (m, 4H).

Example 2-3

(S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H ,12H -benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropionamide 3-A

(R)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H ,12H -benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropionamide 3-B

To 1b (5.0 mg, 9.41 μmol) were added 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide, and the mixture was cooled to 0-5 °C in an ice water bath, followed by dropwise addition of 0.3 mL of N-methylmorpholine. The reaction mixture was stirred until it became clear. To the reaction mixture were added sequentially 3,3,3-trifluoro-2-hydroxypropionic acid 3a (4.1 mg, 28.4 μmol, purchased from Alfa), 1-hydroxybenzotriazole (3.8 mg, 28.1 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.4 mg, 28.2 μmol). After addition, the reaction mixture was stirred at 0-5 °C for 10 min. The ice water bath was removed and the reaction mixture was heated to 30 °C, stirred for 8 h and concentrated under reduced pressure. The obtained crude compound 3 was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5 μm 19 × 250 mm; mobile phase: A - water (10 mmol NH ₄ OAc), B - acetonitrile, gradient elution, flow rate: 18 mL/min), and the appropriate fractions were collected and concentrated under reduced pressure to give the title product (1.5 mg, 1.5 mg).

MS m/z (ESI): 561.9 [M+1].

Single configuration connection (lower retention time)

UPLC analysis: retention time: 1.11 min; purity: 88% (chromatographic column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm; mobile phase: A - water (5 mmol NH ₄ OAc), B - acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.94 (d, 1H), 7.80 (d, 1H), 7.32 (s, 1H), 7.20 (d, 1H), 6.53 (s, 1H), 5.61-5.55 (m, 1H), 5.45-5.23 (m, 3H), 5.15-5.06 (m, 1H), 4.66-4.57 (m, 1H), 3.18-3.12 (m, 1H), 2.40 (s, 3H) , 2.26-2.20 (m, 1H), 2.16-2.08 (m, 1H), 2.02-1.94 (m, 1H), 1.89-1.82 (m, 1H), 1.50-1.40 (m, 1H), 0.87 (t, 3H).

Single configuration connection (longer retention time)

UPLC analysis: retention time: 1.19 min; purity: 90% (chromatographic column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm; mobile phase: A - water (5 mmol NH ₄ OAc), B - acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.97 (d, 1H), 7.80 (d, 1H), 7.31 (s, 1H), 7.16 (d, 1H), 6.53 (s, 1H), 5.63-5.55 (m, 1H), 5.45-5.20 (m, 3H), 5.16-5.07 (m, 1H), 4.66-4.57 (m, 1H), 3.18-3.12 (m, 1H), 2.40 (s, 3H) , 2.22-2.14 (m, 1H), 2.04-1.95 (m, 2H), 1.89-1.82 (m, 1H), 1.50-1.40 (m, 1H), 0.87 (t, 3H).

Example 2-4

N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopentane-1-carboxamide 4

To 1b (3.0 mg, 5.64 μmol) was added 1 mL of N,N-dimethylformamide. The mixture was cooled to 0-5 °C in an ice water bath and triethylamine was added dropwise. The reaction mixture was stirred until it became clear. To the reaction mixture were added 1-hydroxy-cyclopentanecarboxylic acid 4a (2.2 mg, 16.9 μmol, prepared as described in patent application "WO2013106717") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) sequentially. After the addition, the reaction mixture was stirred at 0-5 °C for 1 h, quenched with 5 mL of water and extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 ml × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography using developing solvent system B to give the title product 4 (2.5 mg, yield 80.9%).

MS m/z (ESI): 548.0 [M+1].

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.73-7.62 (m, 2H), 5.75-5.62 (m, 1H), 5.46-5.32 (m, 2H), 5.26-5.10 (m, 1H), 3.30-3.10 (m, 1H), 2.43 (s, 3H), 2.28-2.20 (m, 2H), 2.08-1.84 (m, 8H), 1.69-1.58 (m, 2H), 1.04-1.00 (m, 2H), 0.89 (t, 3H).

Example 2-5

N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclopropane-1-carboxamide 5

To 1b (2.0 mg, 3.76 μmol) was added 1 mL of N,N-dimethylformamide. The mixture was cooled to 0-5 °C in an ice water bath and triethylamine was added dropwise. The reaction mixture was stirred until it became clear. To the reaction mixture were added 1-(hydroxymethyl)-cyclopentanecarboxylic acid 5a (0.87 mg, 7.5 μmol, prepared as described in patent application "WO201396771") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2 mg, 7.24 μmol) sequentially. After the addition, the reaction mixture was stirred at 0-5 °C for 2 h, quenched with 5 mL of water and extracted with ethyl acetate (8 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 ml × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography using developing solvent system B to give the title product 5 (1.0 mg, yield 50%).

MS m/z (ESI): 533.9 [M+1].

^1H NMR (400 MHz, CDCl ₃ ): δ 8.07 (s, 1H), 7.23-7.18 (m, 2H), 6.71-6.64 (m, 1H), 6.55 -6.51 (m, 1H), 5.36-5.27 (m, 2H), 4.67-4.61 (m, 2H), 3.53-3.48 (m, 1H), 3.30-3.22 (m, 2H), 3.18-3.13 (m, 1H), 2.71-2.61 ( m, 2H), 2.35-2.28 (m, 1H), 2.04-1.91 (m, 4H), 1.53-1.40 (m, 3H), 0.91-0.75 (m, 4H).

Example 2-6

N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benz o[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclobutane-1-carboxamide 6

To 1b (3.0 mg, 5.64 μmol) was added 1 mL of N,N-dimethylformamide. The mixture was cooled to 0-5 °C in an ice water bath and triethylamine was added dropwise. The reaction mixture was stirred until it became clear. To the reaction mixture were added sequentially 1-(hydroxymethyl)cyclobutane-1-carboxylic acid 6a (2.2 mg, 16.9 μmol, prepared as described in "Journal of the American Chemical Society, 2014, vol. 136, #22, p.8138-8142") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol). After addition, the reaction mixture was stirred at 0-5°C for 1 h, quenched with 5 mL of water and extracted with ethyl acetate (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride solution (5 mL × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography using developing solvent system B to give the title product 6 (2.1 mg, yield 67.9%).

MS m/z (ESI): 548.0 [M+1].

^1H NMR (400 MHz, DMSO- _d6 ): δ 7.85-7.62 (m, 1H), 6.88 (br, 1H), 5.87-5.48 (m, 2H), 5 .47-5.33 (m, 1H), 5.31-5.06 (m, 1H), 4.25-3.91 (m, 2H), 3.25 (br, 1H), 2.60-2.32 (m, 3H), 2.23 (t, 1H), 2.15-1.95 (m, 3H), 1.70-1.56 (m, 2H), 1.41-1.17 (m, 9H), 1.03 (s, 1H), 0.95-0.80 (m, 2H).

Example 2-7

N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopentane-1-carboxamide 7

To 1b (3.0 mg, 5.64 μmol) were added 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide, and the mixture was cooled to 0-5 °C in an ice water bath, followed by dropwise addition of 0.3 mL of N-methylmorpholine. The reaction mixture was stirred until it became clear. To the reaction mixture were added sequentially 1-hydroxycyclobutanecarboxylic acid 7a (2.0 mg, 17.22 μmol, purchased from PharmaBlock), 1-hydroxybenzotriazole (2.3 mg, 17.0 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.2 mg, 16.7 μmol). After addition, the reaction mixture was stirred at 0-5 °C for 10 min. The ice water bath was removed and the reaction mixture was stirred at room temperature for 2 hours and concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developing solvent system B to give the title product 7 (2.5 mg, yield 83.1%).

MS m/z (ESI (electrospray ionization): 534.0 [M+1].

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.28 (d, 1H), 7.75 (d, 1H), 7.29 (s, 1H), 6.51 (s, 1H), 6.12 (s, 1H), 5.59-5.51 (m, 1H), 5.41 (s, 2H), 5.20-5.01 (m, 2H), 3.27-3.17 (m, 1H), 3.15-3.05 (m, 1H), 2.71-2.63 (m, 1H ), 2.37 (s, 3H), 2.12-2.05 (m, 1H), 2.03-1.94 (m, 2H), 1.92-1.78 (m, 4H), 1.50-1.42 (m, 1H), 0.90-0.83 (m, 4H).

Example 2-8

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazeicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 8

Stage 1

Benzyl 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropane-1-carboxylate 8c

Benzyl 1-hydroxycyclopropane-1-carboxylate 8a (104 mg, 0.54 mmol; prepared as described in patent application "US2005/20645") and 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate 8b (100 mg, 0.27 mmol; prepared as described in patent application "CN105829346A") were added to the reaction flask and 5 mL of tetrahydrofuran was added. The system was purged with argon three times and the mixture was cooled to 0-5 °C in an ice water bath, followed by the addition of potassium tert-butoxide (61 mg, 0.54 mmol). The ice bath was removed and the reaction mixture was warmed to room temperature and stirred for 10 min, followed by the addition of 20 ml of ice water and extraction with ethyl acetate (5 ml × 2) and chloroform (5 ml × 5). The organic phases were combined and concentrated. The resulting residue was dissolved in 3 ml of 1,4-dioxane, followed by the addition of 0.6 ml of water, sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorenylmethyl chloroformate (70 mg, 0.27 mmol). The mixture was stirred at room temperature for 1 h. 20 ml of water was added, followed by extraction with ethyl acetate (8 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, and the resulting residue was purified by silica gel column chromatography using developing solvent system B to give the title product 8c (100 mg, yield 73.6%).

MS m/z (ESI): 501.0 [M+1].

Stage 2

1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropane-1-carboxylic acid 8d

8c (50 mg, 0.10 mmol) was dissolved in 3 ml of a solvent mixture of tetrahydrofuran and ethyl acetate (V:V=2:1) and palladium on carbon (25 mg, 10% loading) 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 washed with tetrahydrofuran. The filtrate was concentrated to give the title product 8d (41 mg, 100% yield).

MS m/z (ESI): 411.0 [M+1].

Stage 3

(9H-fluoren-9-yl)methyl(2-(((1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3 ,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano [3',4':6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropoxy)methyl)amino)-2-oxoethyl)carbamate 8e

1b (7 mg, 0.013 mmol) was added to the reaction flask and 1 ml of N,N-dimethylformamide was added. The system was purged with argon three times and the mixture was cooled to 0-5 °C in an ice-water bath, followed by the addition of a drop of triethylamine, a solution of 8d (7 mg, 0.017 mmol) in 0.5 ml of N,N-dimethylformamide and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (7 mg, 0.026 mmol). The reaction mixture was stirred in an ice bath for 35 min. 10 ml of water was added, followed by extraction with ethyl acetate (5 ml × 3). The organic phases were combined, washed with saturated sodium chloride solution (10 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was purified by thin layer chromatography using developing solvent system B to give the title product 8e (8.5 mg, yield 78.0%).

MS m/z (ESI): 828.0 [M+1].

Stage 4

1-((2-aminoacetylamino)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10, 13, 15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropan-1-carboxamide 8f

8e (4 mg, 4.84 μmol) was dissolved in 0.2 ml of dichloromethane and 0.1 ml of diethylamine was added. The reaction mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. 2 ml of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and the upper n-hexane layer was removed; the procedures were repeated three times. The suspension was concentrated under reduced pressure to give the crude title product 8f (2.9 mg), which was used directly in the next step without purification.

MS m/z (ESI): 606.0 [M+1].

Stage 5

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2,5,8,11,14-pentaazeicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 8

Crude 8f (2.9 mg, 4.84 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide. The system was purged with argon three times and the solution was cooled to 0-5 °C in an ice water bath. A solution of (S)-2(-2-(-2-(6-(2,5-dioxo-1H-pyrrol-1-yl)hexanamido)acetylamino)acetylamino)-3-phenylpropionic acid 8 g (2.7 mg, 5.80 μmol, prepared as described in patent application "EP2907824") in 0.3 mL of N,N-dimethylformamide was added, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol). The reaction mixture was stirred in an ice bath for 30 min. Then the ice bath was removed and the reaction mixture was warmed to room temperature, stirred for 15 min and purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5 μm 19 × 250 mm; mobile phase: A - water (10 mmol NH ₄ OAc), B - acetonitrile, gradient elution, flow rate: 18 mL/min). The appropriate fractions were collected and concentrated under reduced pressure to give the title product 8 (2 mg, yield 39.0%).

MS m/z (ESI): 1060.0 [M+1].

^1H NMR (400 MHz, DMSO- _d6 ): δ 9.01 (d, 1H), 8.77 (t, 1H), 8.21 (t, 1H), 8.08-7.92 (m , 2H), 7.73 (d, 1H), 7.28 (s, 1H), 7.24-7.07 (m, 4H), 6.98 (s, 1H), 6.50 (s, 1H), 5.61 (q, 1H), 5.40 (s, 2H), 5.32 (t, 1H), 5. 12 (q, 2H), 4.62 (t, 1H), 4.52 (t, 1H), 4.40-4.32 (m, 1H), 3.73-3.47(m, 8H), 3.16-3.04 (m, 2H), 2.89 (dd, 1H), 2.69-2.55 (m, 2H), 2.37-2.23 (m, 4H) , 2.12-1.93 (m, 4H), 1.90-1.74 (m, 2H), 1.52-1.38 (m, 4H), 1.33-1.11 (m, 5H), 0.91-0.81 (m, 4H).

Example 2-9

N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2 ,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A

N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2 ,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B

Stage 1

Benzyl 2-cyclopropyl-2-hydroxyacetate 9a

2a (1.3 g, 11.2 mmol; prepared as described in patent application "WO2013/106717") was dissolved in 50 mL of acetonitrile and potassium carbonate (6.18 g, 44.8 mmol), benzyl bromide (1.33 mL, 11.2 mmol) and tetrabutylammonium iodide (413 mg, 1.1 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 48 h and filtered through celite, and the filter cake was washed with ethyl acetate (10 mL). The filtrates were combined and concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with developing solvent system C to give the title product 9a (2 g, 86.9% yield).

Stage 2

Benzyl 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oate 9b

9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol) were added to the reaction flask and 4 mL of tetrahydrofuran was added. The system was purged with argon three times and the reaction mixture was cooled to 0-5 °C in an ice water bath followed by the addition of potassium tert-butoxide (109 mg, 0.98 mmol). The ice bath was removed and the reaction mixture was warmed to room temperature and stirred for 40 min followed by the addition of 10 mL of ice water and extraction with ethyl acetate (20 mL×2) and chloroform (10 mL×5). The organic phases were combined and concentrated. The obtained 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 thereto. 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 obtained residue was purified by silica gel column chromatography using a C developing solvent system to give the title product 9b (48 mg, yield 19%).

MS m/z (ESI): 515.0 [M+1].

Stage 3

10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid 9c

9b (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 substance) 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 washed with ethyl acetate. The filtrate was concentrated to give the crude title product 9c (13 mg), which was directly used in the next step without purification.

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

Stage 4

(9H-fluoren-9-yl)methyl(2-(((1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13- dioxo-2,3,9,10,13,15-hexa hydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2- oxoethyl)carbamate 9d

1b (10 mg, 18.8 mmol) was added to the reaction flask and 1 ml of N,N-dimethylformamide was added. The system was purged with argon three times and the mixture was cooled to 0-5 °C in an ice water bath, followed by the addition of a drop of crude triethylamine 9c (13 mg, 30.6 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (16.9 mg, 61.2 μmol). The reaction mixture was stirred in an ice bath for 40 min. 10 ml of water was added, followed by extraction with ethyl acetate (10 ml×3). The organic phases were combined, washed with saturated sodium chloride solution (10 ml×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developing solvent system B to give the title product 9d (19 mg, yield 73.6%).

MS m/z (ESI): 842.1 [M+1].

Stage 5

2-((2-aminoacetamido)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-diox o-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl) acetamide 9e

9d (19 mg, 22.6 μmol) was dissolved in 2 ml of dichloromethane and 1 ml of diethylamine was added. The reaction mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. 1 ml of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was suspended with 3 ml of n-hexane and left to stand. Then, the supernatant was removed and a solid was left. The solid residue was concentrated under reduced pressure and dried with an oil pump to give the crude title product 9e (17 mg), which was directly used in the next step without purification.

MS m/z (ESI): 638.0 [M+18].

Stage 6

N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2 ,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A

N-((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2 ,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7] indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B

Crude 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide. The system was purged with argon three times and the solution was cooled to 0-5 °C in an ice water bath. A solution of 8 g (21.2 mg, 44.8 μmol) in 0.3 mL of N,N-dimethylformamide was added followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (18.5 mg, 67.3 μmol). The reaction mixture was stirred in an ice bath for 10 min. Then the ice bath was removed and the reaction mixture was warmed to room temperature and stirred for 1 h to give compound 9. The reaction mixture was purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19×250 mm; mobile phase: A - water (10 mmol NH ₄ OAc), B - acetonitrile, gradient elution, flow rate: 18 mL/min). The appropriate fractions were collected and concentrated under reduced pressure to give the title products (9-A: 2.4 mg, 9-B: 1.7 mg).

MS m/z (ESI): 1074.4 [M+1].

Compound with single configuration 9-A (lower retention time):

UPLC analysis: retention time: 1.14 min; purity: 85% (chromatographic column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm; mobile phase: A - water (5 mmol NH ₄ OAc), B - acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.60 (t, 1H), 8.51-8.49 (d, 1H), 8.32-8.24 (m, 1H), 8 ,13-8.02 (m, 2H), 8.02-7.96 (m, 1H), 7.82-7.75 (m, 1H), 7.31 (s, 1H), 7.26-7.15 (m, 4H), 6.99 (s, 1H), 6.55-6.48 (m, 1H), 5.65-5.54 (m, 1H), 5.41 (s, 2H), 5.35-5.15 (m, 3H), 4.74-4.62 (m, 1H), 4.54-4.40 (m, 2H), 3.76-3.64 (m, 4H), 3.62-3.48 (m, 2H), 3.20-3.07 (m, 2H ), 3.04-2.94 (m, 1H), 2.80-2.62 (m, 1H), 2.45-2.30 (m, 3H), 2.25-2.15 (m, 2H), 2.04-2.15 (m, 2H), 1.93-1.78 (m, 2H), 1.52-1.39 ( m, 3H), 1.34-1.12 (m, 5H), 0.87 (t, 3H), 0.64-0.38 (m, 4H).

Compound with single configuration 9-B (longer retention time):

UPLC analysis: retention time: 1.16 min; purity: 89% (chromatographic column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm; mobile phase: A - water (5 mmol NH ₄ OAc), B - acetonitrile).

^1H NMR (400 MHz, DMSO- _d6 ): δ 8.68-8.60 (m, 1H), 8.58-8.50 (m, 1H), 8.32-8.24 (m, 1H), 8.13-8.02 (m, 2H), 8.02-7.94 (m, 1H), 7.82-7.75 (m, 1H), 7.31 (s, 1H), 7.26-7.13 (m, 3H), 6.99 (s, 1H), 6.55-6, 48 (m, 1H), 5.60-5.50 (m, 1H), 5.41 (s, 2H), 5.35-5.15 (m, 2H), 4.78-4.68 (m, 1H), 4.60-4.40 (m, 2H), 3.76-3.58 (m, 4H), 3.58-3.48 (m, 1H), 3.20-3.10 ( m, 2H), 3.08-2.97 (m, 2H), 2.80-2.72 (m, 2H), 2.45-2.30 (m, 3H), 2.25-2.13 (m, 2H), 2.13-2.04 (m, 2H), 2.03-1.94 (m, 2H), 1.91-1 .78 (m, 2H), 1.52-1.39 (m, 3H), 1.34-1.12 (m, 4H), 0.91-0.79 (m, 3H), 0.53-0.34 (m, 4H).

3. Preparing ADC

Example 3-1 ADC-1

To the aqueous PBS buffer of PD3 antibody (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/ml, 4.0 ml, 270 nmol) prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 67.5 μl, 675 nmol) was added at 37°C. The reaction mixture was shaken on a shaking water bath at 37°C for 3 h before stopping the reaction. The reaction mixture was cooled to 25°C on a water bath.

Compound 9-A (2.9 mg, 2700 nmol) was dissolved in 180 μL of DMSO (dimethyl sulfoxide), and the resulting solution was added to the above reaction mixture, which was then shaken in a shaking water bath at 25 °C for 3 h before stopping the reaction. 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 obtain the type product ADC-1 PD3-9-A in PBS buffer (1.93 mg/mL, 18.4 mL), which was then stored at 4 °C.

Average value calculated using the UV-Vis method: n=3.77.

Example 3-2 ADC-2

To the aqueous PBS buffer of PD3 antibody (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 4.0 mL, 270 nmol) prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 143.1 μL, 1431 nmol) was added at 37°C. The reaction mixture was shaken on a shaking water bath at 37°C for 3 h before stopping the reaction. The reaction mixture was cooled to 25°C on a water bath.

Compound 9-A (4.35 mg, 4050 nmol) was dissolved in 270 μL of DMSO and the resulting solution was added to the above reaction mixture, which was then shaken in a shaking water bath at 25 °C for 3 h before stopping the reaction. 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 obtain the type product ADC-2 PD3-9-A in PBS buffer (1.69 mg/mL, 17.8 mL), which was then stored at 4 °C.

Average value calculated by UV-Vis method: n=6.59.

Example 3-3 ADC-3

To the aqueous PBS buffer of PD3 antibody (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/ml, 0.3 ml, 20.3 nmol) at 37°C was added prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 5.1 μl, 51 nmol). The reaction mixture was shaken on a shaking water bath at 37°C for 3 h before stopping the reaction. The reaction mixture was cooled to 25°C on a water bath.

Compound 12H (0.194 mg, 203 nmol, obtained with reference to WO2017063509) was dissolved in 20 μL of acetonitrile, and the resulting solution was added to the above reaction mixture, which was then shaken in a water bath by shaking at 25 °C for 3 hours before stopping the reaction. 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 obtain the title product ADC-3 in PBS buffer (4.3 mg/mL, 0.6 mL), which was then stored at 4 °C.

Average value calculated by UV-Vis method: n=4.14.

Example 3-4 ADC-4

The synthesis was carried out with reference to example 19 in WO2015098099.

To the aqueous PBS buffer of TINA antibody (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/mL, 2.0 mL, 135.4 nmol) at 37°C was added prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 33.8 μL, 338 nmol). The reaction mixture was shaken on a shaking water bath at 37°C for 3 h before stopping the reaction. The reaction mixture was cooled to 25°C on a water bath.

Compound 58 (1.4 mg, 1354 nmol) was dissolved in 70 μL of DMSO and the resulting solution was added to the above reaction mixture, which was then shaken on a shaker with a water bath at 25 °C for 3 h before the reaction was stopped. 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 typical title product ADC-4 TINA-58 in PBS buffer (1.13 mg/mL, 15.1 mL), which was then stored at 4 °C.

Average value calculated by UV-Vis method: n=3.99.

Example 3-5 ADC-5

To the aqueous PBS buffer of PD3 antibody (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/ml, 1.5 ml, 101.4 nmol) prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 25.3 μl, 253 nmol) was added at 37°C. The reaction mixture was shaken on a shaking water bath at 37°C for 3 h before stopping the reaction. The reaction mixture was cooled to 25°C on a water bath.

Compound 58 (1.05 mg, 1014 nmol, see Example 58 on page 163 of patent CN104755494A) was dissolved in 60 μL of DMSO and the resulting solution was added to the above reaction mixture, which was then shaken in a shaking water bath at 25 °C for 3 hours before stopping the reaction. The reaction mixture was desalted and purified on a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5) to give an example of the title product ADC-5 PD3-58 in PBS buffer (0.82 mg/mL, 13.5 mL), which was then stored at 4 °C.

Average value calculated by UV-Vis method: n=3.88.

Example 3-6 ADC-6

To the aqueous PBS buffer of hRS7 antibody (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/ml, 1.4 ml, 94.60 nmol) at 37°C was added prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 50.1 μl, 501 nmol). The reaction mixture was shaken on a shaking water bath at 37°C for 3 h before stopping the reaction. The reaction mixture was cooled to 25°C on a water bath.

Compound SN38 (synthesized with reference to Example 1 on page 59 of patent CN105407891A, 2.1 mg, 1419 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 shaking water bath at 25°C for 3 hours before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5) to give the typical title product ADC-6 from hRS7-SN38 in PBS buffer (1.03 mg/mL, 11.5 mL), which was then stored at 4°C.

Average value calculated by CE-SDS method: n=7.56.

Example 3-7 ADC-7

To the aqueous PBS buffer of hRS7 antibody (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/ml, 2.18 ml, 147.3 nmol) at 37°C was added prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 36.8 μl, 368 nmol). The reaction mixture was shaken on a shaking water bath at 37°C for 3 h before stopping the reaction. The reaction mixture was cooled to 25°C on a water bath.

Compound 9-A (1.58 mg, 1471 nmol) was dissolved in 100 μL of DMSO and the resulting solution was added to the above reaction mixture, which was then shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5) to give the illustrative title product ADC-6 from hRS7-9-A in PBS buffer (1.10 mg/mL, 16.4 mL), which was then stored at 4 °C.

Average value calculated by UV-Vis method: n=3.72.

Example 3-8 ADC-8

To the aqueous PBS buffer of hRS7 antibody (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/ml, 2.18 ml, 147.3 nmol) at 37°C was added prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 36.8 μl, 368 nmol). The reaction mixture was shaken on a shaking water bath at 37°C for 3 h before stopping the reaction. The reaction mixture was cooled to 25°C on a water bath.

Compound 58 (1.52 mg, 1473 nmol, see Example 58 on page 163 of patent CN104755494A) was dissolved in 100 μL of DMSO and the resulting solution was added to the above reaction mixture, which was then shaken in a shaking water bath at 25 °C for 3 hours before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5) to give the typical title product ADC-8 from hRS7-58 in PBS buffer (1.02 mg/mL, 16.8 mL), which was then stored at 4 °C.

Average value calculated by UV-Vis method: n=3.93.

Example 3-9 ADC-9

To the aqueous PBS buffer of TINA antibody (0.05 M aqueous PBS buffer at pH 6.5; 10.0 mg/ml, 0.95 ml, 64.2 nmol) at 37°C was added prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 16.0 μl, 160 nmol). The reaction mixture was shaken on a shaking water bath at 37°C for 3 h before stopping the reaction. The reaction mixture was cooled to 25°C on a water bath.

Compound 9-A (0.69 mg, 642 nmol) was dissolved in 30 μL of DMSO and the resulting solution was added to the above reaction mixture, which was then shaken on a shaking water bath at 25 °C for 3 h before stopping the reaction. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5) to obtain the illustrative title product ADC-9 from TINA-9-A in PBS buffer (0.99 mg/mL, 7.0 mL), which was then stored at 4 °C.

Average value calculated by UV-Vis method: n=3.99.

Drug loading analysis of ADC stock solution

ADC is a cross-linked antibody drug, and its mechanism of treating diseases is to transport toxin molecules into cells depending on the targeting efficiency of the antibody so as to kill the cells. The drug load plays a decisive role in the effectiveness of the drug.

1. UV-Vis calculation method

The drug loading of the ADC stock solution was determined by UV method.

Experimental procedures

Cuvettes containing sodium succinate buffer solution were placed in the reference and sample wells, and the absorbance of the blank solvent was subtracted. A cuvette containing the test solution was then placed in the sample well, and the absorbance values at 280 nm and 370 nm were determined.

Calculating results

The loading capacity of the ADC stock solution was determined using ultraviolet spectrophotometry (device: Thermo nanodrop2000 ultraviolet spectrophotometer), based on the principle that the total absorbance of the ADC stock solution at a given wavelength is the sum of the absorbance values of the cytotoxic drug and the monoclonal antibody at that wavelength, namely:

(1) A _{280 nm} = ε _mab-280 bC _mab + ε _drug-280 bC _drug

ε _drug-280 : the mean molar extinction coefficient of the drug at 280 nm was 5100;

C _{of drug} : concentration of drug;

ε _mab-280 : the mean molar extinction coefficient of the stock solution of monoclonal antibody at 280 nm is 214600;

C _mab : concentration of the stock solution of monoclonal antibody;

b: optical path length, which is 1 cm.

Similarly, the equation for the total absorbance of a sample at 370 nm can be written as:

(2) A _{370 nm} = ε _mab-370 bC _mab + ε _drug-370 bC _drug

ε _drug-370 : the mean molar attenuation coefficient of the drug at 370 nm was 19000;

C _{of drug} : concentration of drug;

ε _mab-370 : the extinction coefficient of the monoclonal antibody stock solution at 370 nm is 0;

C _mab : concentration of the stock solution of monoclonal antibody;

b: The optical path length is 1 cm.

The drug loading can be calculated using both equations (1) and (2) and the attenuation coefficients of the monoclonal antibody and drug at both wavelengths and their concentrations.

Drug loading = C _drug /C _mab .

2. CE-SDS calculation method

Reagents and instruments

The SDS-Mw assay kit manufactured by Beckman, Cat. No. 390953, containing SDS-MW gel separation buffer, SDS-MW sample buffer, acid cleaning solution (0.1 mol/L hydrochloric acid solution), basic cleaning solution (0.1 mol/L sodium hydroxide solution), and internal standard substance (10 kDa) was used. The SDS kit manufactured by Beijing Institute of Technology BioCEart, Cat. No. BSYK018, containing CE-SDS gel buffer and CE-SDS sample buffer was also used.

Alkylation solution (0.25 mol iodoacetamide solution): About 0.046 g iodoacetamide was weighed, 1 ml of ultrapure water was added to dissolve and mixed well, and the resulting solution was stored at 2-8°C for 7 days in the dark.

Capillary electrophoresis apparatus: PA800plus from SCIEX.

Capillary: Uncoated fused silica capillary (50 µm ID) cut to a total length of 30.2 cm and an effective high resolution separation length of 20 cm.

Preparation of the test sample solution

The test sample was diluted to 1 mg/mL with SDS (sodium dodecyl sulfate) sample buffer. 95 μL of the test solution (1 mg/mL) was taken out and 5 μL of 0.8 mol/L aqueous iodoacetamide solution was added, the resulting solution was shaken and mixed well. 95 μL of the blank control was taken out and 5 μL of 0.8 mol/L aqueous iodoacetamide solution was added, the resulting solution was shaken and mixed well. 75 μL of samples were taken out of the sample tubes, added to the sample vials, and analyzed immediately.

Determination method

1) Pre-treatment of capillary: 0.1mol/L sodium hydroxide solution was washed under 60 psi for 3 min, then washed with 0.1mol/L hydrochloric acid solution under 60 psi for 2 min, and finally washed with pure water under 70 psi for 1 min. The above pre-treatment should be performed before each operation.

2) Pre-filling of capillary: SDS gel separation buffer was washed at 50 psi for 15 minutes. The above pre-treatment should be performed before each operation.

Sample injection: Electrokinetic sample injection was performed at 10 kV reverse polarity and the reduced sample was injected for 20 s.

Separation: Separation was performed at 15 kV reverse polarity for 40 min.

Temperature in the sample chamber: from 18 to 25°C.

Capillary temperature: from 18 to 25°C.

Analysis of results

The data were analyzed using Beckman software based on the binding of the corresponding drug to the sulfhydryl released from the open disulfide bond in the antibody, and the proportion of the adjusted peak area, such as heavy chain, non-glycosylated heavy chain, and light chain, was calculated from the sum of all adjusted peak areas. The calculation formula is: DAR=[4 × peak area of heavy chain (H)+2 × peak area of half antibody (H-L)+4 × peak area of dual heavy chain (H-H)+2 × peak area of heavy-heavy-light chain (H-H-L)]/[peak area of heavy chain (H)/2+peak area of half antibody (H-L)/2+peak area of dual heavy chain (H-H)+peak area of heavy-heavy-light chain (H-H-L)+peak area of full antibody], and then the weighted average ADC was calculated.

The activity of the antibodies of the present invention was validated using a biochemical assay.

Test Example 1: Antibody Protein Level Binding Assay

The hTROP-2 protein was diluted to 1 μg/ml with PBS buffer at pH 7.4 (Shanghai BasalMedia Technologies Co., LTD., B320), added to a 96-well microplate at 100 μl per well, and incubated at 4°C overnight. After the liquid was discarded, 300 μl of 5% skim milk (BD, 232100) diluted with PBS was added to each well for blocking, and incubated at 37°C for 2 hours. After blocking was completed, the blocking solution was discarded; after the plate was washed 3 times with PBST buffer (pH 7.4, PBS containing 0.1% Tween 20), 100 μl of gradient-diluted antibody solution was added to each well, and incubated at 37°C for 1 hour. After completion of the incubation, the plate was washed 3 times with PBST buffer, and 100 μl of 1:8000 diluted mouse anti-human IgG (H+L) antibody (Jackson ImmunoResearch, 209-035-088) was added to each well and incubated at 37° _C for 1 hour. After the plate was washed 3 times with PBST buffer, 100 μl of TMB chromogenic substrate (KPL, 5120-0077) was added to each well and incubated at room temperature for 10-15 minutes, and the reaction was stopped by adding 50 μl of 1 _MH2SO4 to each well. The optical density at 450 nm was estimated using a microplate reader, the binding curves of antibody and antigen were matched using the software, and the _EC50 value was calculated. The binding activity of antibodies to proteins is shown in Table 2.

Table 2. Binding activity at the antibody protein level Antibodies PD3 hRS7 TINA E _max (optical density value) 1.40 1.37 1.39 EC ₅₀ (nM) 13.6 18.29 16,14

The result shows that the anti-PD3 antibody in the present application has higher binding activity to the hTROP-2 protein.

Test Example 2: Cellular Level Antibody Binding Assay

Stably transfected CHOK1 cells expressing TROP-2 were suspended in FACS buffer (2% fetal bovine serum (Gibco, 10099141), pH 7.4 PBS (Sigma, P4417-100TAB)) to obtain a suspension of 1× ¹⁰⁶ cells/ml, which was then added to a 96-well round-bottomed plate (Corning, 3795) at 100 μl/well. After centrifugation and removal of the supernatant, the test antibody, which was diluted with FACS buffer to different concentrations, was added at 50 μl/well. The plate was incubated in the dark in a refrigerator at 4°C for 1 hour. The plate was washed three times with FACS buffer by centrifugation at 300 g and goat anti-human IgG Alexa Fluor 488 (H+L) (Invitrogen, A-11013) was added at a working concentration. The plate was incubated in the dark in a refrigerator at 4 °C for 40 min. The plate was washed three times with FACS buffer by centrifugation at 300 g and tested on a BD FACS CantoII flow cytometer for geometric mean fluorescence intensity. The EC ₅₀ value of antibody binding to stably transfected cells expressing TROP-2 was calculated. The binding activity of antibodies to cells is shown in Fig. 1 and Table 3.

Table 3. Binding activity of antibodies at the cellular level Antibodies PD3 hRS7 TINA E _max (MFI) (mean fluorescence intensity) 16693 16050 14217 EC ₅₀ (nM) 1.57 2.31 3.55

The result shows that the anti-PD3 antibody in the present application has higher binding activity to cells expressing hTROP-2 protein.

Test Example 3: Antibody Endocytosis Assay

The aim of the assay is to ensure that activated DT (diphtheria toxin) kills cells after DT3C protein enters the cells, indirectly reflecting endocytosis of the TROP-2 antibody. The in vitro endocytic activity of the antibody was assessed according to EC ₅₀ and E _max .

DT3C is a recombinantly expressed fusion protein and is formed by fusing diphtheria toxin fragment A (toxic part only) and group G streptococcus fragment 3C (IgG binding part). The protein can have high affinity for the IgG part of the antibody, penetrate into the cells together with the IgG part during endocytosis of the antibody, and release toxic DT by the action of intracellular furin protease. DT can inhibit the ribosylation activity of EF2-ADP, block the process of protein translation, and finally cause cell death. DT3C, which does not penetrate into the cell, does not have cell killing activity. The endocytic activity of the antibody was evaluated based on cell killing.

Cell suspensions were prepared with fresh cell medium containing 20% low-IgG FBS at a cell density of 2 × ¹⁰⁴ cells/mL and added to cell culture plates at 50 μL/well and incubated with 5% carbon dioxide at 37°C for 16 h. DT3C at 4× was prepared in serum-free medium and filtered through a 0.22 μm filter to obtain a sterile solution. Antibody at 4× was prepared in serum-free medium, and 80 μL DT3C and 80 μL antibody were mixed at a 1:1 volume and incubated at room temperature for 30 min. 50 μL of the diluted antibody-DT3C mixture was added to 50 μL of cells and incubated in an incubator for three days. 50 μl of CTG was added to each well and incubated for 10 min at room temperature in the dark, and chemiluminescence values were detected using Victor3. The endocytic activity of the antibody is shown in Table 4.

Table 4. Endocytic activity of antibodies Antibodies PD3 hRS7 TINA E _max 99.1% 98.5% 98.7% EC ₅₀ (nM) 0.15 0.18 0.18

The results show that the anti-PD3 antibody in the present application has a higher endocytosis efficiency.

Test Example 4: Antibody Affinity Assay

The affinity of the antibody to TROP-2 was detected in the form of a capture antibody. The antibody was affinity captured with Protein A (Cat.#29127556, GE) using a biosensor chip conjugated with anti-human IgG antibody (Cat.#BR-1008-39, Lot#10260416, GE), then hTROP-2 antigen was flowed over the surface of the chip and a Biacore T200 was used to detect the reaction signals in real time to obtain association and dissociation curves. After dissociation was complete for each assay cycle, the chip was washed and regenerated with Glycine1.5 Regeneration Buffer (Cat.#BR100354, GE) or 3 M _MgCl2 (from the Human Antibody Capture Kit, Cat.#BR100839, GE). After analysis was completed, the data were refined with a Langmuir model (1:1) using GE Biacore T200 Evaluation version 3.0 to obtain affinity values. The antibody affinity for proteins is shown in Table 5.

Table 5. Antibody affinity determined by Biacore Antibodies PD3 hRS7 TINA KD (equilibrium dissociation constant) (M) 6.86E-10 9.87E-10 2,15E-8

The results show that the PD3 antibody in the present application has a higher affinity for hTROP-2.

Test Example 5: Analysis of Cellular Activity of ADC Molecules

The cells used in this assay were as follows: FaDu (+++), purchased from ATCC, Cat. No. HTB-43™; HCC827(+++), purchased from ATCC, Cat. No. CRL-2868; Colo205(++), purchased from Cell Bank, Chinese Academy of Sciences, Cat. No. TCHu102; DMS53 (++), purchased from ATCC, Cat. No. CRL-2062™; SK-OV-3(+), purchased from ATCC, Cat. No. HTB-77; CHO-K1(-), purchased from ATCC, Cat. No. CCL-61™; where "+" represents the expressed amount of TROP-2 in the cell population, more "+" means the expressed amount of TROP-2 is higher and "-" means TROP-2 is not expressed.

Cell suspensions were prepared with fresh cell medium containing 10% FBS at a density of 3703 cells/mL and added to a 96-well cell culture plate at 135 μL/well and incubated with 5% carbon dioxide at 37°C for 16 h. ADC samples were formulated at a concentration of 5 μM with PBS. 5 μM stock concentration was diluted fivefold with PBS for a total of 8 concentrations. 15 μL of the above ADC solution was added to each well. The plate was cultured at 37°C in 5% carbon dioxide for 6 days. 70 μL of CTG was added to each well and incubated for 10 min at room temperature in the dark, chemiluminescence values were detected using Victor3 and data analysis was performed using GraphPad Prism software.

The results show that ADC-1 has a stronger cell killing effect, and the killing effect is positively correlated with the expression level of TROP-2 on the surface of tumor cell.

Table 6-1. Cytotoxicity of ADCs to Cells with Different Levels of TROP-2 Expression FaDu HCC827 Colo205 DMS53 SK-OV-3 CHO-K1 Samples EC ₅₀
(nM) E _max
(%) EC ₅₀
(nM) E _max
(%) EC ₅₀
(nM) E _max
(%) EC ₅₀
(nM) E _max
(%) EC ₅₀
(nM) E _max
(%) EC ₅₀
(nM) E _max
(%) ADC-1 0.23 100.42 0.14 89.64 39.23 99.38 42,46 89.39 n.d. 61.91 n.d. 9.31 ADC-4 0.53 101.25 0.27 88.16 59.8 99.66 75.58 89.83 n.d. 49.15 n.d. 1.71 ADC-7 0.31 100.75 0.18 89.33 52.75 99.52 72.92 88.74 n.d. 66.11 n.d. 8.69

The second parallel comparative analysis was carried out in the manner described above and the results obtained were as follows:

Table 6-2. Cytotoxicity of ADCs to Cells with Different Levels of TROP-2 Expression FaDu HCC827 Colo205 DMS53 Samples EC ₅₀ (nM) E _max (%) EC ₅₀ (nM) E _max (%) EC ₅₀ (nM) E _max (%) EC ₅₀ (nM) E _max (%) ADC-1 0.08 101.6 0.06 89.57 12.07 100.34 15.08 93.24 ADC-5 0.14 101.58 0.13 88.23 25.63 99.64 25.71 92.47

Test Example 6: Non-specific Cytotoxicity Study

BxPC3 (human pancreatic cancer cells, ATCC, CRL-1687) and MiaPaCa2 (human pancreatic cancer cells, Biocytogen, B-HCL-014) cells were cultured with RPMI1640 and 10% FBS and DMEM (Dulbecco's modified Eagle's minimum essential medium)/high glucose and 10% FBS, respectively; cells were trypsinized, neutralized with fresh medium, and centrifuged at 1000 rpm for 3 min; the supernatant was discarded, and the cells were resuspended with RPMI1640 and 10% FBS. After cell counting, BxPC3 cells were adjusted to 6 × 10 ⁴ cells/mL and MiaPaCa2-luc cells were adjusted to 1.5 × 10 ⁴ cells/mL. 500 μl BxPC3 cells and 500 μl MiaPaCa2-luc cells were added to each well of 12-well plate 1. 500 μl MiaPaCa2-luc cells and 500 μl RPMI1640 medium containing 10% serum-FBS were added to 12-well plate 2. The plate was cultured at 37°C in 5% carbon dioxide for 24 h.

ADC samples were prepared as 40× concentration of intermediate solutions (0.2 μM). 25 μL of the above samples were collected and added to the corresponding well of a 12-well plate. A solvent control group was set. The plate was cultured at 37°C in 5% carbon dioxide for 6 days. The cells in the 12-well plate were trypsinized, neutralized with fresh medium, and centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and the cells were resuspended in 1 mL FACS buffer (PBS and 2.5% FBS). 20 μL of cells were collected and stained with 20 μL trypan blue and counted. The cells in plate 1 were centrifuged at 1000 rpm for 3 min, the supernatant was discarded, the cells were resuspended in 100 μl FACS buffer, 2 μl TROP-2 (EGP-1) monoclonal antibody (MR54) were added, and the cells were incubated on ice for 30 min. The cells were centrifuged at 2000 rpm at 4°C for 1 min, the supernatant was discarded, and the cells were resuspended in 150 μl FACS buffer. Detection was performed using BD FACSVerse. Data were analyzed by Flowjo 7.6. The results of the non-specific killing activity assay are shown in Fig. 2.

The results show that the ADC-1 of the present invention has a clear killing side effect, and the ADC does not kill TROP-2-negative MiaPaCa2 cells, but the ADC-1 kills TROP-2-negative cells when TROP-2-expressing BxPC3 cells are mixed with negative MiaPaCa2 cells.

Biological assessment of in vivo activity

Test Example 7: In vivo Efficacy Assessment in Fadu Cells, a CDX Mouse Model

Fadu cells (3 × 10 ⁶ ) were subcutaneously inoculated into the right flank of Balb/c nude mice, 10 days after inoculation, nude mice with heavy weight, too large tumor and too small tumor were removed after the tumor volume was about 245 mm ³ , and the remaining mice were randomly divided into 5 groups of 8 mice according to tumor volume.

Each mouse was injected intraperitoneally with ADC at a concentration of 0.1 ml/10 g body weight on days 0 and 8 for a total of 2 injections at a dose of 1 mg/kg. Tumor volumes and body weights were measured twice a week and the results were recorded.

The data were recorded using Excel statistical software: SD (standard deviation) values were calculated as the mean; SD (standard error) values were calculated as STDEV (square root of the sample variance); SEM (standard error of the mean) values were calculated as STDEV/SQRT (number of animals per group); GraphPad Prism software was used to plot the graph, and statistical analysis of the data was performed using two-way ANOVA or one-way ANOVA.

Tumor volume (V) was calculated as: V=1/2 × L _long × L _short ²

Relative tumor proliferation rate T/C(%)=(T - _T0 )/(C - _C0 ) × 100, where T and C are the tumor volumes of animals at the end of the experiment in the treatment group and control group, respectively; _T0 and _C0 are the tumor volumes of animals at the beginning of the experiment in the treatment group and control group, respectively.

Tumor inhibition rate TGI (%) = 1 - T/C (%).

The results are shown in Table 7 and Fig. 3, which show that ADC-1 has a strong inhibitory effect on FaDu xenograft tumors at a dose of 1 mg/kg.

Table 7. Efficacy of ADC on FaDu xenograft tumors in tumor-bearing mice Group Average tumor volume ( ^mm3 ) Average tumor volume ( ^mm3 ) Degree of tumor inhibition
TGI (%) D0 SEM D21 SEM "Empty" control 246.8 29.04 2245,04 275.14 ND ADC-4 1 mg/kg 244.41 29.62 1180.96 193.55 53 ADC-1 1 mg/kg 251.19 23.45 238.54 107.62 101 ADC-2 1 mg/kg 270.48 21.36 0 0 114 PD3 30 mg/kg 246.02 25 2407.37 207.71 -8

Test Example 8: In vivo efficacy evaluation in SKOV3 cells, a CDX mouse model

SKOV3 cells (5× ¹⁰⁶ ) were subcutaneously inoculated into the right flank of Balb/c nude mice, 23 days after inoculation, nude mice with heavy weight, too large tumor and too small tumor were removed after the tumor volume was about ^180mm3 , and the remaining mice were randomly divided into 5 groups of 8 mice according to tumor volume.

Each mouse was intraperitoneally injected with ADC at a concentration of 0.1 ml/10 g body weight for a total of 2 injections, and the dose is shown in the following table. Tumor volumes and body weights were measured twice a week and the results were recorded. The data were recorded using Excel statistical software: SD values were calculated as the mean; SD values were calculated as STDEV (square root of sample variance); SEM values were calculated as STDEV/SQRT (number of animals per group); GraphPad Prism software was used to plot the graph, and statistical analysis of the data was performed using two-way ANOVA or one-way ANOVA.

Tumor volume (V) was calculated as: V=1/2 × L _long × L _short ²

Relative tumor proliferation rate T/C(%)=(T _-T0 )/(C - _C0 ) × 100, where T and C are the tumor volumes of animals at the end of the experiment in the treatment group and control group, respectively; _T0 and _C0 are the tumor volumes of animals at the beginning of the experiment in the treatment group and control group, respectively.

Tumor inhibition rate TGI (%) = 1 - T/C (%).

The results are shown in Table 8 and Fig. 4, which show that ADC-1 has a strong inhibitory effect on SKOV3 xenograft tumors at different doses, and the effect is dose-dependent and inhibitory.

The effect depends on the dose.

Table 8. Efficacy of ADC on SKOV3 xenograft tumors in tumor-bearing mice Group Average tumor volume ( ^mm3 ) Average tumor volume ( ^mm3 ) Degree of tumor inhibition
TGI (%) D0 SEM D21 SEM "Empty" control 183.26 7.6 1081.36 132.88 ND ADC-1 10 mg/kg 185.48 9.62 302.93 39.34 87 ADC-1 3 mg/kg 185.09 10.07 519.9 38.93 63 ADC-1 1 mg/kg 183.29 8.87 795.34 124.81 32 ADC-4 10 mg/kg 183.83 8.9 938.99 148.98 16 ADC-4 3 mg/kg 184.17 8.2 873.38 50.84 23 ADC-4 1 mg/kg 185.17 8.38 1110,52 159.73 -3 PD3 30 mg/kg 182.85 8.38 1384.28 93.87 -34

Test Example 9: In vivo Efficacy Assessment in Colo205 Cells, a CDX Mouse Model

Colo205 cells (5× ¹⁰⁶ ) were subcutaneously inoculated into the right flank of Balb/c nude mice, 10 days after inoculation, nude mice with heavy weight, too large tumor and too small tumor were removed after the tumor volume was about ^245mm3 , and the remaining mice were randomly divided into 6 groups of 8 mice according to tumor volume.

Each mouse was injected intraperitoneally with ADC at a concentration of 0.1 ml/10 g body weight on day 0 (D0) and day 8 (D8) for a total of 2 injections at a dose of 1 mg/kg. Tumor volumes and body weights were measured twice a week and the results were recorded for 28 days (D28).

The data were recorded using Excel statistical software: SD (standard deviation) values were calculated as the mean; SD (standard error) values were calculated as STDEV (square root of the sample variance); SEM (standard error of the mean) values were calculated as STDEV/SQRT (number of animals per group); GraphPad Prism software was used to plot the graph, and statistical analysis of the data was performed using two-way ANOVA or one-way ANOVA.

Tumor volume (V) was calculated as: V=1/2 × L _long × L _short ²

Relative tumor proliferation rate T/C(%)=(TT ₀ )/(CC ₀ )×100, where T and C are the tumor volumes of animals at the end of the experiment in the treatment group and control group, respectively; T ₀ and C ₀ are the tumor volumes of animals at the beginning of the experiment in the treatment group and control group, respectively.

Tumor inhibition rate TGI (%) = 1 - T/C (%).

The results are shown in Table 9 and Fig. 5, which show that ADC-9 and ADC-7 conjugated with compound 9-A exert potent inhibitory effect on Colo205 xenograft tumors at a dose of 1 mg/kg/L.

Table 9. Efficacy of ADC on FaDu xenograft tumors in tumor-bearing mice Group Average tumor volume ( ^mm3 ) Average tumor volume ( ^mm3 ) Degree of tumor inhibition
TGI (%) D0 SEM D28 SEM "Empty" control 210.30 24.91 1355,56 117.46 ND ADC-6 10 mg/kg 194.93 26.56 1344.84 95,93 0 ADC-7 10 mg/kg 194.64 21.03 35.13 12.80 113 ADC-8 10 mg/kg 199.09 23.41 475.66 175.24 76 ADC-9 10 mg/kg 206.70 21,21 99.86 32.55 109 ADC-4 10 mg/kg 201.52 27.85 513.83 118.53 73

--->

SEQUENCE LIST

<110>JIANGSU HANGRUI MEDICAL CO., LTD.

SHANGHAI HENGRUI PHARMACEUTICAL CO., LTD.

<120>CONJUGATE OF ANTIBODY TO TROP-2 AND EXATECAN ANALOGUE AND ITS

MEDICAL USE

<130>721008CPCT

<140>PCT/CN2021/073279

<141>2021-01-22

<150>202010073438.8

<151>2020-01-22

<160>18

<170>Patent-In 3.5

<210>1

<211>323

<212>Protein

<213>Homo sapiens

<400>1

Met Ala Arg Gly Pro Gly Leu Ala Pro Pro Pro Leu Arg Leu Pro Leu

1 5 10 15

Leu Leu Leu Val Leu Ala Ala Val Thr Gly His Thr Ala Ala Gln Asp

20 25 30

Asn Cys Thr Cys Pro Thr Asn Lys Met Thr Val Cys Ser Pro Asp Gly

35 40 45

Pro Gly Gly Arg Cys Gln Cys Arg Ala Leu Gly Ser Gly Met Ala Val

50 55 60

Asp Cys Ser Thr Leu Thr Ser Lys Cys Leu Leu Leu Lys Ala Arg Met

65 70 75 80

Ser Ala Pro Lys Asn Ala Arg Thr Leu Val Arg Pro Ser Glu His Ala

85 90 95

Leu Val Asp Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Pro Glu Gly

100 105 110

Arg Phe Lys Ala Arg Gln Cys Asn Gln Thr Ser Val Cys Trp Cys Val

115 120 125

Asn Ser Val Gly Val Arg Arg Thr Asp Lys Gly Asp Leu Ser Leu Arg

130 135 140

Cys Asp Glu Leu Val Arg Thr His His Ile Leu Ile Asp Leu Arg His

145 150 155 160

Arg Pro Thr Ala Gly Ala Phe Asn His Ser Asp Leu Asp Ala Glu Leu

165 170 175

Arg Arg Leu Phe Arg Glu Arg Tyr Arg Leu His Pro Lys Phe Val Ala

180 185 190

Ala Val His Tyr Glu Gln Pro Thr Ile Gln Ile Glu Leu Arg Gln Asn

195 200 205

Thr Ser Gln Lys Ala Ala Gly Asp Val Asp Ile Gly Asp Ala Ala Tyr

210 215 220

Tyr Phe Glu Arg Asp Ile Lys Gly Glu Ser Leu Phe Gln Gly Arg Gly

225 230 235 240

Gly Leu Asp Leu Arg Val Arg Gly Glu Pro Leu Gln Val Glu Arg Thr

245 250 255

Leu Ile Tyr Tyr Leu Asp Glu Ile Pro Pro Lys Phe Ser Met Lys Arg

260 265 270

Leu Thr Ala Gly Leu Ile Ala Val Ile Val Val Val Val Val Ala Leu

275 280 285

Val Ala Gly Met Ala Val Leu Val Ile Thr Asn Arg Arg Lys Ser Gly

290 295 300

Lys Tyr Lys Lys Val Glu Ile Lys Glu Leu Gly Glu Leu Arg Lys Glu

305 310 315 320

Pro Ser Leu

<210>2

<211>329

<212>Protein

<213>Artificial sequence

<220>

<223>Amino acid sequence of Trop2-His

<400>2

Met Ala Arg Gly Pro Gly Leu Ala Pro Pro Pro Leu Arg Leu Pro Leu

1 5 10 15

Leu Leu Leu Val Leu Ala Ala Val Thr Gly His Thr Ala Ala Gln Asp

20 25 30

Asn Cys Thr Cys Pro Thr Asn Lys Met Thr Val Cys Ser Pro Asp Gly

35 40 45

Pro Gly Gly Arg Cys Gln Cys Arg Ala Leu Gly Ser Gly Met Ala Val

50 55 60

Asp Cys Ser Thr Leu Thr Ser Lys Cys Leu Leu Leu Lys Ala Arg Met

65 70 75 80

Ser Ala Pro Lys Asn Ala Arg Thr Leu Val Arg Pro Ser Glu His Ala

85 90 95

Leu Val Asp Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Pro Glu Gly

100 105 110

Arg Phe Lys Ala Arg Gln Cys Asn Gln Thr Ser Val Cys Trp Cys Val

115 120 125

Asn Ser Val Gly Val Arg Arg Thr Asp Lys Gly Asp Leu Ser Leu Arg

130 135 140

Cys Asp Glu Leu Val Arg Thr His His Ile Leu Ile Asp Leu Arg His

145 150 155 160

Arg Pro Thr Ala Gly Ala Phe Asn His Ser Asp Leu Asp Ala Glu Leu

165 170 175

Arg Arg Leu Phe Arg Glu Arg Tyr Arg Leu His Pro Lys Phe Val Ala

180 185 190

Ala Val His Tyr Glu Gln Pro Thr Ile Gln Ile Glu Leu Arg Gln Asn

195 200 205

Thr Ser Gln Lys Ala Ala Gly Asp Val Asp Ile Gly Asp Ala Ala Tyr

210 215 220

Tyr Phe Glu Arg Asp Ile Lys Gly Glu Ser Leu Phe Gln Gly Arg Gly

225 230 235 240

Gly Leu Asp Leu Arg Val Arg Gly Glu Pro Leu Gln Val Glu Arg Thr

245 250 255

Leu Ile Tyr Tyr Leu Asp Glu Ile Pro Pro Lys Phe Ser Met Lys Arg

260 265 270

Leu Thr Ala Gly Leu Ile Ala Val Ile Val Val Val Val Val Ala Leu

275 280 285

Val Ala Gly Met Ala Val Leu Val Ile Thr Asn Arg Arg Lys Ser Gly

290 295 300

Lys Tyr Lys Lys Val Glu Ile Lys Glu Leu Gly Glu Leu Arg Lys Glu

305 310 315 320

Pro Ser Leu His His His His His

325

<210>3

<211>121

<212>Protein

<213>Artificial sequence

<220>

<223>PD3 heavy chain variable region

<400>3

Glu Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Gln Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>4

<211>107

<212>Protein

<213>Artificial sequence

<220>

<223>PD3 light chain variable region

<400>4

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys

100 105

<210>5

<211>5

<212>Protein

<213>Artificial sequence

<220>

<223>PD3HCDR1

<400>5

Asn Tyr Gly Met Asn

1 5

<210>6

<211>17

<212>Protein

<213>Artificial sequence

<220>

<223>PD3HCDR2

<400>6

Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Gln Asp Phe Lys

1 5 10 15

Gly

<210>7

<211>12

<212>Protein

<213>Artificial sequence

<220>

<223>PD3HCDR3

<400>7

Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val

1 5 10

<210>8

<211>11

<212>Protein

<213>Artificial sequence

<220>

<223>PD3 LCDR1

<400>8

Lys Ala Ser Gln Asp Val Ser Ile Ala Val Ala

1 5 10

<210>9

<211>7

<212>Protein

<213>Artificial sequence

<220>

<223>PD3 LCDR2

<400>9

Ser Ala Ser Tyr Arg Tyr Thr

1 5

<210>10

<211>9

<212>Protein

<213>Artificial sequence

<220>

<223>PD3 LCDR3

<400>10

Gln Gln His Tyr Ile Thr Pro Leu Thr

1 5

<210>11

<211>330

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<213>Homo sapiens

<400>11

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>12

<211>107

<212>Protein

<213>Homo sapiens

<400>12

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210>13

<211>451

<212>Protein

<213>Artificial sequence

<220>

<223>Heavy Chain PD3

<400>13

Glu Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Gln Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210>14

<211>214

<212>Protein

<213>Artificial sequence

<220>

<223>PD3 Light Chain

<400>14

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>15

<211>451

<212>Protein

<213>Artificial sequence

<220>

<223>Heavy Chain hRS7

<400>15

Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210>16

<211>214

<212>Protein

<213>Artificial sequence

<220>

<223>Light chain hRS7

<400>16

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>17

<211>451

<212>Protein

<213>Artificial sequence

<220>

<223>TINA Heavy Chain

<400>17

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ala

20 25 30

Gly Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe

50 55 60

Lys Gly Arg Val Thr Ile Ser Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210>18

<211>214

<212>Protein

<213>Artificial sequence

<220>

<223>TINA Light Chain

<400>18

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<---

Claims (79)

1. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof, Where: Y is selected from the group consisting of -O-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-; R ^a and R ^b represent hydrogen; R ¹ is C _3-6 cycloalkyl or C _3-6 cycloalkyl C _1-6 alkyl; R ² is hydrogen; m represents 0 or 1; n is a decimal or integer number between 1 and 10; L is a linker moiety; Rc is an antibody to TROP-2 or an antigen-binding fragment thereof, wherein the antibody to TROP-2 or an antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively. 2. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the antibody to TROP-2 is a mouse antibody, a chimeric antibody, a humanized antibody, or a human antibody. 3. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the antibody to TROP-2 or an antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence presented in SEQ ID NO: 3 or has at least 90% identity thereto, and/or the light chain variable region has the amino acid sequence presented in SEQ ID NO: 4 or has at least 90% identity thereto. 4. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein the antibody to TROP-2 or its antigen-binding fragment comprises a heavy chain variable region presented in SEQ ID NO: 3 and a light chain variable region presented in SEQ ID NO: 4. 5. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein the antibody to TROP-2 or an antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region of an antibody; wherein the heavy chain constant region is selected from the group consisting of the constant regions of human IgG1, IgG2, IgG3 and IgG4, and the light chain constant region is selected from the group consisting of the constant regions of the κ and λ chains of a human antibody. 6. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, wherein the antibody to TROP-2 comprises a heavy chain constant region as presented in SEQ ID NO: 11 and a light chain constant region as presented in SEQ ID NO: 12. 7. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any one of claims 1-6, wherein the antibody to TROP-2 comprises a heavy chain as presented in SEQ ID NO: 13 and a light chain as presented in SEQ ID NO: 14. 8. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, where n is a decimal or integer number from 2 to 10. 9. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, where n is a decimal or integer number from 4 to 8. 10. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any one of claims 1-9, wherein Y is selected from the group consisting of: where the O-terminus of Y is attached to the linker fragment -L-. 11. A ligand-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of claims 1-10, wherein the linker unit -L- is -L ¹ -L ² -L ³ -L ⁴ -, where L ¹ is selected from the group consisting of -(succinimidyl-3-yl-N)-W-C(O)-, where W is selected from the group consisting of C _1-8 alkyl, C _1-8 alkyl-C _3-6 cycloalkyl; L ² is selected from the group consisting of -NR ⁴ (CH ₂ CH ₂ O) pCH ₂ CH ₂ C(O)- and a chemical bond, where p is an integer from 1 to 20; L ³ is a peptide residue consisting of 2-7 amino acids, wherein the amino acids are selected from the group consisting of amino acid residues formed from the amino acids phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid; L ⁴ is selected from the group consisting of -NR ⁵ (CR ⁶ R ⁷ ) _t -, where t is an integer from 1 to 6; R ⁴ and R ⁵ represent hydrogen; R ⁶ and R ⁷ represent hydrogen. 12. A ligand-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of claims 1-11, wherein the linker unit -L- is -L ¹ -L ² -L ³ -L ⁴ -, where L ¹ is and s ¹ is an integer from 2 to 8; L ² is a chemical bond; L ³ is a tetrapeptide residue; L ⁴ is -NR ⁵ (CR ⁶ R ⁷ )t-, where R ⁵ , R ⁶ and R ⁷ are hydrogen and t is 1 or 2; where the L ¹ end is attached to Pc and the L ⁴ end is attached to Y. 13. A ligand-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of claims 1-12, wherein L ³ is a residue of the tetrapeptide GGFG. 14. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any one of claims 1-13, where -L- is: 15. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any one of claims 1-14, where -L-Y- is optionally selected from the group consisting of: 16. A ligand-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of claims 1-11, wherein the ligand-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof is a ligand-drug conjugate of the general formula (Pc-L _a -YD) or a pharmaceutically acceptable salt thereof, where Pc, n, m, W, ^L2 , ^L3 , ^R1 , ^R2 , ^R5 , ^R6 and ^R7 are as defined in any of paragraphs 1-11. 17. A ligand-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of claims 1-12 and 16, which is a ligand-drug conjugate of the general formula (Pc-L _b -YD) or a pharmaceutically acceptable salt thereof, Where: s ¹ is an integer between 2 and 8; Rc, R ¹ , R ² , R ⁵ -R ⁷ , m and n are as defined in any of paragraphs 1-12. 18. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any one of claims 1-17, which is selected from the group consisting of: where Pc and n are as defined in any of paragraphs 1-17. 19. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any one of claims 1-18, which is: Where: n is a decimal or integer number from 1 to 8; RS is an antibody to TROP-2 comprising the heavy chain shown in SEQ ID NO: 13 and the light chain shown in SEQ ID NO: 14. 20. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any one of claims 1-19, where n is a decimal or integer number from 4 to 8. 21. A ligand-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any one of claims 1-20, where n is a decimal or integer number from 4 to 6. 22. An antibody to TROP-2 or an antigen-binding fragment thereof, wherein the variable region of the heavy chain comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, respectively, and the variable region of the light chain comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively. 23. An antibody to TROP-2 or an antigen-binding fragment thereof according to claim 22, which is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. 24. An antibody to TROP-2 or an antigen-binding fragment thereof according to claim 22 or 23, comprising a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region has the amino acid sequence presented in SEQ ID NO: 3 or has at least 90% identity thereto, and the light chain variable region has the amino acid sequence presented in SEQ ID NO: 4 or has at least 90% identity thereto. 25. An antibody to TROP-2 or an antigen-binding fragment thereof according to claims 22-24, which comprise a heavy chain constant region and a light chain constant region of an antibody; wherein the heavy chain constant region is selected from the group consisting of the constant regions of human IgG1, IgG2, IgG3 and IgG4, and the light chain constant region is selected from the group consisting of the constant regions of the κ and λ chains of a human antibody. 26. An antibody to TROP-2 or an antigen-binding fragment thereof according to claims 22-25, wherein the antibody to TROP-2 comprises a heavy chain constant region as presented in SEQ ID NO: 11 and a light chain constant region as presented in SEQ ID NO: 12. 27. An antibody to TROP-2 or an antigen-binding fragment thereof according to claims 22-26, which comprises a heavy chain as presented in SEQ ID NO: 13 and a light chain as presented in SEQ ID NO: 14. 28. A nucleic acid molecule encoding an antibody to TROP-2 or an antigen-binding fragment thereof according to any one of claims 22-27. 29. A host cell containing a nucleic acid molecule according to claim 28. 30. A method for producing a ligand-drug conjugate of the general formula (Pc-L _a -YD) or a pharmaceutically acceptable salt thereof, comprising the following steps: subjecting the reduced Pc to a coupling reaction with a compound of the general formula (L _a -YD) to obtain a compound of the general formula (Pc-L _a -YD); Where: RS is an antibody to TROP-2 or an antigen-binding fragment thereof according to any one of claims 22-27; n, m, W, L ² , L ³ , R ¹ , R ² , R ⁵ , R ⁶ and R ⁷ are as defined in paragraph 16. 31. A pharmaceutical composition for the treatment of a disease or condition mediated by TROP-2, comprising: a therapeutically effective dose of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-21, or an anti-TROP-2 antibody or antigen-binding fragment thereof according to any one of claims 22-27, and one or more pharmaceutically acceptable excipients, diluents or carriers. 32. Use of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-21, an antibody to TROP-2 or an antigen-binding fragment thereof according to any one of claims 22-27, or a pharmaceutical composition according to claim 31 for the preparation of a medicament for the treatment of a disease or condition mediated by TROP-2. 33. The use of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 21, an antibody to TROP-2 or an antigen-binding fragment thereof according to any one of claims 22 to 27, or a pharmaceutical composition according to claim 31 for the treatment of tumors, wherein the tumors are head and neck cancer, neuroglioma, esophageal cancer, thyroid cancer, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, rectal cancer, kidney cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer or urothelial carcinoma. 34. Use of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 21, an antibody to TROP-2 or an antigen-binding fragment thereof according to any one of claims 22 to 27, or a pharmaceutical composition according to claim 31 for the preparation of a medicament for the treatment and/or prevention of tumors and cancer, where the tumors and cancer are head and neck cancer, neuroglioma, esophageal cancer, thyroid cancer, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, gastric cancer, rectal cancer, kidney cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer or urothelial carcinoma. 35. The use according to claim 34, wherein the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer, and the head and neck cancer is squamous cell carcinoma of the head and neck.