HK40082496B - Anti-psma antibody-exatecan analogue conjugate and medical use thereof - Google Patents

Description

Anti-PSMA antibody-ecetane analogue conjugate and its pharmaceutical uses

This application claims priority to Chinese patent application filed on March 25, 2020 (application number CN202010218297.4).

Technical Field

This disclosure relates to anti-PSMA antibodies, anti-PSMA antibody-eczema analog conjugates, methods of their preparation, pharmaceutical compositions comprising the same, and their use in the preparation of medicaments for treating PSMA-mediated diseases or conditions; particularly in the preparation of anticancer medicaments.

Background Technology

The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

According to statistics from the American Cancer Society in 2019, prostate cancer ranked first among new cancer cases in men in the United States, with 174,650 cases (CA CANCER J CLIN 2019; 69: 7-34). For localized clinical disease, surgery and radiation therapy are usually chosen. For locally advanced or metastatic disease, androgen deprivation (ADT) therapy is usually used first, either surgically or chemically. For early-stage castration-resistant PC, Sipuleucel-T-cell immunotherapy can be used. For patients in the metastatic castration-resistant stage (mCRPC), androgen inhibitors, androgen receptor antagonists, radiotherapy drugs targeting bone metastases, and chemotherapy drugs acting on microtubules can be used as appropriate. However, each treatment can only prolong survival by a few months, and it is necessary to seek effective treatment methods (European Urology, 66(6), 1190-1193; Journal of Nuclear Medicine, 59(2), 177-182.).

Prostate-specific membrane antigen (PSMA) is expressed not only by prostate epithelial cells but also by non-prostate tissues such as the small intestine, proximal renal tubules, and salivary glands, but at levels far lower than in prostate tissue. PSMA is highly expressed in prostate cancer cells, especially in metastatic, hormone-resistant, and high-grade lesions. In addition, PSMA is also highly expressed in the endothelial cells of angiogenesis in all solid tumors, but it is not expressed in normal blood vessels, so it has also been used as a target for solid tumor therapy (Clinical Cancer Research, Vol. 3, 81-85, January 1997; Urologic Oncology: Seminars and Original Investigations, 1(1), 18-28.; Clin Cancer Res. 2010 November 15; 16(22): 5414-5423.). Both androgen deprivation and androgen receptor antagonist therapy can upregulate the expression of prostate-specific membrane antigen (PSMA) (J Nucl Med 2017; 58: 81-84), which provides a basis for the combined treatment of targeted therapy and traditional hormone therapy.

PSMA belongs to glutamate carboxypeptidase II (GCPII). In the nervous system, it acts as NAALDase, enzymatically hydrolyzing NAAG to obtain glutamate, and is involved in glutamate neurotransmission. In the small intestine, it acts as FOLH1, breaking down folyl-poly-γ-glutamate (FPGn) to obtain folic acid, and is involved in folic acid metabolism (Curr Med Chem. 2012; 19(6): 856-870). In the prostate, it is expressed as PSMA on epithelial cells and is associated with the occurrence of prostate cancer. PSMA consists of 750 amino acids, with 19 intracellular, 24 transmembrane, and 707 extracellular. Crystallization results show that the extracellular portion consists of three domains: a protease-like domain, a apical domain, and a C-terminal domain. All three participate in substrate binding. The first two bind directly to the substrate, while the C-terminal domain plays a role in the formation of PSMA dimers (The EMBO Journal (2006) 25, 1375-1384). Research on glutamate type II carboxypeptidase (GCPII) and its splice variants and paralogs is limited. Splice variants, represented by PSM', are mostly found intracellularly in normal prostate tissue cells. The most thoroughly studied GCPII homologs, GCPIII or NAALADase II, serve as effective drug targets, compensating for the insufficient enzymatic activity of normal GCPII, and share 68% sequence similarity with GCPII (Current Medicinal Chemistry, 2012, 19, 1316-1322; Frontiers in Bioscience, Landmark, 24, 648-687, March 1, 2019).

Antibody-drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxic agents via adaptor compounds. This fully leverages the specificity of antibodies in binding to antigens on the surface of normal and tumor cells, as well as the high efficiency of cytotoxic agents, while avoiding the drawbacks of low efficacy of antibodies and excessive toxic side effects of cytotoxic agents. This means that, compared to traditional chemotherapy drugs, antibody-drug conjugates can more precisely kill tumor cells and reduce the impact on normal cells.

Currently, several ADC drugs are used in clinical trials or research, such as Kadcyla, an ADC drug formed by trastuzumab targeting Her2 and DM1. Meanwhile, there are also ADC drugs targeting PSMA used in clinical treatment research. Cytogen's PSMA-ADC is in Phase II clinical trials, while MedImmune's MEDI-3726 and BZL Biologics Inc.'s MLN-2704 were discontinued after Phase I clinical trials due to poor efficacy. Developing new ADC drugs using different strategies shows great promise.

Summary of the Invention

This disclosure relates to anti-PSMA antibody-ADCs and their uses, providing ADC drugs conjugated with an anti-PSMA antibody or antigen-binding fragment and a cytotoxic substance eczema analogue.

This disclosure provides an antibody-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof:

in:

Y is selected from -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)-;

^Ra and ^Rb may be the same or different, and each is independently selected from hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuteralkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl and heterocyclic group; or ^Ra and ^Rb together with the carbon atom they are attached to form cycloalkyl or heterocyclic group;

^R1 is selected from halogens, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl, and heteroaryl; ^R2 is selected from hydrogen atoms, halogens, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl, and heteroaryl; or, ^R1 and ^R2 together with the carbon atom they are attached to form a cycloalkyl or heterocyclic group;

Alternatively, ^Ra and ^R2 together with the carbon atom attached to them form cycloalkyl or heterocyclic groups;

m is an integer from 0 to 4; non-restrictive examples include m selected from 0, 1, 2, 3, and 4;

n is 1 to 10, where n is a decimal or an integer; preferably, n is 1-8, more preferably 3-8, most preferably 3-7, and even more preferably 6-7.

L represents the connector unit;

Pc is an anti-PSMA antibody or its antigen-binding fragment, preferably, the anti-PSMA antibody or its antigen-binding fragment specifically binds to the PSMA extracellular domain.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the previously described general formula (Pc-L-Y-D), wherein the anti-PSMA antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 with the same sequence as the heavy chain variable region shown in SEQ ID NO: 1, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 with the same sequence as the light chain variable region shown in SEQ ID NO: 2.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the previously described general formula (Pc-L-Y-D), wherein the anti-PSMA antibody or its antigen-binding fragment 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: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the previously described general formula (Pc-L-Y-D), is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the previously described general formula (Pc-L-Y-D), wherein the anti-PSMA antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1 or has at least 90%-100% sequence identity with it, 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% sequence identity; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 2 or has at least 90%-100% sequence identity with it, 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% sequence identity.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the previously described general formula (Pc-L-Y-D), wherein the anti-PSMA antibody or its antigen-binding fragment comprises a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO: 2.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the previously described general formula (Pc-L-Y-D), wherein the anti-PSMA antibody comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and their conventional variants, and the light chain constant region is selected from the constant regions of human antibody κ and λ chains and their conventional variants.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the previously described general formula (Pc-L-Y-D), wherein the anti-PSMA antibody comprises a heavy chain as shown in SEQ ID NO: 9 and a light chain as shown in SEQ ID NO: 10.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the previously described general formula (Pc-L-Y-D), is used, wherein n is 1 to 8, preferably 3-8, more preferably 3-7, and n is a decimal or an integer.

In some embodiments, antibody-drug conjugates or pharmaceutically acceptable salts thereof, as shown in the previously described general formula (Pc-L-Y-D),

in:

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

^Ra and ^Rb may be the same or different, and each is independently selected from hydrogen atoms, deuterium atoms, halogens and _C1-6 alkyl groups;

^R1 is a haloalkyl or _C3-6 cycloalkyl;

^R2 is selected from hydrogen atoms, haloalkyl groups, and _C3-6 cycloalkyl groups;

Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-6 cycloalkyl group;

m is 0 or 1.

In some embodiments, the antibody-drug conjugate, as shown in the previously described general formula (Pc-L-Y-D), or a pharmaceutically acceptable salt thereof, wherein Y is selected from:

The O end of Y is connected to the connector unit L.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the previously described general formula (Pc-LYD), wherein the linker unit -L- is ^{-L1 -L2 -L3 -L4} -,

^L1 is selected from -(succinimide-3-yl-N)-WC(O)-, _-CH2 -C(O) ^-NR3- WC(O)-, or -C(O)-WC(O)-, wherein W is selected from _C1-8 alkyl, _C1-8 alkyl-cycloalkyl, and straight-chain heteroalkyl of 1 to 8 atoms, wherein the heteroalkyl contains 1 to 3 heteroatoms selected from N, O, and S, wherein the _C1-8 alkylene, _C1-8 alkyl-cycloalkyl, and straight-chain heteroalkyl are each optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteryl, alkoxy, and cycloalkyl;

^L2 is selected from ^-NR4 ( _CH2CH2O ) _p1CH2CH2C (O)-, ^-NR4 ( _{CH2CH2O)p1CH2C (} O)-, ^-S ( _CH2 ) _p2C (O)- and chemical ^bonds , where ^p1 is an integer from ¹ _{to 20} ;

^L3 is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid is selected from amino acids formed from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E) and aspartic acid (D), and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

^L4 is selected from ^-NR5 ( ^CR6R7 ) _t- , -C(O) ^NR5 , -C(O) ^NR5 ( _CH2 ) _t- and chemical bonds, where ^t is an integer from 1 to 6;

^R3 , ^R4 and ^R5 may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl, deuteralkyl and hydroxyalkyl;

^R6 and ^R7 may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the previously described general formula (Pc-LYD), wherein the linker unit -L- is ^{-L1 -L2 -L3 -L4} -,

^L1 is an integer from 2 to 8 for ^s1 ;

^L2 is a chemical bond;

^L3 is a tetrapeptide residue; preferably, ^L3 is a tetrapeptide residue of GGFG (SEQ ID NO: 13) (glycine-glycine-phenylalanine-glycine);

^L4 is ^-NR5 ( ^CR6R7 )t-, where ^R5 , ^R6 , or ^R7 may be the same or different, and each is independently a hydrogen atom or an alkyl group, and t is 1 or 2;

The ^L1 terminal is connected to Pc, and the ^L4 terminal is connected to Y.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the previously described general formula (Pc-L-Y-D), wherein -L- is:

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the previously described general formula (Pc-L-Y-D), wherein -L-Y- is optionally selected from:

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt represented by the previously described general formula (Pc-LYD) is an antibody-drug conjugate or its pharmaceutically acceptable salt represented by the general formula (Pc- _La -YD):

in:

W, ^L2 , ^L3 , ^R5 , ^R6 , and ^R7 are as defined in the previously described connector unit-L-.

Pc, n, ^R1 , ^R2 , m are as defined in the general formula (Pc-LYD);

Specifically, Pc is an anti-PSMA antibody or its antigen-binding fragment;

m is an integer from 0 to 4; non-restrictive examples include m selected from 0, 1, 2, 3, and 4;

n is 1 to 10, where n is a decimal or an integer; preferably, n is 1-8, more preferably 3-8, most preferably 3-7, and more preferably 6-7;

^R1 is selected from halogens, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl, and heteroaryl; ^R2 is selected from hydrogen atoms, halogens, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl, and heteroaryl; or, ^R1 and ^R2 together with the carbon atom they are attached to form a cycloalkyl or heterocyclic group;

W is selected from _C1-8 alkyl, _C1-8 alkyl-cycloalkyl, or straight-chain heteroalkyl with 1 to 8 atoms, wherein the heteroalkyl contains 1 to 3 heteroatoms selected from N, O, or S, wherein each of the _C1-8 alkyl, cycloalkyl, and straight-chain heteroalkyl is optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy, and cycloalkyl;

^L2 is selected from ^-NR4 ( _CH2CH2O ) _p1CH2CH2C (O)-, ^-NR4 ( _CH2CH2O ) _p1CH2C (O)-, ^-S _{( CH2} ) ^p1C (O)- and chemical ^bonds , where ^p1 is an integer from ₁ to ₂₀ ;

^L3 is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acids formed from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E), and aspartic acid (D), and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy, and cycloalkyl;

R ⁵ is selected from hydrogen atoms, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups;

^R6 and ^R7 may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups.

In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt represented by the general formula (Pc-LYD) as described in any of the preceding embodiments is an antibody-drug conjugate or its pharmaceutically acceptable salt represented by the general formula (Pc- _Lb -YD):

in:

^s1 is an integer from 2 to 8;

Pc, ^R1 , ^R2 , ^R5 , ^R6 , ^R7 , m and n are as defined in the general formula (Pc-L _a -YD).

In some embodiments, the antibody-drug conjugate represented by the general formula (Pc-L-Y-D) as described in any of the preceding embodiments, or a pharmaceutically acceptable salt thereof, is selected from:

Wherein Pc and n are as defined in the general formula (Pc-L-Y-D), specifically, Pc is an anti-PSMA antibody or its antigen-binding fragment, or an anti-PSMA antibody as described in any of the preceding items;

n is 1 to 10, where n is a decimal or an integer; preferably, n is 1-8, more preferably 3-8, most preferably 3-7; and most preferably 6-7.

In some embodiments, the antibody-drug conjugate, or a pharmaceutically acceptable salt thereof, representing the general formula (Pc-L-Y-D) as described in any of the preceding claims, is:

in:

n is 1 to 8, preferably 3-8, and n is a decimal or an integer;

PM is an anti-PSMA antibody that contains a heavy chain as shown in SEQ ID NO: 9 and a light chain as shown in SEQ ID NO: 10.

This disclosure further provides a method for preparing an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as shown in the general formula (Pc-La _- YD), comprising the following steps:

Pc' is the reduced Pc, which undergoes a coupling reaction with a compound of general formula (La _- YD) to obtain a compound of general formula (Pc- _La -YD).

in:

Pc is an anti-PSMA antibody or its antigen-binding fragment;

n, m, W, ^L2 , ^L3 , ^R1 , ^R2 , ^R5 , ^R6 and ^R7 are as defined in the aforementioned general formula (Pc-L _a -YD).

This disclosure further provides a method for preparing an antibody-drug conjugate as shown in the general formula (Pc-L’-D), comprising the following steps:

In the above reaction, Pc’ undergoes a coupling reaction with the compound represented by formula (L’-D) to obtain the compound represented by the general formula (Pc-L’-D); wherein:

Pc is the anti-PSMA antibody or its antigen-binding fragment as described above; Pc’ is the reduced Pc.

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

This disclosure further provides a method for preparing an antibody-drug conjugate as shown in PM-9-A, comprising the following steps:

PM' undergoes a coupling reaction with the compound represented by formula 9-A to give the compound represented by the general formula (PM-9-A); wherein:

n can be 1 to 8, preferably 3 to 8, and n can be a decimal or an integer;

PM is an anti-PSMA antibody, which contains a heavy chain with the sequence shown in SEQ ID NO: 9 and a light chain with the sequence shown in SEQ ID NO: 10;

PM' is obtained after reducing PM.

In some embodiments, n represents the drug loading of the antibody-drug conjugate, also known as the DAR value, which can be determined by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA, and HPLC. In some embodiments, n is an average of 0-10, preferably 1-10, more preferably 1-8, or 2-8, or 2-7, or 2-4, or 3-8, or 3-7, or 3-6, or 4-7, or 4-6, or 4-5. In some embodiments, n is an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

On the other hand, this disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in this disclosure, and one or more pharmaceutically acceptable excipients, diluents, or carriers. In some embodiments, a unit dose of the pharmaceutical composition contains 0.1 mg to 3000 mg or 1 mg to 1000 mg of the anti-PSMA antibody or the antibody-drug conjugate as described above.

On the other hand, this disclosure provides the use of the antibody-drug conjugate or its pharmaceutically acceptable salt or pharmaceutical composition comprising the present disclosure as a medicament.

On the other hand, this disclosure provides the use of the antibody-drug conjugate or its pharmaceutically acceptable salt or pharmaceutical composition comprising the present disclosure in the preparation of a medicament for treating PSMA-mediated diseases or conditions, wherein the PSMA-mediated diseases or conditions are preferably PSMA-high, medium or low expression cancers.

On the other hand, this disclosure provides the use of antibody-drug conjugates or pharmaceutically acceptable salts thereof or pharmaceutical compositions comprising the present disclosure in the preparation of medicaments for treating or preventing cancer, wherein the cancer is preferably squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal 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, bone marrow cancer. The lymphoma is selected from: multiple myeloma, myelodysplastic syndrome, Kuckenberg tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, urothelial carcinoma, and Merkel cell carcinoma, preferably prostate cancer; more preferably, the lymphoma is selected from: Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells/histocytes, and lymphoplasmacytic lymphoma; the lung cancer is selected from: non-small cell lung cancer and small cell lung cancer; the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.

On the other hand, this disclosure further relates to a method for treating and/or preventing tumors, the method comprising administering to a subject in need a therapeutically effective dose of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the present disclosure; preferably wherein the tumor is a cancer associated with high PSMA expression, moderate PSMA expression, or low PSMA expression.

On the other hand, this disclosure further relates to a method for treating or preventing tumors or cancer, the method comprising administering to a subject in need a therapeutically effective dose of an antibody-drug conjugate according to this disclosure or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the conjugate thereof; wherein the tumors and cancers are preferably squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumors, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal 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. Lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Kuckenberg tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, urothelial carcinoma, and Merkel cell carcinoma; preferably prostate cancer; more preferably, the lymphoma is selected from: Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/histiocytic rich large B-cell lymphoma, and lymphoplasmacytic lymphoma; the lung cancer is selected from: non-small cell lung cancer and small cell lung cancer; the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia.

On the other hand, this disclosure further provides the aforementioned anti-PSMA antibody or antibody-drug conjugate thereof as a drug, preferably as a drug for treating cancer or tumors, and more preferably as a drug for treating PSMA-mediated cancers.

The active compound (e.g., a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to this disclosure) can be formulated in a form suitable for administration via any appropriate route, preferably in a unit dose manner or in a manner that allows the subject to self-administer a single dose. The unit dose manner of the active compound or composition described in this disclosure can be in the form of tablets, capsules, sachets, bottled liquids, powders, granules, lozenges, suppositories, regenerated powders, or liquid formulations.

The dosage of the active compound or composition used in the treatment methods disclosed herein will generally vary depending on the severity of the disease, the subject's weight, and the relative efficacy of the active compound. As a general guideline, a suitable unit dose may be 0.1 mg to 1000 mg.

In addition to the active compound, the pharmaceutical compositions disclosed herein may contain one or more excipients selected from the following: fillers, diluents, binders, wetting agents, disintegrants, or excipients. Depending on the method of administration, the composition may contain 0.1 to 99% by weight of the active compound.

The PSMA antibody and antibody-drug conjugate disclosed herein have good affinity for cell surface antigens, good endocytosis efficiency and strong tumor inhibition efficiency, and have a wider drug application window, higher tumor inhibition effect and therapeutic activity, better safety, pharmacokinetic properties and drugability (such as stability), making them more suitable for clinical drug application.

Attached Figure Description

Figure 1A: In vitro binding ability of the ADC or antibody of this disclosure to cellular MDAPCa.

Figure 1B: In vitro binding ability of the ADC or antibody of this disclosure to cellular LNCaP.

Figure 1C: In vitro binding ability of the ADC or antibody of this disclosure to cell 22Rv1.

Figure 1D: The in vitro binding ability of the ADC or antibody of this disclosure to PC-3 cells.

Figure 1E: In vitro binding ability of the ADC or antibody of this disclosure to DU 145 cells.

Figure 2A: In vitro endocytosis experiment of antibody PM in LNCaP cells; 2K cells, 10% U-L IgG FBS.

Figure 2B: In vitro endocytosis of antibody PM in 22Rv1 cells; 2K cells, 10% U-L IgG FBS.

Figure 3A: Killing effects of different ADCs of this disclosure on LNCaP cells, using 2K cells, 4.5% FBS.

Figure 3B: Killing effect of the toxin (compound 2-B) of this disclosure on LNCaP cells, using 2K cells, 4.5% FBS.

Figure 3C: Killing effect of different ADCs of this disclosure on 22Rv1 cells, using 4K cells, 4.5% FBS.

Figure 3D: Killing effect of the toxin (compound 2-B) of this disclosure on 22Rv1 cells, using 4K cells, 4.5% FBS.

Figure 3E: Killing effect of different ADCs of this disclosure on PC-3 cells, using 4K cells, 4.5% FBS.

Figure 3F: Killing effect of the toxin (compound 2-B) of this disclosure on PC-3 cells, using 2K cells, 4.5% FBS.

Figure 4A: Inhibitory activity of different doses of ADC on human prostate cancer cell 22Rv1 xenografts in nude mice.

Figure 4B: Changes in mouse body weight during the test of the inhibitory activity of different doses of ADC on human prostate cancer cell xenografts in nude mice.

Figure 5A: Inhibitory activity of different doses of ADC on human prostate cancer cell 22Rv1 xenografts in nude mice.

Figure 5B: Changes in mouse body weight during the test of the inhibitory activity of different doses of ADC on human prostate cancer cell 22Rv1 xenografts in nude mice.

Figure 6A: Inhibitory activity of different doses of ADC on human prostate cancer LNCap xenografts in SCID Beighe mice.

Figure 6B: Changes in body weight of mice during the test of the inhibitory activity of different doses of ADC on human prostate cancer LNCap xenografts in SCID Beighe mice.

Figure 7: Pharmacokinetic stability of ADC-2 disclosed herein, wherein the concentration of ADC is 100 μg/ml.

Figure 8: Plasma stability of ADC-5 of this disclosure, wherein the concentration of ADC is 100 μg/ml.

Figure 9: Efficacy of ADC administration on 22Rv1 xenografts in tumor-bearing nude mice.

Detailed Implementation

the term

Unless otherwise specified, all technical and scientific terms used herein are consistent with the common understanding of one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used to practice or test this disclosure, preferred methods and materials are described herein. In describing and claiming protection for this disclosure, the following terms are used in accordance with the definitions below.

When a trade name is used in this disclosure, it is intended to include the formulation of the product under that trade name, the pharmaceutical product under that trade name, and the active pharmaceutical ingredient portion thereof.

Unless otherwise stated, the terms used in the specification and claims have the following meanings.

The term "drug" or "toxin" refers to cytotoxic drugs, which are chemical molecules that strongly disrupt the normal growth of tumor cells. In principle, cytotoxic drugs can kill cells at sufficiently high concentrations; however, due to their lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, leading to serious side effects. This term includes small molecule toxins or enzyme-active toxins from bacterial, fungal, plant, or animal sources; radioactive isotopes (e.g., At ^-211 , I ^-131 , I ^-125 , Y ^-90 , Re ^-186 , Re ^-188 , Sm ^-153 , Bi- ²¹² , P- ³² , and Lu radioactive isotopes); chemotherapeutic agents; antibiotics; and ribolysins.

The terms “connector unit”, “connector”, “linking unit”, or “linking fragment” refer to a chemical structural fragment or bond that is linked at one end to a ligand (antibody in this disclosure) and at the other end to a drug. It may also be linked to other connectors before being linked to a ligand or drug.

The linker may comprise one or more linker elements. Exemplary linker elements include 6-maleiminohexanoyl (“MC”), maleiminopropionyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimino-4-(2-pyridylthio)valerate (“SPP”), N-succinimino-4-(N-maleiminomethyl)cyclohexane-1-carboxylate (“SMCC”, also referred to herein as “MCC”), and N-succinimino-4-iodo-acetyl)aminobenzoate (“SIAB”). The linker may comprise one or more, or combinations thereof, of the following elements: extensions, spacers, and amino acid units, synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker may be a “cleavable linker” that facilitates the release of a drug into cells. For example, acid-labile adapters (e.g., hydrazone), protease-sensitive (e.g., peptidase-sensitive) adapters, light-labile adapters, dimethyl adapters, or disulfide-containing adapters can be used (Chari et al., Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020).

Connector components include, but are not limited to:

MC = 6-maleiminohexanoyl, with the following structure:

Val-Cit or “vc” = valine-citrulline (an example dipeptide in a protease-cleavable linker);

Citrulline = 2-amino-5-ureaporic acid;

PAB = p-aminobenzyloxycarbonyl (an example of a "self-sacrificing" connector element);

Me-Val-Cit = N-methyl-valine-citrulline (where the linker peptide bond has been modified to prevent it from being cleaved by cathepsin B);

MC(PEG)6-OH = Maleiminohexanoyl-polyethylene glycol (can attach to antibody cysteine);

SPP = N-succinimino-4-(2-pyridylthio)valerate;

SPDP = N-succinimino-3-(2-pyridyl dithio)propionate;

SMCC = succinimino-4-(N-maleiminomethyl)cyclohexane-1-carboxylic acid ester;

IT = iminothione.

IT = iminothione.

The term "antibody-drug conjugate" refers to an antibody linked to a biologically active drug via a linker unit. In this disclosure, "antibody-drug conjugate" (ADC) refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug via a linker unit. The antibody can be directly or via a linker to the drug. n is the average number of drug modules per antibody, which can be an integer or a decimal, and its range can be: for example, about 0 to about 20 drug modules per antibody, in some embodiments 1 to about 10 drug modules per antibody, in some embodiments 1 to about 8 drug modules per antibody, such as 2, 3, 4, 5, 6, 7, or 8 drug modules. The antibody-drug conjugate mixtures of this disclosure, wherein the average drug load per antibody is about 1 to about 10, including but not limited to about 3 to about 7, about 3 to about 6, about 3 to about 5, about 1 to about 9, about 7, or about 4.

The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

The term "antibody" refers to immunoglobulin. A complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins are classified into five classes, or isotypes, based on the amino acid composition and sequence of their heavy chain constant regions: IgM, IgD, IgG, IgA, and IgE, with corresponding heavy chains of μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further lead to subclasses; 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 their constant regions. Each of the five classes of Ig can possess either a κ or λ chain.

The sequence of approximately 110 amino acids near the N-terminus of both the heavy and light chains of a full-length antibody varies considerably and is known as the variable region (Fv region). The remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). The three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

The terms "fully human antibody," "fully human antibody," "human antibody," or "fully human monoclonal antibody" refer to antibodies whose variable and constant regions are both human-derived, eliminating immunogenicity and toxicity. Related technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology.

The term "antigen-binding fragment" refers to one or more segments of an antibody that maintain its ability to specifically bind to antigens. Fragments of full-length antibodies can be used to perform the antigen-binding function of antibodies. The binding fragments included in the “antigen-binding fragment” are selected from Fab, Fab′, F(ab′)2, single-chain antibody (scFv), dimerized V region (biantibody), disulfide bond-stabilized V region (dsFv), and antigen-binding fragments of peptides containing CDRs. Examples include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab′) ₂ fragments, bivalent fragments consisting of two Fab fragments connected by disulfide bridges on hinge regions; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains of a single arm of an antibody; (v) single-domain or dAb fragments (Ward et al., (1989) Nature 341: 544-546) consisting of VH domains; and (vi) separate complementarity-determining regions (CDRs) or (vii) combinations of two or more separate CDRs optionally connected by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by synthetic linkers using recombinant methods, thereby enabling the production of a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as a single-chain Fv (scFv); see, for example, 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 in the term "antigen-binding fragment" of antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

Typically, Fab is an antibody fragment with a molecular weight of about 50,000 and antigen-binding activity obtained by treating an IgG antibody molecule with the protease papain (e.g., cleaving amino acid residue at position 224 of the H chain), wherein the N-terminal portion of the H chain and the L chain are linked together by disulfide bonds.

Typically, F(ab′)2 is obtained by digesting the portion below the disulfide bond in the hinge region of IgG with the enzyme pepsin. It is an antibody fragment with a molecular weight of approximately 100,000, possessing antigen-binding activity, and containing two Fab regions connected at the hinge position.

Typically, Fab′ is an antibody fragment with a molecular weight of approximately 50,000 and possessing antigen-binding activity, obtained by cleaving the disulfide bonds in the hinge region of the aforementioned F(ab′)2.

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

The terms “single-chain antibody,” “single-chain Fv,” or “scFv” refer to molecules containing a variable domain (or VH) of the antibody heavy chain and a variable domain (or VL) of the antibody light chain linked by a linker. Such scFv molecules may have a general structure: NH₂ _- VL-linker-VH-COOH or NH₂ _- VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example, using variants with 1–4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444–6448). Other connectors that may be used in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immuno 1.31: 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 the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDRI, LCDR2, LCDR3) in each light chain variable region. The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (see Al-Lazikani et al., (1997) JMB 273: 927-948), and the ImMunoGenTics (IMGT) numbering rule (Lefranc M.P., Immunologist, 7, 132-136 (1999)). Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003), etc. For example, for the classical format, following Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following Chothia rules, the CDR amino acids 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 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 95-102 (HCDR3). The CDRs are defined by combining the CDR definitions from Kabat and Chothia. The CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) from human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) from human VL. Following IMGT rules, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), while those in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). Following IMGT rules, the CDR region of an antibody can be defined using the IMGT/DomainG protocol. apAlign is used to determine the CDRs. Unless otherwise specified, the six CDRs described in this disclosure are obtained according to the Kabat numbering rules ((Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242)). According to the Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Other numbering rules in the art include Chothia, IMGT, etc.

The term "antibody framework region" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

The term "epitope" or "antigenic determinant" refers to a site on an antigen that is bound by an immunoglobulin or antibody. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE. Morris, Ed. (1996).

The terms "specific binding,""selectivebinding,""selectivebinding," and "specific binding" refer to the binding of an antibody or antigen-binding fragment to a pre-defined epitope on an antigen. Typically, antibody or antigen-binding fragments bind with an affinity (KD) of approximately less than ^10⁻⁷ M, such as approximately less than ^10⁻⁸ M, ^10⁻⁹ M, or ^10⁻¹⁰ M or less.

The term "KD" refers to the dissociation equilibrium constant of antibody-antigen interactions. Typically, the antibody or antigen-binding fragments of this disclosure bind to PSMA or its epitopes with a dissociation equilibrium constant (KD) of less than about ^10⁻⁷ M, for example less than about ^10⁻⁸ M or ^10⁻⁹ M. For example, in this disclosure, the affinity of the antibody for cell surface antigens is determined using the FACS method to determine the KD value.

The term "nucleic acid molecule" refers to either a DNA molecule or an RNA molecule. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

Amino acid sequence “sequence identity” refers to the percentage of amino acid residues in a first sequence that are identical to those in a second sequence during amino acid sequence alignment, where gaps are introduced where necessary to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be performed in a variety of ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

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 DNA loop to which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells that have been introduced into them (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-episodic mammalian vectors).

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include microorganisms (such as bacteria), plant cells, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, 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 NSO cells.

The engineered antibody or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or GSepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

The term "peptide" refers to a molecule composed of two or more amino acid molecules linked together by peptide bonds; it is a structural and functional segment of a protein.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms (including 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 their various branched isomers, etc. More preferably, lower alkyl groups containing 1 to 6 carbon atoms are used. 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, etc. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any usable connection point. The substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

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

The term "alkylene" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. It is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, more preferably containing 1 to 6 carbon atoms (including 1, 2, 3, 4, 5 or 6 carbon atoms). Non-limiting examples of alkylene groups include, but are not limited to, methylene ( _-CH2- ), 1,1-ethylene (-CH( _{CH3 )} -), 1,2-ethylene ( _{-CH2CH2 )} -, 1,1-propylene (-CH( _CH2CH3 )-), 1,2-propylene ( _{-CH2CH ( CH3} )-), 1,3-propylene ( _{-CH2CH2CH2- )} , _1,4 -butylene _{( -CH2CH2CH2CH2-} ) _, and _{1,5 - butylene ( -CH2CH2CH2CH2CH2-} ). The alkylene group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably independently selected independently from one or more substituents chosen from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

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

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 8 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

The cycloalkyl ring includes cycloalkyl groups (including monocyclic, spirocyclic, fused, and bridged rings) fused to an aryl, heteroaryl, or heterocyclic alkyl ring as described above, wherein the ring connected to the parent structure is a cycloalkyl group. Non-limiting examples include indanyl, tetrahydronaphthyl, and benzocycloheptyl, etc.; preferably phenylcyclopentyl and tetrahydronaphthyl.

The cycloalkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably independently selected independently from one or more substituents chosen from hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent comprising 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) _P (where p is an integer from 0 to 2), but excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon. Preferably, it comprises 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it comprises 3 to 8 ring atoms, of which 1 to 3 are heteroatoms; even more preferably, it comprises 3 to 6 ring atoms, of which 1 to 3 are heteroatoms; most preferably, it comprises 5 or 6 ring atoms, of which 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, tetrahydropyranyl, 1,2,3,6-tetrahydropyridyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups.

The heterocyclic ring comprises a heterocyclic group (including monocyclic, spirocyclic, fused heterocyclic, and bridged heterocyclic rings) fused to an aryl, heteroaryl, or cycloalkyl ring as described above, wherein the ring connected to the parent structure is a heterocyclic group, and non-limiting examples include:

The heterocyclic group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably independently selected independently from one or more substituents chosen from hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. The aryl ring comprises an aryl ring fused to a heteroaryl, heterocyclic, or cycloalkyl ring as described above, wherein the ring connected to the parent structure is an aryl ring, and non-limiting examples include:

The aryl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably independently selected independently from one or more substituents chosen from hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 10-membered, more preferably 5- or 6-membered, such as furanyl, thiophene, pyridinyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, etc. The heteroaryl ring comprises a heteroaryl group fused to an aryl, heterocyclic, or cycloalkyl ring as described above, wherein the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples include:

The heteroaryl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably independently selected independently from one or more substituents chosen from hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl and heteroaryl.

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

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

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

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

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

The term "amino" refers to _-NH2 .

The term "nitro" refers to _-NO2 .

The term "cyano" refers to -CN.

"Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the possibility or absence of the event or environment. For example, "optionally alkyl-substituted heterocyclic group" means that the alkyl group may but does not have to be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

"Substituted" refers to one or more hydrogen atoms in a group, preferably up to five, more preferably one, two, or three hydrogen atoms that are independently substituted by a substituent. Substituents are only considered in their possible chemical positions, and those skilled in the art can determine (experimentally or theoretically) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

The term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein or their physiologically/pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically/pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

The term "pharmaceutically acceptable salt" or "medicinal salt" refers to the salts of the antibody-drug conjugates disclosed herein, which are safe and effective when used in subjects and have the intended biological activity. The antibody-drug conjugates disclosed herein contain at least one amino group and can therefore form salts with acids. Non-limiting examples of medicinal salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

"Drug loading" or "average drug load," also known as drug-to-antibody ratio (DAR), refers to the average number of drugs conjugated to each antibody in an ADC. It can range from about 1 to about 10 drugs per antibody, and in some embodiments, from about 1 to about 8 drugs per antibody, preferably from the ranges of 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8, and 6-8. Exemplarily, the drug loading can be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The general formula of the ADC disclosed herein includes a set of antibody-drug conjugates within the aforementioned range of drug loading. In embodiments of this disclosure, the drug loading is expressed as n, also referred to as the DAR value, exemplarily an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Drug loading can be determined using conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA, and HPLC.

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

(1) Control the molar ratio of the ligation reagent and the monoclonal antibody.

(2) Control the reaction time and temperature.

(3) Choose different reaction reagents.

For the preparation of conventional pharmaceutical compositions, please refer to the Chinese Pharmacopoeia.

The term "carrier" is used in the context of the drugs disclosed herein, referring to a system that can alter the way a drug enters the body and its distribution within the body, control the rate of drug release, and deliver the drug to a target organ. Drug carrier release and targeting systems can reduce drug degradation and loss, decrease side effects, and improve bioavailability. For example, polymeric surfactants, due to their unique amphiphilic structure, can self-assemble to form various forms of aggregates, preferred examples being micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules while also exhibiting good membrane permeability, making them excellent drug carriers.

The term "excipient" refers to any additive in a pharmaceutical preparation other than the active pharmaceutical ingredient (API). Examples of excipients include binders, fillers, disintegrants, and lubricants in tablets; the base portion in semi-solid preparations such as ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solvents, osmotic pressure regulators, and colorants in liquid preparations.

The term "diluent," also known as a filler, is primarily used to increase the weight and volume of tablets. The addition of diluents not only ensures a specific volume but also reduces dosage deviations of the main components and improves the compressibility of the drug. When the tablet contains oily components, absorbents are added to absorb the oil and maintain a "dry" state, facilitating tablet formation. Examples of absorbents include starch, lactose, inorganic salts of calcium, and microcrystalline cellulose.

The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Water, Ringer's solution, and isotonic sodium chloride solution may be used as acceptable solvents and media. The sterile injectable formulation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then treated with a mixture of water and glycerol to form a microemulsion. The injection solution or microemulsion can be injected into the bloodstream of the subject by local large-volume injection. Alternatively, the solution and microemulsion are preferably administered in a manner that maintains a constant circulating concentration of the compound disclosed herein. To maintain such a constant concentration, a continuous intravenous delivery device may be used. An example of such a device is the Deltec CADD-PLUS™ 5400 intravenous infusion pump.

The pharmaceutical composition may be in the form of a sterile injectable aqueous or oil suspension for intramuscular and subcutaneous administration. This suspension may be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. The sterile injectable formulation may also be a sterile injectable solution or suspension prepared in a non-toxic, parenteral-acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol. Furthermore, a sterile fixative oil may be conveniently used as a solvent or suspension medium. For this purpose, any blended fixative oil, including synthetic mono- or diglycerides of glycerol, may be used. Additionally, fatty acids such as oleic acid may also be used to prepare the injectable formulation.

Synthesis method

To achieve the synthesis objective, the following synthesis technique was adopted:

A method for preparing the compound represented by general formula (PM-9-A) includes the following steps:

PM is reduced to give PM', and PM' is coupled with general formula (9-A) to give the compound shown in general formula (PM-9-A); the reducing agent is preferably TCEP, and in particular, the reducing agent is preferably the reducing agent for reducing the disulfide bonds on the antibody;

in:

PM is an anti-PSMA antibody or its antigen-binding fragment;

n is between 1 and 10, and n can be a decimal or an integer.

Details of one or more embodiments of this disclosure are set forth in the foregoing description. While any methods and materials similar to or the same as those described herein may be used to practice or test this disclosure, preferred methods and materials are described below. Other features, objects, and advantages of this disclosure will be apparent from the description and claims. In the description and claims, the singular form includes plural references unless clearly indicated otherwise in the context. Unless otherwise defined, all technical and scientific terms used herein have their general meaning as understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications referenced in the description are incorporated herein by reference. The following embodiments are presented to illustrate preferred embodiments of this disclosure more fully. These embodiments should not be construed in any way as limiting the scope of this disclosure, which is defined by the claims.

Experimental methods not specifically described in the embodiments or test examples of this disclosure are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. See Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory; Methods of Modern Molecular Biology, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. Reagents not specifically named are commercially available, conventional reagents.

Example 1. Preparation of Antibody

(I) Preparation of PSMA Antibody

1.1 Antibody Sequence

The antibody disclosed herein was prepared according to WO2003034903A2, wherein the amino acid sequence of the variable region of AB-PG1-XG1-006 is as follows:

AB-PG1-XG1-006 Heavy Chain Variable Region:

AB-PG1-XG1-006 Light Chain Variable Region:

Note: The underlined part is the CDR area determined according to the Kabat numbering rules.

Table 1. CDR region of AB-PG1-XG1-006 antibody

Antibody AB-PG1-XG1-006 Heavy chain CDR1 RYGMH (SEQ ID NO: 3) Heavy chain CDR2 VIWYDGSNKYYADSVKG(SEQ ID NO：4) Heavy chain CDR3 GGDFLYYYYYGMDV(SEQ ID NO: 5) Light chain CDR1 RASQGISNYLA (SEQ ID NO: 6) Light chain CDR2 EASTLQS (SEQ ID NO: 7) Light chain CDR3 QNYNSAPFT (SEQ ID NO: 8)

1.2 Construction of full-length antibody

Based on the above sequence, primers were designed and PCR was constructed to obtain the VH/VK gene fragment, thus obtaining the variable region.

The variable region of the antibody is then homologously recombinated with the constant region gene (CH1-Fc/CL) fragment to construct the complete antibody VH-CH1-Fc/VK-CL.

The complete full-length antibody PM sequence is constructed as follows:

Heavy chain (IgG1) amino acid sequence:

Light chain (Kappa) amino acid sequence:

1.3 Expression and purification of full-length antibodies

Plasmids expressing the light and heavy chains of the antibodies were transfected into HEK293E cells. After 6 days, the supernatant was collected, centrifuged at high speed to remove impurities, and purified using a Protein A column. The column was washed with PBS until the A280 reading returned to baseline. The target protein was eluted with acidic elution buffer (pH 3.0-3.5) and neutralized with 1M Tris-HCl (pH 8.0-9.0). After appropriate concentration, the eluted sample was further purified using PBS-equilibrated Superdex 200 (GE) gel chromatography to remove aggregates. The monomer peaks were collected and aliquoted for later use.

(II) Preparation of control antibody Lmab

The control antibody labetuzumab (Lmab) was prepared according to WHO Drug Information Vol. 30, No. 1, 2016, and its heavy and light chain amino acid sequences are as follows:

> Antibody heavy chain sequence Lmab-HC

> Antibody light chain sequence Lmab-LC

Example 2. Preparation of the compound

Experimental methods not specifying specific conditions in the embodiments of this disclosure are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents not specifying their source are commercially available, conventional reagents.

The structures of the compounds were determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform ( _CDCl3 ), and deuterated methanol ( _CD3OD ). The internal standard was tetramethylsilane (TMS). Chemical shifts are given in units of ^10⁻⁶ (ppm).

MS measurements were performed using a Finnigan LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQadvantage MAX).

UPLC measurements were performed using a Waters Acquity UPLC SQD liquid chromatography-mass spectrometry system.

HPLC determinations were performed using an Agilent 1200DAD high-performance liquid chromatograph (Sunfire C18 150×4.6mm column) and a Waters 2695-2996 high-performance liquid chromatograph (Gimini C18 150×4.6mm column).

The UV-HPLC determination was performed using a Thermo nanodrop2000 UV spectrophotometer.

The proliferation inhibition rate and _IC50 value were determined using a Phera starFS microplate reader (BMG GmbH, Germany).

Thin-layer chromatography (TLC) uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The silica gel plates used in TLC are 0.15mm to 0.2mm in diameter, while those used for TLC separation and purification are 0.4mm to 0.5mm in diameter.

Column chromatography typically uses 200-300 mesh silica gel from Yantai Huanghai as the carrier.

The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc., and Darui Chemicals.

Unless otherwise specified in the examples, the reactions were carried out under an argon or nitrogen atmosphere.

Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

A hydrogen atmosphere refers to a reaction vessel connected to a hydrogen balloon with a volume of approximately 1L.

The pressurized hydrogenation reaction was performed using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated three times.

The microwave reaction was performed using a CEM Discover-S 908860 microwave reactor.

Unless otherwise specified in the examples, the solution in the reaction refers to an aqueous solution.

Unless otherwise specified in the examples, the reaction temperature is room temperature.

Room temperature is the optimal reaction temperature, with a range of 20℃ to 30℃.

Preparation of PBS buffer solution with pH=6.5 in the example: Take 8.5g _{of KH2PO4} , 8.56g _{of K2HPO4 ·} 3H2O, 5.85g of NaCl and 1.5g of EDTA and place them in a bottle, make up to 2L, sonicate to dissolve completely, and shake well to obtain the solution.

The eluent systems for column chromatography and the developing solvent systems for thin-layer chromatography used to purify the compounds include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, and C: petroleum ether and ethyl acetate system. The volume ratio of the solvents is adjusted according to the polarity of the compounds, and small amounts of triethylamine and acidic or basic reagents can also be added for adjustment.

Some of the compounds disclosed herein were characterized by Q-TOF LC/MS. The Q-TOF LC/MS was performed using an Agilent 6530 Precision Mass Number Quadrupole-Time-of-Flight Mass Spectrometer and an Agilent 1290-Infinity Ultra-High Performance Liquid Spectrometer (Agilent Poroshell 300SB-C85μm, 2.1×75mm column).

The Y-D drug portion of this disclosed antibody-drug conjugate is referenced in PCT/CN2019/107873, and the related compound synthesis and testing are cited in this patent. The non-limiting synthetic examples are cited below:

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]indolazino[1,2-b]quinoline-1-yl)-1-hydroxycyclopropane-1-carboxamide

Add 1 mL of N,N-dimethylformamide to eczema sulfonate 1b (2.0 mg, 3.76 μmol, prepared by the method disclosed in patent application "EP0737686A1"), cool to 0-5 °C in an ice-water bath, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-hydroxycyclopropylformic 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-methylmorpholine chloride (3.8 mg, 13.7 μmol) to the reaction solution in sequence. After the addition is complete, stir the reaction solution at 0-5 °C for 2 hours. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution was extracted with ethyl acetate (8 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with developing solvent system B to give the title product 1 (1.6 mg, yield: 82.1%).

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

¹ H NMR (400MHz, CDCl ₃ ): δ7.90-7.84(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-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-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-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)-2-hydroxyacetamide 2-B

Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to 1b (4 mg, 7.53 μmol), purge three times with argon, cool to 0-5 °C in an ice-water bath, add 0.3 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add 2-cyclopropyl-2-hydroxyacetic acid 2a (2.3 mg, 19.8 μmol, prepared using the method disclosed in patent application "WO2013106717"), 1-hydroxybenzotriazole (3 mg, 22.4 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction mixture at 0-5 °C for 1 hour. Remove the ice-water bath, heat to 30 °C, and stir for 2 hours. The reaction solution was concentrated under reduced pressure, and the crude compound 2 was purified by high performance liquid chromatography (separation conditions: column: XBridgePrep C18 OBD 5μm 19*250mm; mobile phase: A-water (10mmol _NH4OAc ), B-acetonitrile, gradient elution, flow rate: 18mL/min). The corresponding fractions were collected and concentrated under reduced pressure to obtain the title product (2-A: 1.5mg, 2-B: 1.5mg).

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

Single-configuration compound 2-B (shorter retention time)

UPLC analysis: retention time 1.06 min, purity: 88% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

^1H NMR (400MHz, DMSO-d6 ₎ ): δ8.37(d, 1H), 7.76(d, 1H), 7.30(s, 1H), 6.51(s, 1H), 5.58-5.56(m, 1 H), 5.48 (d, 1H), 5.41 (s, 2H), 5.32-5.29 (m, 2H), 3.60 (t, 1H), 3.19-3.1 3(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 2-A with a single configuration (longer retention time)

UPLC analysis: retention time 1.10 min, purity: 86% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

^1H NMR (400MHz, DMSO-d6 ₎ ): δ8.35(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]indolazino[1,2-b]quinoline-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]indolazino[1,2-b]quinoline-1-yl)-3,3,3-trifluoro-2-hydroxypropionamide 3-B

Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to 1b (5.0 mg, 9.41 μmol), cool in an ice-water bath to 0–5 °C, add 0.3 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add 3,3,3-trifluoro-2-hydroxypropionic acid 3a (4.1 mg, 28.4 μmol, supplier Alfa), 1-hydroxybenzotriazole (3.8 mg, 28.1 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.4 mg, 28.2 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction mixture at 0–5 °C for 10 minutes. Remove the ice-water bath, heat to 30 °C, and stir for 8 hours. The reaction solution was concentrated under reduced pressure, and the crude compound 3 was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18mL/min). The corresponding fractions were collected and concentrated under reduced pressure to obtain the title product (1.5mg, 1.5mg).

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

Single-configuration compounds (shorter retention time)

UPLC analysis: retention time 1.11 min, purity: 88% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

^1H NMR (400MHz, DMSO-d6 ₎ ): δ8.94(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 compounds (longer retention time)

UPLC analysis: retention time 1.19 min, purity: 90% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.97 (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).

Examples 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]indolazino[1,2-b]quinoline-1-yl)-1-hydroxycyclopentane-1-carboxamide 4

Add 1 mL of N,N-dimethylformamide to 1b (3.0 mg, 5.64 μmol), cool in an ice-water bath to 0-5 °C, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-hydroxy-cyclopentanecarboxylic acid 4a (2.2 mg, 16.9 μmol, prepared using the method disclosed in patent application "WO2013106717") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (4.7 mg, 16.9 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction solution at 0-5 °C for 1 hour. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution was extracted with ethyl acetate (10 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with 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 (400MHz, CDCl ₃ ): δ7.73-7.62(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).

Examples 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]indolazino[1,2-b]quinoline-1-yl)-1-(hydroxymethyl)cyclopropane-1-carboxamide 5

Add 1 mL of N,N-dimethylformamide to 1b (2.0 mg, 3.76 μmol), cool in an ice-water bath to 0-5 °C, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-(hydroxymethyl)-cyclopentanecarboxylic acid 5a (0.87 mg, 7.5 μmol, prepared using the method disclosed in patent application "WO201396771") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (2 mg, 7.24 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction solution at 0-5 °C for 2 hours. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution was extracted with ethyl acetate (8 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with developing solvent system B to give the title product 5 (1.0 mg, yield: 50%).

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

¹ H NMR (400MHz, CDCl ₃ ): δ8.07 (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).

Examples 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-benzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)-1-(hydroxymethyl)cyclobutane-1-carboxamide 6

Add 1 mL of N,N-dimethylformamide to 1b (3.0 mg, 5.64 μmol), cool in an ice-water bath to 0-5 °C, add one drop of triethylamine, and stir until the reaction solution becomes clear. Add 1-(hydroxymethyl)cyclobutane-1-carboxylic acid 6a (2.2 mg, 16.9 μmol; prepared according to the method disclosed in the literature "Journal of the American Chemical Society, 2014, vol. 136, #22, pp. 8138-8142") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (4.7 mg, 169 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction solution at 0-5 °C for 1 hour. The reaction was quenched by adding 5 mL of water to the reaction solution. The reaction solution was extracted with ethyl acetate (10 mL × 3). The organic phases were combined and washed with saturated sodium chloride solution (5 mL × 2). The organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by thin-layer chromatography with developing solvent system B to give the title product 6 (2.1 mg, yield: 67.9%).

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

¹ H NMR (400MHz, DMSO-d ₆ ): δ7.85-7.62(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, 1 H), 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).

Examples 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]indolazino[1,2-b]quinoline-1-yl)-1-hydroxycyclobutane-1-carboxamide 7

Add 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide to 1b (3.0 mg, 5.64 μmol), cool in an ice-water bath to 0–5 °C, add 0.3 mL of N-methylmorpholine dropwise, and stir until the reaction solution becomes clear. Add 1-hydroxycyclobutanecarboxylic acid 7a (2.0 mg, 17.22 μmol, supplier's reagent), 1-hydroxybenzotriazole (2.3 mg, 17.0 μmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.2 mg, 16.7 μmol) sequentially to the reaction solution. After the addition is complete, stir the reaction solution at 0–5 °C for 10 minutes. Remove the ice-water bath and stir at room temperature for 2 hours. Concentrate the reaction solution under reduced pressure, and purify the residue by thin-layer chromatography using developing solvent system B to give title product 7 (2.5 mg, yield: 83.1%).

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

^1H NMR (400MHz, DMSO-d6 ₎ ): δ8.28(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),3.1 5-3.05(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).

Examples 2-8

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)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]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide 8

first step

1-((2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropane-1-carboxylic acid benzyl ester 8c

1-Hydroxycyclopropane-1-carboxylic acid benzyl ester 8a (104 mg, 0.54 mmol; prepared by the method disclosed in patent application "US2005/20645") and 2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate 8b (100 mg, 0.27 mmol; prepared by the method disclosed in patent application "CN105829346A") were added to a reaction flask, 5 mL of tetrahydrofuran was added, the mixture was purged with argon three times, the temperature was lowered to 0-5 °C in an ice-water bath, potassium tert-butoxide (61 mg, 0.54 mmol) was added, the ice bath was removed, the mixture was heated to room temperature and stirred for 10 minutes, 20 mL of ice water was added, and the mixture was extracted with ethyl acetate (5 mL × 2) and chloroform (5 mL × 5). The organic phases were combined and concentrated. The residue was dissolved in 3 mL of 1,4-dioxane, and 0.6 mL of water was added. Sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorene methyl chloroformate (70 mg, 0.27 mmol) were added, and the mixture was stirred at room temperature for 1 hour. 20 mL of water was added, and the mixture was extracted with ethyl acetate (8 mL × 3). The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using solvent system B to give the title product 8c (100 mg, yield: 73.6%).

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

Step 2

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

Dissolve 8c (50 mg, 0.10 mmol) in 3 mL of a mixture of tetrahydrofuran and ethyl acetate (V:V = 2:1), add palladium on carbon (25 mg, 10%), purge three times with hydrogen, and stir at room temperature for 1 hour. Filter the reaction solution with diatomaceous earth, wash the filter cake with tetrahydrofuran, concentrate the filtrate to give the title product 8d (41 mg, yield: 100%).

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

Step 3

(9H-fluorene-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]indolazino[1,2-b]quinoline-1-yl)aminocarbonyl)cyclopropoxy)methyl)amino)2-oxoethyl)carbamate 8e

Add 1b (7 mg, 0.013 mmol) to the reaction flask, add 1 mL of N,N-dimethylformamide, purge three times with argon, cool to 0-5 °C in an ice-water bath, add one drop of triethylamine, add 0.5 mL of 8d (7 mg, 0.017 mmol) in N,N-dimethylformamide solution, add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (7 mg, 0.026 mmol), and stir in an ice bath for 35 minutes. Add 10 mL of water, extract with ethyl acetate (5 mL × 3), wash the organic phase with saturated sodium chloride solution (10 mL), dry to anhydrous sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the residue by thin-layer chromatography with developing solvent system B to give the title product 8e (8.5 mg, yield 78.0%).

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

Step 4

1-((2-aminoacetamido)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]indolazino[1,2-b]quinoline-1-yl)cyclopropane-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 mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and 2 mL of toluene was added for further concentration under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added and the mixture was stirred. The upper n-hexane layer was decanted. This process was repeated three times. The crude product 8f (2.9 mg) was concentrated under reduced pressure. The product was used directly in the next reaction without purification.

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

Step 5

1-(((S)-7-benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12,15-pentoxo-2,5,8,11,14-pentazaeicosyl)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]indolazino[1,2-b]quinoline-1-yl)cyclopropane-1-carboxamide 8

Crude product 8f (2.9 mg, 4.84 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide, purged three times with argon, and cooled to 0-5 °C in an ice-water bath. 0.3 mL of N,N-dimethylformamide solution containing 8 g (2.7 mg, 5.80 μmol, prepared using the method disclosed in patent application "EP2907824") of (S)-2(-2-(-2-(6-(2,5-dioxo-1H-pyrrolo-1-yl)hexamylamino)acetamido)acetamido)-3-phenylpropionic acid was added. 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (2.7 mg, 9.67 μmol) was added. The mixture was stirred in an ice bath for 30 minutes, then the ice bath was removed, and the mixture was heated to room temperature and stirred for 15 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18mL/min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product 8 (2mg, yield: 39.0%).

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

¹ H NMR (400MHz, DMSO-d ₆ ): δ9.01 (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, 4 H), 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, 1 H), 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), 2.37-2.2 3(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).

Examples 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]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexamamide 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]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexamamide 9-B

first step

2-Cyclopropyl-2-hydroxyacetic acid benzyl ester 9a

2a (1.3 g, 11.2 mmol; prepared by the method disclosed 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 solution was stirred at room temperature for 48 hours, filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (10 mL). The filtrates were combined and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with solvent system C to give the title product 9a (2 g, yield: 86.9%).

Step 2

10-Cyclopropyl-1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazabut-11-acid benzyl ester 9b

Add 9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol) to the reaction flask, add 4 mL of tetrahydrofuran, purge three times with argon, cool to 0-5 °C in an ice-water bath, add potassium tert-butoxide (109 mg, 0.98 mmol), remove the ice bath, raise to room temperature and stir for 40 minutes, add 10 mL of ice water, and extract with ethyl acetate (20 mL × 2) and chloroform (10 mL × 5). Combine the organic phases and concentrate. Dissolve the residue in 4 mL of dioxane, add 2 mL of water, add sodium bicarbonate (49.2 mg, 0.586 mmol) and fluorene methyl chloroformate (126 mg, 0.49 mmol), and stir at room temperature for 2 hours. Add 20 mL of water, extract with ethyl acetate (10 mL × 3), wash the organic phase with saturated sodium chloride solution (20 mL), dry to anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. The residue was purified by silica gel column chromatography using solvent system C to give the title product 9b (48 mg, yield: 19%).

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

Step 3

10-Cyclopropyl-1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-acid 9c

9b (20 mg, 0.038 mmol) was dissolved in 4.5 mL of a mixture of tetrahydrofuran and ethyl acetate (V:V = 2:1), and palladium on carbon (12 mg, 10% purity, dry type) was added. The mixture was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction solution was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated to obtain the crude product 9c (13 mg). This product was used directly in the next step of the reaction without further purification.

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

Step 4

(9H-fluorene-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-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)2-oxoethyl)carbamate 9d

Add 1b (10 mg, 18.8 μmol) to the reaction flask, add 1 mL of N,N-dimethylformamide, purge three times with argon, cool to 0-5 °C in an ice-water bath, add one drop of triethylamine, add crude product 9c (13 mg, 30.6 μmol), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (16.9 mg, 61.2 μmol), and stir in an ice bath for 40 minutes. Add 10 mL of water, extract with ethyl acetate (10 mL × 3), and combine the organic phases. Wash the organic phase with saturated sodium chloride solution (10 mL × 2), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the residue by thin-layer chromatography using developing solvent system B to give the title product 9d (19 mg, yield: 73.6%).

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

Step 5

2-((2-aminoacetamido)methoxy)-2-cyclopropyl-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]indolazino[1,2-b]quinoline-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 mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and 1 mL of toluene was added and concentrated under reduced pressure. This process was repeated twice. 3 mL of n-hexane was added to the residue and stirred. After standing, the supernatant was decanted, and the solid was retained. The solid residue was concentrated under reduced pressure and dried using an oil pump to obtain the crude product 9e (17 mg). The product was used directly in the next reaction without purification.

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

Step 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]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexamamide 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]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexet-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexamamide 9-B

Crude product 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide, purged three times with argon, cooled to 0-5 °C in an ice-water bath, and 8 g (21.2 mg, 44.8 μmol) of 0.3 mL of N,N-dimethylformamide solution was added. Then, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (18.5 mg, 67.3 μmol) was added. The mixture was stirred in an ice bath for 10 minutes, then the ice bath was removed, and the mixture was stirred at room temperature for 1 hour to produce compound 9. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5μm 19*250mm; mobile phase: A-water (10mmol _NH4OAc ): B-acetonitrile, gradient elution, flow rate: 18mL/min), and the corresponding components were collected and concentrated under reduced pressure to obtain the title product (9-A: 2.4mg, 9-B: 1.7mg).

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

Single-configuration compound 9-A (shorter retention time)

UPLC analysis: retention time 114 min, purity: 85% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.60 (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), 2.15-2.04(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).

The single-configuration compound 9-B (with a longer retention time)

UPLC analysis: retention time 1.16 min, purity: 89% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol _NH4OAc ), B-acetonitrile).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.68-8.60 (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), 7.2 6-7.13 (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).

Example 3. Preparation of ADC

ADC drug loading analysis

Experimental Objectives and Principles

The ADC load was determined using ultraviolet spectrophotometry (UV-Vis). Instrument: Thermo Nanodrop 2000 UV-Vis spectrophotometer. The principle is that the total absorbance of the ADC at a certain wavelength is equal to the sum of the absorbance of the drug and the monoclonal antibody at that wavelength.

Experimental methods

After placing cuvettes containing sodium succinate buffer solution into the reference absorption cell and the sample absorption cell, respectively, and subtracting the solvent blank, cuvettes containing the test solution were placed into the sample absorption cell, and absorbance was measured at 280 nm and 370 nm.

Result calculation:

(1)A _280nm =ε _mab-280 bC _mab +ε _Drug-280 bC _Drug

ε _Drug-280 : The drug has an average molar extinction coefficient of 5100 at 280 nm;

C _Drug : Drug concentration;

ε _mab-280 : The average molar extinction coefficient of the monoclonal antibody at 280 nm is 214,600;

C _mab : Concentration of monoclonal antibody;

b: The optical path length is 1cm.

Similarly, the equation for the total absorbance of the sample at 370 nm can be obtained:

(2)A _370nm =ε _mab-370 bC _mab +ε _Drug-370 bC _Drug

ε _Drug-370 : The drug has an average molar extinction coefficient of 19000 at 370 nm;

C _Drug : Drug concentration;

ε _mab-370 : The monoclonal antibody has an extinction coefficient of 0 at 370 nm;

C _mab : Concentration of monoclonal antibody;

b: The optical path length is 1cm.

The drug loading in the ADC can be calculated by combining equations (1) and (2) with the extinction coefficients and concentration data of the monoclonal antibody and drug at two detection wavelengths.

Drug load = C_{_Drug} / C_{_mab} .

Examples of preparation of PSMA antibody-drug conjugate PM-9-A with different DAR values

The following examples illustrate the preparation process of the ADCs disclosed herein. In Examples 3-4, 3-5, 3-7, and 3-11, antibody PM was coupled with drug 9-A containing a linker unit via a thiol group on cysteine residues to prepare antibody-drug conjugate PM-9-A (DAR = approximately 3-4). In Examples 3-1 to 3-3 and 3-6, antibody PM was coupled with drug 9-A containing a linker unit via a thiol group on cysteine residues to prepare PSMA ADC molecule PM-9-A (DAR = approximately 6-7). Examples 3-8 to 3-10 served as controls, where the thiol group on the cysteine residues of antibody PM was reacted with drug VcMMAE (Hanxiang Biotechnology, CAS 646502-53-6) containing a linker unit to prepare conjugate molecule PM-VcMMAE.

By adjusting the ratio of antibody to drug, the scale of the reaction, and other conditions, antibody-drug conjugates with different DAR values (n) can be obtained. The preferred DAR value is 1-8, more preferably 3-8, and most preferably 3-7.

(I) Preparation of antibody-drug conjugates with PM-9-A with different DAR values

Example 3-1 ADC-1

At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 9.8 μL, 98 nmol) was added to the PBS buffered aqueous solution of antibody PM (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/mL, 0.27 mL, 18 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (0.3 mg, 277 nmol) was dissolved in 20 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (0.18 mg/mL, 11 mL) of the exemplary product ADC-1, a conjugate of formula PM-9-A, which was stored at 4 °C.

UV-Vis calculated average: n = 6.31.

Example 3-2 ADC-2

At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 128.9 μL, 1289 nmol) was added to the PBS buffered aqueous solution of antibody PM (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/mL, 3.6 mL, 243 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (3.93 mg, 3649 nmol) was dissolved in 200 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.93 mg/mL, 15.4 mL) of the exemplary product ADC-2, a conjugate of formula PM-9-A, which was stored at 4 °C.

UV-Vis calculated average: n = 6.63.

Example 3-3 ADC-3

At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 358.1 μL, 3581 nmol) was added to the PBS buffered aqueous solution of antibody PM (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/mL, 10 mL, 676 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (10.91 mg, 10135 nmol) was dissolved in 480 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (3.47 mg/mL, 25 mL) of the exemplary product ADC-3, the conjugate of formula PM-9-A, which was stored at 4 °C.

UV-Vis calculated average: n = 6.9.

Example 3-4 ADC-4

At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 3.5 μL, 35 nmol) was added to the PBS buffered aqueous solution of antibody PM (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/mL, 0.21 mL, 14 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (0.15 mg, 139 nmol) was dissolved in 20 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (0.28 mg/mL, 4.6 mL) of the exemplary product ADC-4, a conjugate of formula PM-9-A, which was stored at 4 °C.

UV-Vis calculated average: n = 3.68.

Example 3-5 ADC-5

At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 84.8 μL, 848 nmol) was added to the PBS buffered aqueous solution of antibody PM (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/mL, 5.23 mL, 353 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (3.8 mg, 3534 nmol) was dissolved in 260 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (2.48 mg/mL, 18.2 mL) of the exemplary product ADC-5, a conjugate of formula PM-9-A, which was stored at 4 °C.

UV-Vis calculated average: n = 3.89.

Example 3-6 ADC-6

At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 125.3 μL, 1253 nmol) was added to the PBS buffered aqueous solution of antibody PM (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/mL, 3.5 mL, 236 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (3.82 mg, 3547 nmol) was dissolved in 150 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.83 mg/mL, 14 mL) of the exemplary product ADC-6, the conjugate of formula PM-9-A, which was stored at 4 °C.

UV-Vis calculated average: n = 6.61.

Example 3-7 ADC-7

At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 238.1 μL, 2381 nmol) was added to the PBS buffered aqueous solution of antibody PM (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/mL, 14.68 mL, 992 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (10.67 mg, 9919 nmol) was dissolved in 420 μl of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain the exemplary product ADC-7 of the conjugate shown as PM-9-A in PBS buffer (3.61 mg/mL, 37 mL), which was stored at 4 °C.

UV-Vis calculated average: n = 3.93.

(II) Preparation of control antibody drug conjugated with PM-VcMMAE (refer to patent WO2007002222A2)

Example 3-8 ADC-8

At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 42.2 μL, 422 nmol) was added to the PBS buffered aqueous solution of antibody PM (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/mL, 2.5 mL, 169 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

The compound VcMMAE (2.22 mg, 1689 nmol) was dissolved in 100 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain the exemplary product ADC-8, a conjugate of the formula PM-VcMMAE, in PBS buffer (1.76 mg/mL, 12 mL), which was stored at 4 °C.

CE-SDS calculated average: n = 4.47.

Example 3-9 ADC-9

At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 55.7 μL, 557 nmol) was added to the PBS buffered aqueous solution of antibody PM (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/mL, 3.3 mL, 223 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

The compound VcMMAE (2.93 mg, 2230 nmol) was dissolved in 200 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain the exemplary product ADC-9 of the conjugate shown in formula PM-VcMMAE in PBS buffer (2.05 mg/mL, 17 mL), which was stored at 4 °C.

CE-SDS calculated average: n = 4.23.

Example 3-10 ADC-10

At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 42.2 μL, 422 nmol) was added to the PBS buffered aqueous solution of antibody PM (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/mL, 2.5 mL, 169 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

The compound VcMMAE (2.22 mg, 1689 nmol) was dissolved in 150 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain the exemplary product ADC-10 of the conjugate shown in formula PM-VcMMAE in PBS buffer (2.2 mg/mL, 13 mL), which was stored at 4 °C.

CE-SDS calculated average: n = 3.92.

Example 3-11 ADC-11

At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 42.2 μL, 422 nmol) was added to the PBS buffered aqueous solution of antibody PM (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/mL, 2.4 mL, 160 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (1.75 mg, 1629 nmol) was dissolved in 100 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.34 mg/mL, 15.5 mL) of the exemplary product ADC-11, a conjugate of formula PM-9-A, which was stored at 4 °C.

UV-Vis calculated average: n = 4.19.

Example 3-12 ADC-12

At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 42.2 μL, 422 nmol) was added to the PBS buffered aqueous solution of antibody PM (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/mL, 2.4 mL, 160 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 58 (prepared according to Example 58 on page 163 of patent CN104755494A, 1.68 mg, 1624 nmol) was dissolved in 100 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (1.45 mg/mL, 14.8 mL) of the exemplary product ADC-12, a conjugate of formula PM-58, and stored at 4 °C.

UV-Vis calculated average: n = 4.16.

(III) Preparation of control antibody drug conjugated with Lmab-9-A

At 37°C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 74.1 μL, 741 nmol) was added to the PBS buffered aqueous solution of antibody Lmab (pH = 6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg/mL, 2.15 mL, 145 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

Compound 9-A (3.20 mg, 2179 nmol) was dissolved in 155 μL of dimethyl sulfoxide and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffer (0.99 mg/mL, 15 mL) of the title product Lmab-9-A, which was stored at 4 °C.

UV-Vis calculated average: n = 7.07.

The activity of the antibodies disclosed herein is verified using the following biochemical assay methods.

Test Example 1: In vitro cell binding experiment

This experiment evaluates antibody binding by detecting the fluorescence signal of cell surface antibodies and assessing the intensity of the fluorescence signal. Prepared ADC-2, ADC-10, the control ADC Lmab-9-A, and antibody PM were used together for in vitro binding detection.

Serially diluted ADC-2, ADC-10, and antibody PM were incubated with 1× ^10⁵ cells (ATCC, MDA PCa 2b/CRL-2422, LNCaP/CRL-1740, 22Rv1/CRL-2505, PC-3/CRL-1435, DU 145/HTB-81) at 4°C for 60 min, followed by washing away excess ADC or antibody. Cells were then incubated with FITC-labeled goat anti-human IgG (H+L) secondary antibody (Jackson ImmunoResearch, 109-095-003) at 4°C for 30 min. After washing away excess antibody, the fluorescence signal on the cell surface was read using BD CantoII (results are shown in Table 2, Figures 1A, 1B, 1C, 1D, and 1E).

Table 2. In vitro cell binding activity ( _EC50 , nM)

MDAPCa 2b LNCaP 22RV1 PC-3 DU145 Antibody PM 1.079 1.421 0.6761 Do not combine Do not combine ADC-2 0.7183 0.9381 0.5714 Do not combine Do not combine ADC-10 0.9537 1.581 0.6894 Do not combine Do not combine Lmab-9-A Do not combine Do not combine Do not combine Do not combine Do not combine

The expression level of the antigen PSMA in the cells was detected as follows: MDA PCa 2b > LNCaP > 22Rv1, while PC-3 and DU 145 did not express PSMA.

The results showed that ADC-2, ADC-10, the control ADCLmab-9-A, and antibody PM had correspondingly strong or weak binding abilities to cells with different PSMA antigen expression levels. The smaller _{the EC50} , the stronger the binding ability.

Test Example 2: In vitro cell endocytosis experiment

DT3C, 70 kDa, is a recombinant fusion protein composed of fragment A (toxin only) of diphtheria toxin and fragment 3C (IgG binding part) of group G streptococcus. This protein has a high affinity for the IgG portion of antibodies and enters the cell along with the antibody during endocytosis. Under the action of intracellular furin protease, it releases the toxic DT3C, which inhibits EF2-ADP ribosylation activity, blocks protein translation, and ultimately leads to cell death. DT3C that does not enter the cell does not possess cell-killing activity. The endocytic activity of the antibody is evaluated based on its cell-killing effect.

Aseptically filtered DT3C (70KD, SB HRS3A, MEI3AU0803) and PM antibody (DT3C molar concentration was 6 times the antibody molar concentration) were mixed at a 1:1 volume ratio and incubated at room temperature for 30 minutes. The mixture was then serially diluted with serum-free medium and added to cells (ATCC, LNCaP/CRL-1740, 22Rv1/CRL-2505) cultured in medium containing 20% low IgG FBS prepared one day prior (2000 cells/well). The cells were incubated at 37°C for three days in a 5% CO2 incubator. CellTiter-Glo (Promega, G7573) was then added and incubated at room temperature in the dark for 10 minutes. Chemiluminescence was read on a Victor3 microarray (results are shown in Table 3, Figure 2A, and Figure 2B).

Table 3. Intracellular endocytic activity of antibodies in different cell types ( _IC50 , nM)

LNCaP 22RV1 Antibody PM+DT3C ~4.641e+012 0.08563 hIgG1 control + DT3C No swallowing No swallowing DT3C only No swallowing No swallowing

The results showed that antibody PM had endocytic ability on cells that positively expressed PSMA antigen.

Test Example 3: Cell Proliferation Inhibition Experiment

This experiment evaluated the inhibitory effect of PSMAADC on cell proliferation (ATCC, LNCaP/CRL-1740, 22Rv1/CRL-2505, PC-3/CRL-1435) by detecting intracellular ATP content and based on _IC50 value.

Samples to be tested: ADC-2, ADC-4, control ADC Lmab-9-A

1. Inhibitory effect on LNCaP cell proliferation

LNCaP cells were cultured in RPMI-1640 medium containing 10% FBS, passaged 2-3 times per week at a passage ratio of 1:3 or 1:6. During passage, the medium was aspirated, the cell layer was washed with 5 mL of 0.25% trypsin, the trypsin was then aspirated, and the cells were digested in an incubator for 3-5 minutes. The cells were then resuspended in fresh medium. 180 μL of the cell suspension was added to each well of a 96-well cell culture plate (2 × ^10³ cells/well), with RPMI-1640 medium containing 4.5% FBS. Only medium was added to the outer edge of the 96-well plate. The culture plates were incubated for 24 hours (37°C, 5% _CO₂ ).

Prepare the target ADCs (ADC-2, ADC-4, Lmab-9-A) to their initial concentrations, and serially dilute them 1:6 with PBS for 9 points, plus a 0 concentration point. Add 20 μL of each dilution to the cell culture plate and incubate for 5 days (37°C, 5% _CO2 ). For compound 2-B (i.e., the free toxin of 9-A), first dilute it with DSMO to prepare a stock solution and prepare a 200 μM solution with PBS as the starting concentration. Then serially dilute it 1:6 with PBS for 9 points, plus a 0 concentration point. Add 1 μL of each stock solution to 20 μL of RPMI-1640 and transfer it to a 96-well cell culture plate. Incubate for 5 days (37°C, 5% _CO2 ). Add 80 μL of CellTiter-Glo reagent to each well of the 96-well cell culture plate, incubate at room temperature in the dark for 10-15 minutes, and read the chemiluminescence signal values in a Victor3. Process the data using GraphPad software. (The results are shown in Table 4, Figure 3A, and Figure 3B).

2. Inhibitory effect on 22Rv1 cell proliferation

22Rv1 cells were cultured in RPMI-1640 medium containing 10% FBS, passaged twice a week at a ratio of 1:3 or 1:6. During passage, the medium was aspirated, the cell layer was washed with 5 mL of 0.25% trypsin, the trypsin was then aspirated, and the cells were digested in an incubator for 3–5 minutes. Fresh medium was added to resuspend the cells. 180 μL of cell suspension was added to each well of a 96-well cell culture plate (4 × ^10³ cells/well), with RPMI-1640 medium containing 4.5% FBS. Only medium was added to the outer edge of the 96-well plate. The plates were incubated for 24 hours (37°C, 5% _CO₂ ). The preparation and experimental methods for the ADC and compound 2-B were the same as above. (Results are shown in Table 4, Figures 3C and 3D).

3. Inhibitory effect on PC-3 cell proliferation

PC-3 cells were cultured in F-12K medium containing 10% FBS, passaged 2-3 times per week at a passage ratio of 1:3 or 1:6. During passage, the medium was aspirated, the cell layer was washed with 5 mL of 0.25% trypsin, the trypsin was then aspirated, and the cells were digested in an incubator for 3-5 minutes. The cells were then resuspended in fresh medium. 180 μL of cell suspension was added to each well of a 96-well cell culture plate (4 × ^10³ cells/well), with the medium being 4.5% FBS in F-12K. Only the medium was added to the outer edge of the 96-well plate. The culture plates were incubated for 24 hours (37°C, 5% _CO₂ ). The preparation and experimental methods for the ADC and compound 2-B were the same as above. (Results are shown in Table 4, Figure 3E, and Figure 3F).

Table 4. Cell-killing effects ( _IC50 , nM)

Compound 2-B ADC-2 ADC-4 Lmab-9-A LNCaP 1.695 0.7988 4.67 ~7.751e+006 22RV1 2.843 1.238 78.34 ~3.509e+007 PC-3 8.328 ~1.256e+006 ~3.140e+006 ~529.9

Conclusion: PSMAADC exhibits cytotoxic activity in LNCaP and 22RV1 cells. Compound 2-B (i.e., the free toxin of 9-A) demonstrates transmembrane cytotoxicity.

Test Example 4: Evaluation of the efficacy of ADC drugs on human prostate cancer cell 22Rv1 xenografts in nude mice

I. Testing Methods

Male nude mice (nu/nu) aged 6-8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Certificate No. 1908120082). The mice were housed in SPF-grade conditions. Human prostate cancer cells 22Rv1 (Chinese Academy of Sciences) were subcutaneously inoculated into the nude mice. When the average tumor volume reached 220 ^mm³ , the animals were randomly divided into groups (D0), with 6 mice in each group. Intraperitoneal injections of the drug were administered twice weekly for a total of 5 weeks. Tumor volume and body weight were measured twice weekly, and data were recorded.

The tumor volume V = 1/2 × a × ^b² , where a and b represent the length and width, respectively.

The relative tumor proliferation rate T/C (%) = (T-T0)/(C-C0) × 100, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.

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

II. Test Subjects

ADC-10: 3mpk, 10mpk;

ADC-2: 3mpk, 10mpk;

Blank control group: PBS buffer at pH 7.4.

III. Antitumor effect of antibody ADCs

By day 17 (D17) after the start of administration, the tumor inhibition rates of 10mpk and 3mpk of positive ADC-10 were 48.9% and 10.0%, respectively, and the tumor inhibition rates of 10mpk and 3mpk of ADC-2 were >100% and 91.5%, respectively, which were significantly better than those of the blank control group and the positive ADC group, and showed a good dose-dependent relationship (Table 5, Figure 4A).

The mice maintained a stable weight during the administration process, indicating that there were no obvious toxic side effects at any dose of ADC-2 (Figure 4B).

Table 5. Efficacy of ADC administration on 22Rv1 xenografts in tumor-bearing nude mice.

vs. blank control group: *p<0.05, ***p<0.001; vs. corresponding dose group of ADC-10: ###p<0.001

Test Example 5: Evaluation of the efficacy of ADC drugs on human prostate cancer cell 22Rv1 xenografts in nude mice

I. Testing Methods

Male nude mice (nu/nu) aged 6-8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Certificate No. 1908120082). The mice were housed in SPF-grade conditions. Human prostate cancer cells 22Rv1 (Chinese Academy of Sciences) were subcutaneously inoculated into the nude mice. When the average tumor volume reached 210 ^mm³ , the animals were randomly divided into groups (D0), with 8 mice in each group. Intraperitoneal injections of the drug were administered twice weekly for a total of 6 weeks. Tumor volume and body weight were measured twice weekly, and data were recorded.

The tumor volume V = 1/2 × a × ^b² , where a and b represent the length and width, respectively.

The relative tumor proliferation rate T/C (%) = (T-T0)/(C-C0) × 100, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.

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

II. Test Subjects

ADC-8: 10mpk;

ADC-6: 3mpk, 6mpk;

ADC-7: 3mpk, 6mpk, 10mpk;

Blank control group: PBS buffer at pH 7.4.

III. Antitumor effect of antibody ADCs

By day 14 (D14) after the start of administration, the tumor inhibition rate of positive ADC-8 at 10 mpk was 81.6%, while the tumor inhibition rates of tested ADC-6 at 6 mpk and 3 mpk were >100% and 97.8%, respectively. The tumor inhibition rates of another tested ADC-7 at 10 mpk, 6 mpk, and 3 mpk were >100%, 88.4%, and 80.5%, respectively. All administration groups were significantly better than the control group. At the same dose, tested ADC-6 was significantly better than tested ADC-7. Both tested ADCs showed a good dose-dependent relationship (Table 6, Figure 5A).

The mice maintained a stable weight during the administration process, indicating that there were no obvious toxic side effects at any of the tested antibody doses (Figure 5B).

Table 6. Efficacy of ADC administration on 22Rv1 xenografts in tumor-bearing nude mice.

vs. control group: ***p<0.001; vs. ADC-6 corresponding dose group: ##p<0.01, ###p<0.001

Test Example 6: Evaluation of the efficacy of ADC drugs on human prostate cancer cell xenografts of LNCap in SCID Beighe mice

I. Testing Methods

Male SCID Beige mice, 6-8 weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Certificate No. 1908120082). The mice were housed in SPF-grade conditions. Human prostate cancer cells (LNCap, ATCC) were subcutaneously inoculated into the mice. When the average tumor volume reached 160 ^mm³ , the animals were randomly divided into groups (D0), with 7 mice in each group. The mice received intraperitoneal injections twice daily on D0 and D4. Tumor volume and body weight were measured and recorded twice weekly.

The tumor volume V = 1/2 × a × ^b² , where a and b represent the length and width, respectively.

The relative tumor proliferation rate T/C (%) = (T-T0)/(C-C0) × 100, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment.

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

II. Test Subjects

ADC-9: 10mpk;

ADC-3: 3mpk, 10mpk;

ADC-5: 3mpk, 6mpk, 10mpk;

Blank control group: PBS buffer at pH 7.4.

III. Antitumor effect of antibody ADCs

Tumors in all treatment groups began to regress after two doses on D0 and D4, and the efficacy was maintained until the experimental endpoint on D18. Both tested ADC-3 and ADC-5 showed some dose dependence, but there was no significant difference between them (Table 7, Figure 6A).

The mice maintained a stable weight during the administration process, indicating that there were no obvious toxic side effects at any of the tested antibody doses (Figure 6B).

Table 7. Efficacy of ADC administration on LNCap xenografts in SCID-bearing Beige mice.

vs. control group: ***p<0.001

Test Example 7: T1/2 Evaluation of PMSAADC SD Rats

Nine male SD rats were divided into three groups of three. They were kept under 12/12-hour light/dark cycles, kept at a constant temperature of 20-26℃ and humidity of 40-70%, and allowed free access to food and water. They were purchased from Zhejiang Vital River Laboratory Animal Co., Ltd. On the day of the experiment, the male SD rats were injected intravenously via the tail vein with the test drugs ADC-2, ADC-5, and ADC-10, respectively, at a dose of 3 mg/kg and an injection volume of 5 mL/kg.

Blood was collected at the following time points: 5 minutes, 8 hours, 24 hours (day 2), 3 days, 5 days, 8 days, 11 days, 15 days, 22 days, and 29 days after drug administration. 300 μL of blood was collected from the fundus vein of rats each time (equivalent to 150 μL of serum). The collected blood samples were left at room temperature for half an hour until agglutination, then centrifuged at 1000×g for 15 minutes at 4°C. The supernatant (serum) was transferred to EP tubes and immediately stored at -80°C. The concentration of PSMA antibody ADC in the serum of SD rats was detected using ELISA.

Test method:

Total antibody detection:

1. Add 100 μL of 1 μg/mL goat anti-human IgG FC (abcam) to each well of an ELISA plate and incubate overnight at 4°C.

2. Rinse three times with PBST washing solution (1×PBS, 0.5% Tween-20 (Sangon Biotech)).

3. Add 200 μL of blocking solution (5% milk, Beyotime) to each well and incubate at 37°C for 1-3 hours.

4. Rinse three times with PBST washing solution.

5. Add 100 μL of standard sample, quality control sample and test sample, and incubate at 37°C for 1-3 hours.

6. Rinse three times with PCB cleaning solution.

7. Add 100 μL of mouse pre-adsorbed goat anti-human IgG light/heavy chain HRP (1:5000, abcam) and incubate at 37°C for 1-1.5 hours.

8. Rinse three times with PCB cleaning solution.

9. Add 100 μL TMB (KPL) and incubate at room temperature in the dark for 10 minutes.

10. Add 100 μL of 1M dilute sulfuric acid (Sinopharm Group) to terminate the process, and read the plate at a wavelength of 450 nm (molecular device, flexstation 3).

Complete ADC (Intact ADC) detection:

1. Add 100 μL of 1 μg/mL goat anti-mouse IgG Fc to each well of an ELISA plate and incubate overnight at 4°C.

2. Rinse three times with PBST washing solution.

3. Add 200 μL of blocking solution to each well and incubate at 37°C for 1-3 hours.

4. Rinse three times with PCB cleaning solution.

5. Add 100 μL of 0.2 μg/mL antitoxin antibody or anti-pab-mmae (Zhongke Yingmu, S-497-8) to each well and incubate at 37℃ for 1-1.5 hours.

6. Rinse three times with PCB cleaning solution.

7. Add 100 μL of standard sample, quality control sample and test sample, and incubate at 37°C for 1-3 hours.

8. Rinse three times with PCB cleaning solution.

9. Add 100 μL of mouse pre-adsorbed goat anti-human IgG light/heavy chain HRP (1:5000) and incubate at 37°C for 1-1.5 hours.

10. Rinse three times with PCB cleaning solution.

11. Add 100 μL of TMB and incubate at room temperature in the dark for 10 minutes.

12. Add 100 μL of 1M dilute sulfuric acid to stop the reaction, and read the plate at a wavelength of 450 nm.

Detection and analysis results:

The concentration of PSMA antibody ADC in serum was detected by ELISA, and pharmacokinetic analysis was performed (the results are shown in Table 8).

Table 8. T1 _/2 of PSMA antibody-conjugate in SD rats

The results showed that after intravenous administration of 3 mg/kg of the test antibody conjugates ADC-10, ADC-2, and ADC-5 to rats, the half-lives of total antibody (antibodies conjugated in ADCs and free antibodies in serum) in rats were approximately 219.8 hours (9.2 days), 171.2 hours (7.1 days), and 172.5 hours (7.2 days), respectively; the half-lives of intact ADCs in rats were approximately 76.7 hours (3.2 days), 153.9 hours (6.4 days), and 137.9 hours (5.7 days), respectively. The T1 _/2 of ADC-2 and ADC-5 was superior to that of ADC-10.

Test Example 8: Stability Study of PSMA ADC (Detection of Free Toxins)

1. Stability of ADC-2 in rat pharmacokinetic samples

Liquid chromatography-mass spectrometry (LC-MS) was used to monitor the release of compound 2-B (a free toxin of 9-A) over 28 days in male rats after intravenous administration, reflecting the stability of the sample under constant concentration conditions. The levels of compound 2-B in rat plasma at different time points (5 minutes, 8 hours, 1 day, 2 days, 4 days, 7 days, 10 days, 14 days, 21 days, and 28 days) over 28 days demonstrated good stability for ADC-2, as shown in Table 9.

The results showed that ADC-2 exhibited good stability in male rats for 28 days.

Table 9 Stability Study of PSMAADC

Blq indicates 1 ng/mL; M indicates that the rat is male.

2. PSMA ADC Plasma Stability Study

Liquid chromatography-mass spectrometry (LC-MS) was used to monitor the release of compound 2-B from sample ADC- ₂ (100 μg/mL) at different time points (0, 7, 14, and 21 days in plasma of five species: human, monkey, dog, rat, and mouse (heparin anticoagulated) and 1% BSA-PBS as a control) in a 37℃ & 5% CO2 incubator. This was to reflect the stability of the sample under specific concentration (100 μg/mL) conditions. The determination of compound 2-B at different time points showed that ADC-2 exhibited good stability, as shown in Figure 7 and Table 10.

The results showed that ADC-2 exhibited good stability in plasmas of different species at certain temperatures and within a certain time period.

Note: The experiment was conducted in a sterile laboratory, and the blank plasma was sterilized by filtration through a 0.22 μm microporous membrane.

Table 10. Content of compound 2-B (ng/mL)

The linear range of compound 2-B is 1–5000 ng/mL; BLQ indicates 1 ng/mL. “-1” and “-2” indicate the first and second replicates.

3. PSMA ADC Plasma Stability Study

Liquid chromatography-mass spectrometry (LC-MS) was used to monitor the amount of compound 2-B lost from sample ADC-5 at different time points (0, 7, 14, and 23 days) in plasma (heparin anticoagulated from human, monkey, dog, rat, and mouse, with 1% BSA-PBS as a control) at 37℃ in a 5% _CO2 incubator. This was used to reflect the stability of the sample under specific concentration (100 μg/mL) conditions. The determination of compound 2-B content at different time points showed that ADC-5 exhibited good stability, as shown in Table 11 and Figure 8.

The results showed that ADC-5 exhibited good stability in plasmas of different species at certain temperatures and within a certain time period.

Note: The experiment was conducted in a sterile laboratory, and the blank plasma was sterilized by filtration through a 0.22 μm microporous membrane.

Table 11. Content of Compound 2-B (ng/mL) - ADC-5 (100 μg/mL) in different plasmas after incubation at 37°C for 23 days.

BSA rats mice people dog monkey 0d-1 BLQ BLQ BLQ BLQ BLQ BLQ 0d-2 BLQ BLQ BLQ BLQ BLQ BLQ 7d-1 BLQ 3.74 4.69 2.46 7.14 BLQ 7d-2 BLQ 3.82 3.88 BLQ 5.89 BLQ 14d-1 BLQ 10.1 9.32 BLQ 14.2 5.09 14d-2 BLQ 10.0 10.6 2.00 13.8 6.34 23d-1 3.32 14.9 16.5 8.57 23.1 9.11

23d is a single well, and the linear range of compound 2-B is 2–5000 ng/mL; BLQ indicates 1 ng/mL.

Test Example 9: Evaluation of the efficacy of ADC drugs on human prostate cancer cell 22Rv1 xenografts in nude mice

I. Testing Methods

Male nude/nude mice, 6-8 weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Certificate No. 1908120082). The mice were housed in SPF-grade conditions. Human prostate cancer cells 22Rv1 (Chinese Academy of Sciences) were subcutaneously inoculated into the nude mice. When the average tumor volume reached 190 ^mm³ , the animals were randomly divided into groups (D0), with 8 mice in each group. Intraperitoneal injections of the drug were initiated twice a week for a total of 4 administrations. Tumor volume and body weight were measured twice weekly, and data were recorded.

The formula for calculating tumor volume (V) is: V = 1/2 × a × b ^2, where a and b represent length and width, respectively.

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

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

II. Test Subjects

ADC-11: 5mpk

ADC-12: 5mpk

Blank control group: PBS buffer at pH 7.4

III. Antitumor effect of antibody ADCs

By day 13 (D13) after the start of administration, the tumor inhibition rates of ADC-11 and ADC-12 at a dose of 5 mpk were 87.63% and 79.82%, respectively, both significantly better than the control group. There was no significant difference between ADC-11 and ADC-12 (Table 12, Figure 9).

The mice maintained a stable weight during the administration process, indicating that the tested antibodies did not have significant toxic side effects.

Table 12. Efficacy of ADC administration on 22Rv1 xenografts in tumor-bearing nude mice.

vs. blank control group: **p<0.01, ***p<0.001.

sequence list

<110> Jiangsu Hengrui Medicine Co., Ltd.

Shanghai Hengrui Medicine Co., Ltd.

<120> Anti-PSMA antibody-ecetane analogue conjugate and its pharmaceutical uses

<130> 721024CPCT

<150> CN202010218297.4

<151> 2020-03-25

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<223> AB-PG1-XG1-006 Heavy Chain Variable Region

<400> 1

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Arg Tyr

20 25 30

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

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Gln Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asp Phe Leu Tyr Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val

100 105 110

Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser Ser

115 120

<210> 2

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<223> AB-PG1-XG1-006 Light Chain Variable Zone

<400> 2

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 Arg Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

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

35 40 45

Tyr Glu Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Asn Tyr Asn Ser Ala Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 3

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<223> AB-PG1-XG1-006 HCDR1

<400> 3

Arg Tyr Gly Met His

1 5

<210> 4

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<223> AB-PG1-XG1-006 HCDR2

<400> 4

Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 5

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<223> AB-PG1-XG1-006 HCDR3

<400> 5

Gly Gly Asp Phe Leu Tyr Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10

<210> 6

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<223> AB-PG1-XG1-006 LCDR1

<400> 6

Arg Ala Ser Gln Gly Ile Ser Asn Tyr Leu Ala

1 5 10

<210> 7

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<223> AB-PG1-XG1-006 LCDR2

<400> 7

Glu Ala Ser Thr Leu Gln Ser

1 5

<210> 8

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<221> DOMAIN

<223> AB-PG1-XG1-006 LCDR3

<400> 8

Gln Asn Tyr Asn Ser Ala Pro Phe Thr

1 5

<210> 9

<211> 453

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<223> PM-HC

<400> 9

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Arg Tyr

20 25 30

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

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Gln Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Asp Phe Leu Tyr Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val

100 105 110

Trp Gly Gln Gly Thr Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Leu Ser Pro Gly Lys

450

<210> 10

<211> 214

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<223> PM-LC

<400> 10

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 Arg Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

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

35 40 45

Tyr Glu Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Asn Tyr Asn Ser Ala Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp 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> 11

<211> 449

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<223> Antibody heavy chain sequence Lmab-HC

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Thr Thr Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys

85 90 95

Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

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

325 330 335

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

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

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

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

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

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 12

<211> 213

<212> PRT

<213> Artificial Sequence

<220>

<221> CHAIN

<223> Antibody light chain sequence Lmab-LC

<400> 12

Asp Ile Gln Leu 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 Gly Thr Ser

20 25 30

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

35 40 45

Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Leu Tyr Arg Ser

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Arg Gly Glu Cys

210

<210> 13

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<221> PEPTIDE

<223> Tetrapeptide Linker

<400> 13

Gly Gly Phe Gly

1

Claims (17)

1. An antibody-drug conjugate of the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof: in: Y is -O-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-; ^Ra and ^Rb may be the same or different, and each is independently selected from hydrogen atoms, deuterium atoms, halogens and _C1-6 alkyl groups; ^R1 is a halogenated _C1-6 alkyl or _C3-6 cycloalkyl; ^R2 is selected from hydrogen atoms, halogenated _C1-6 alkyl groups, and _C3-6 cycloalkyl groups; Alternatively, ^R1 and ^R2 together with the carbon atoms they are attached to form a _C3-6 cycloalkyl group; m is 0 or 1; n is between 1 and 10, and n is a decimal or an integer; L represents the connector unit - ^L1 - ^L2 - ^L3 - ^L4 -, ^L1 is selected from -(succinimide-3-yl-N)-WC(O)-, _-CH2 -C(O) ^-NR3- WC(O)- and -C(O)-WC(O)-, wherein W is selected from _C1-8 alkyl and _C1-8 alkyl- _C3-6 cycloalkyl; ^L2 is selected from ^-NR4 ( _CH2CH2O ) _p1CH2CH2C (O)-, ^-NR4 ( _CH2CH2O ) _p1CH2C (O)-, ^-S _{( CH2} ) ^p1C (O)- and chemical ^bonds , where ^p1 is an integer from ₁ to ₂₀ ; ^L3 is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acids formed from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid. ^L4 is selected from ^-NR5 ( ^CR6R7 ) _t- , -C(O) ^NR5 , -C(O) ^NR5 ( _CH2 ) _t- and chemical bonds, where ^t is an integer from 1 to 6; ^R3 , ^R4 and ^R5 may be the same or different, and each is independently selected from hydrogen atoms, _C1-6 alkyl, halo- _C1-6 alkyl, deuterated _C1-6 alkyl and _C1-6 hydroxyalkyl; ^R6 and ^R7 may be the same or different, and each is independently selected from hydrogen atoms, halogens, _C1-6 alkyl, halo- _C1-6 alkyl, deuterated _C1-6 alkyl and _C1-6 hydroxyalkyl; Pc is an anti-PSMA antibody or its antigen-binding fragment, containing a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 respectively. 2. The antibody-drug conjugate of the general formula (Pc-L-Y-D) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the anti-PSMA antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. 3. The antibody-drug conjugate of the general formula (Pc-L-Y-D) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the anti-PSMA antibody or its antigen-binding fragment comprises a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO: 2. 4. The antibody-drug conjugate of the general formula (Pc-L-Y-D) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the anti-PSMA antibody comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and their conventional variants, and the light chain constant region is selected from the constant regions of human antibody κ and λ chains and their conventional variants. 5. The antibody-drug conjugate of the general formula (Pc-L-Y-D) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the anti-PSMA antibody comprises a heavy chain as shown in SEQ ID NO: 9 and a light chain as shown in SEQ ID NO: 10. 6. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-L-Y-D) according to claim 1, wherein n is 1 to 8, and n is a decimal or an integer. 7. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-L-Y-D) according to claim 1, wherein n is 3 to 8, and n is a decimal or an integer. 8. The antibody-drug conjugate of the general formula (Pc-L-Y-D) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Y is selected from: The O end of Y is connected to the connector unit L. 9. The antibody-drug conjugate of the general formula (Pc-LYD) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the linker unit L is ^{-L1 -L2 -L3 -L4} -, ^L1 is an integer from 2 to 8 for ^s1 ; ^L2 is a chemical bond; ^L3 is a tetrapeptide residue; ^L4 is ^-NR5 ( ^CR6R7 )t-, ^{where R5} , ^R6 , or ^R7 may be the same or different, and each is independently a hydrogen atom or a _C1-6 alkyl group, and t is 1 or 2; The ^L1 terminal is connected to Pc, and the ^L4 terminal is connected to Y. 10. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-L-Y-D) according to claim 1, wherein L is: 11. The antibody-drug conjugate of the general formula (Pc-L-Y-D) according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is selected from: in Pc is an anti-PSMA antibody or its antigen-binding fragment, containing a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 respectively; n is between 1 and 10, and n can be a decimal or an integer. 12. The antibody-drug conjugate of the general formula (Pc-L-Y-D) according to claim 11, or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is: in: n is between 1 and 8, where n is a decimal or an integer; PM is an anti-PSMA antibody that contains a heavy chain as shown in SEQ ID NO: 9 and a light chain as shown in SEQ ID NO: 10. 13. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of the general formula (Pc-L-Y-D) according to claim 12, wherein n is 3 to 8, and n is a decimal or an integer. 14. A pharmaceutical composition comprising an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, and one or more pharmaceutically acceptable excipients, diluents or carriers. 15. Use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, or the pharmaceutical composition according to claim 14, in the preparation of a medicament for the treatment and/or prevention of tumors and cancer. 16. The use according to claim 15, wherein the tumor and cancer are selected from squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastrointestinal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, leukemia, lymphoma, bone cancer, myeloma, myelodysplastic syndrome, Kuckenberg tumor, myeloproliferative neoplasm, squamous cell carcinoma, urothelial carcinoma, and Merkel cell carcinoma. 17. The use according to claim 16, wherein the lymphoma is selected from: Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells/histocytes, and lymphoplasmacytic lymphoma; the lung cancer is selected from: non-small cell lung cancer and small cell lung cancer; the leukemia is selected from: chronic myeloid leukemia, acute myeloid leukemia, etc. Leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia; the gastrointestinal cancer is selected from gastric cancer, intestinal cancer, and colorectal cancer; the kidney cancer is clear cell renal cell carcinoma; the skin cancer is melanoma; the bone cancer is chondrosarcoma and Ewing's sarcoma; the myeloma is multiple myeloma; the pharyngeal cancer is nasopharyngeal carcinoma; the brain cancer is selected from glioma and glioblastoma multiforme.