Dual-ligand drug conjugate and its use

Abstract

A conjugate compound or a pharmaceutically acceptable salt thereof, wherein the conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload and two targeting molecules. Also relates to a pharmaceutical composition, wherein the pharmaceutical composition comprises the conjugate compound or a pharmaceutically acceptable salt thereof. In addition, it relates to a method for delivering a payload to a subject in need thereof and a method for treating a disease, wherein the method comprises administering a therapeutically effective amount of the conjugate compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition to the subject.

Description

Technical Field

[0001] The present application relates to the field of biomedical chemistry. More specifically, the present application relates to a dual-ligand drug conjugate, a pharmaceutical composition comprising the dual-ligand drug conjugate, a method of using the dual-ligand drug conjugate to deliver a payload to a subject in need thereof, and a method of using the dual-ligand drug conjugate to treat a disease. Background Art

[0002] Generally, the pathological and physiological characteristics of diseased cells are significantly different from those of normal cells. One of them is that the surface of diseased cells has specific or overexpressed substances (e.g., antigens, chemical signals, receptors, etc.), which are not expressed or expressed at a low level in normal cells. Based on this principle, antibody-drug conjugates (ADCs) and peptide-drug conjugates (PDCs) have been developed to treat diseases. At present, although some ADC and PDC drugs have been launched or entered clinical research, due to the design principles of these drugs, ADC and PDC have great limitations in clinical applications.

[0003] Due to the complexity and large molecular weight of ADC, its development faces many difficulties, including lack of suitable targets, production difficulties and poor drug stability. Currently, ADC is mainly used in the field of tumor treatment. In some cases, the affinity of targeted antibodies for cancer cell surface antigens can be as high as 10 -9 ~10 -12 (Kd, mol / L), so while having high specificity for target cells, ADC also has high specificity for normal cells with the same targeting receptor as target cells. At the same time, since ADC has a long metabolism time in the body (1 to 3 weeks), it will continue to kill normal cells during this period, thus greatly increasing the toxic side effects of ADC. Therefore, the more ideal indication for ADC should be a disease characterized by a very different amount of cell surface antigens in tumors and normal cells. However, there are very few diseases that meet this strict requirement.

[0004] PDC is used to treat a variety of diseases in clinical or preclinical studies, but these are simply to connect chemotherapy drugs with peptides, or to add peptides to nanoparticles or polymer materials that encapsulate chemotherapy drugs. This will prevent most peptides from entering cells due to their large molecular weight and charge. Therefore, most of these PDCs are currently only suitable for extracellular treatment, which severely limits the scope of application and efficacy of PDC.

[0005] Drug conjugate compounds can also be ligand-drug conjugates (LDCs), in which the ligands can be peptides or small molecules. However, there are many problems with the application of LDCs in terms of bioavailability, stability, efficacy, and toxicity. For example, many ligands cannot enter cells due to their large molecular weight, lipophilicity, or other properties, which limits their therapeutic applications. In addition, if the ligand is coupled to conventional chemotherapeutic drugs (e.g., doxorubicin, paclitaxel, etc.), the efficacy is usually low, and if it is coupled to highly effective drug molecules (e.g., MMAE, DM1), the toxicity is relatively large, and may even cause animal poisoning and death before reaching the therapeutically effective amount for tumor treatment.

[0006] Therefore, there is an urgent need in the field to obtain improved LDCs that can act on the widely expressed receptors on the surface of diseased cells, broaden the targeting range and therapeutic window, and enhance drug efficacy and avoid drug side effects. Summary of the invention

[0007] One aspect of the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, wherein the conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and a prostate-specific membrane antigen ligand portion, respectively.

[0008] Another aspect of the present application discloses a coupled compound or a pharmaceutically acceptable salt thereof, wherein the coupled compound or a pharmaceutically acceptable salt thereof comprises a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and a ligand portion having formula (I):

[0009]

[0010] Another aspect of the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, wherein the conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and P10, respectively, and the payload is camptothecin and any derivative thereof.

[0011] In some embodiments, the prostate-specific membrane antigen ligand contained in the conjugate compound or a pharmaceutically acceptable salt thereof comprises the following structure:

[0012]

[0013] In some embodiments, the prostate-specific membrane antigen ligand contained in the conjugate compound or a pharmaceutically acceptable salt thereof comprises the following structure:

[0014]

[0015] In some embodiments, the prostate-specific membrane antigen ligand contained in the conjugate compound or a pharmaceutically acceptable salt thereof comprises the following structure:

[0016]

[0017] In some embodiments, the prostate-specific membrane antigen ligand contained in the conjugate compound or a pharmaceutically acceptable salt thereof comprises the following structure:

[0018]

[0019] In some embodiments, the two targeting molecules contained in the conjugate compound or a pharmaceutically acceptable salt thereof are different.

[0020] In some embodiments, the synergistic molecule contained in the conjugate compound or a pharmaceutically acceptable salt thereof is a cell-interacting molecule.

[0021] In some embodiments, the conjugate compound or a pharmaceutically acceptable salt thereof comprises two targeting molecules that interact with different cellular molecules.

[0022] In some embodiments, the synergistic molecule comprised by the conjugate compound or a pharmaceutically acceptable salt thereof is an endocytosis molecule capable of mediating endocytosis.

[0023] In some embodiments, the synergistic molecule contained in the conjugate compound or its pharmaceutically acceptable salt binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), GNRHR, Her2, Trop2, Her3, NECTIN4, LRP1, GLUT1, EGFR1, AXL, CA9, CD44, Claudin18.2, APN, DLL3, CEACAM5, FZD10, TFRC, MET, IGFR1, SSTR2, CCKBR, LFA1, ICAM, GPR87, GM-CSF, GM-CSFR, TIM3, TLR family, CD40, CD40L, OX40, OX40L, GITRL, GITR, 4-BBL, 4 -1BB, CD70, CD27, ICOSL, ICOS, HHLA2, CD28, CD86 / 80, CD28, MHCII antigens, TCR, CTLA-4, CD155, CD122, CD113, IGIT, PD-L1, PD1, Galectin-9, TIM-3, HVEM, BTLA, CD160, VISTA, B7-H4, B7-H3, phosphatidylserine, HHLA2, LAG3, Galectin-3, LILRB4, SIGLEC15, NKG2A, NKG2D, SLAMF7, KIR2DL1, KIR2DL2, KIR2DL3, FGFR1, FGFR2, FGFR4, NeuGcGM3, and CXCR4.

[0024] In some embodiments, the conjugate compound or a pharmaceutically acceptable salt thereof comprises a prostate-specific membrane antigen ligand portion and a synergistic molecule portion that binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), SSTR2, and GNRHR.

[0025] In some embodiments, the conjugate compound or a pharmaceutically acceptable salt thereof comprises a ligand portion represented by formula (I) and a synergistic molecule portion, wherein the synergistic molecule portion binds to a molecule selected from the group consisting of FOLR1, TRPV6, SSTR2 and GNRHR.

[0026] In yet other embodiments, the conjugate compound or a pharmaceutically acceptable salt thereof comprises P10 and a synergistic molecule portion that binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA) and GNRHR.

[0027] In some embodiments, the synergistic molecule contained in the conjugate compound or its pharmaceutically acceptable salt is folic acid or its analog. In some embodiments, the folic acid analog is selected from the group consisting of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate and 5,10-methylenetetrahydrofolate.

[0028] In some embodiments, the conjugate compound or a pharmaceutically acceptable salt thereof comprises one, two, three, four or more payloads. In some embodiments, the payload is selected from the group consisting of small molecule compounds, nucleotides, peptides, and proteins. In some embodiments, the payload is a small molecule compound. In some embodiments, the small molecule compound is selected from the group consisting of camptothecin and any derivative thereof, auristatin and any derivative thereof, maytansine and any derivative thereof, radionuclide complexes, cyclooxygenase-2 inhibitors, paclitaxel and any derivative thereof, epothilone and any derivative thereof, bleomycin and any derivative thereof, dactinomycin and any derivative thereof, plicamycin and any derivative thereof, and mitomycin C. In some embodiments, the small molecule compound is camptothecin and any derivative thereof, auristatin and any derivative thereof, maytansine and any derivative thereof, radionuclide complexes, or cyclooxygenase-2 inhibitors.

[0029] In some embodiments, the effective load contained in the conjugate compound of the present application or a pharmaceutically acceptable salt thereof is connected to at least one of the targeting molecules via a linker.

[0030] In some embodiments, the linker contained in the conjugate compound of the present application or a pharmaceutically acceptable salt thereof is a peptide linker, a disulfide linker, a pH-dependent linker, or a combination of the above linkers.

[0031] In some embodiments, the peptide linker can be cleaved by protease or reductive cleavage under specific physiological conditions. In some embodiments, the peptide linker is selected from the group consisting of cysteine, lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-lysine, cysteine-glutamic acid, aspartic acid-aspartic acid and aspartic acid-aspartic acid-lysine, and optionally, the carboxylic acid in the above amino acids is amidated.

[0032] In some embodiments, the disulfide linker is selected from the group consisting of DMDS, MDS, DSDM, and NDMDS.

[0033] In some embodiments, the pH-dependent linker is cis-aconitic anhydride.

[0034] In some embodiments, the linker of the conjugate compound of the present application or a pharmaceutically acceptable salt thereof comprises the following structure:

[0035]

[0036]

[0037]

[0038] Alternatively, the linker is a combination of the above structure and a peptide linker.

[0039] In some embodiments, the two targeting molecules contained in the conjugate compound of the present application or its pharmaceutically acceptable salt are connected by a spacer. In some embodiments, the spacer described in the present application comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14, Arg-Arg, Ala-Ser-Asn, Ala-Ala-Ala, Ser-Ser-Arg, Pro-Arg and Pro-Leu-Gly.

[0040] In some embodiments, the conjugate compound of the present application is CB-20B, and its structural formula is as follows:

[0041]

[0042] In some embodiments, the conjugate compound of the present application is CB-20BK, and its structural formula is as follows:

[0043]

[0044] In some embodiments, the conjugate compound of the present application is CB-60S, and its structural formula is as follows:

[0045]

[0046] In some embodiments, the conjugate compound of the present application is CB-60SK, and its structural formula is as follows:

[0047]

[0048] In some embodiments, the conjugate compound of the present application is CB-20C, and its structural formula is as follows:

[0049]

[0050] In some embodiments, the conjugate compound of the present application is CB-1020, and its structural formula is as follows:

[0051]

[0052] In some embodiments, the conjugate compound of the present application is CB-1320, and its structural formula is as follows:

[0053]

[0054] In some embodiments, the conjugate compound of the present application is CB-1820, and its structural formula is as follows:

[0055]

[0056] In some embodiments, the conjugate compound of the present application is CR19428, and its structural formula is as follows:

[0057]

[0058] In some embodiments, the conjugate compound of the present application is 20R-SM09, and its structural formula is as follows:

[0059]

[0060] In some embodiments, the conjugate compound of the present application is CB-20R, and its structural formula is as follows:

[0061]

[0062] Where M is the radionuclide.

[0063] In some embodiments, the conjugate compound of the present application is CB-18G, and its structural formula is as follows:

[0064]

[0065] In some embodiments, the conjugate compound of the present application is CR19426, and its structural formula is as follows:

[0066]

[0067] In some embodiments, the conjugate compound of the present application is CB-10S, and its structural formula is as follows:

[0068]

[0069] In some embodiments, the conjugate compound of the present application is CR19425, and its structural formula is as follows:

[0070]

[0071] In some embodiments, the conjugate compound of the present application is CB-50S, and its structural formula is as follows:

[0072]

[0073] Another aspect of the present application discloses a pharmaceutical composition, which comprises the conjugate compound of the present application or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0074] In some embodiments, the composition is for intravenous, subcutaneous, oral, intramuscular, or intraventricular administration.

[0075] Another aspect of the present application discloses a method for delivering a payload to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the conjugate compound described in the present application or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described in the present application.

[0076] Another aspect of the present application discloses a method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the conjugate compound described in the present application or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described in the present application.

[0077] In some embodiments, the method for treating a disease in a subject of the present application further comprises administering one or more therapeutic agents in combination with the conjugate compound or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition.

[0078] Another aspect of the present application discloses a use of the conjugate compound described in the present application or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described in the present application in the preparation of a drug for treating a disease in a subject.

[0079] Another aspect of the present application discloses a conjugate compound described in the present application or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described in the present application, for treating a disease in a subject.

[0080] In some embodiments, the disease is selected from the group consisting of cancer, immune disease, cardiovascular disease, metabolic disease, and neurological disease.

[0081] In some embodiments, the cancer is selected from the group consisting of prostate cancer, breast cancer, lung cancer, kidney cancer, leukemia, ovarian cancer, stomach cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, skin cancer, lymphoma, and multiple myeloma.

[0082] In some embodiments, the immune disease is an autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of connective tissue disease, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

[0083] In some embodiments, the cardiovascular disease is selected from the group consisting of angina, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmia, and congenital heart disease.

[0084] In some embodiments, the metabolic disease is selected from the group consisting of diabetes, gout, obesity, hypoglycemia, hyperglycemia, and dyslipidemia.

[0085] In some embodiments, the neurological disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, head injury, multiple sclerosis, vertigo, coma, and epilepsy. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 The chemical structures of the coupled compounds CB-20B, CB-20BK, CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB-1820, CR19428, 20R-SM09, CB-20R, CB-18G, CR19426, CB-10S, CR19425 and CB-50S are shown.

[0087] Figure 2A The graph shows the time course of the binding and internalization of Cy5-pep-20BK to different cells (from top to bottom: LNCaP cells, SKOV3 cells, DU145 cells, and NCI-H460 cells). Figure 2B The graph shows the time course of the binding and internalization of Cy5-pep-20AK to different cells (from top to bottom: LNCaP cells, SKOV3 cells, DU145 cells, and NCI-H460 cells).

[0088] Figure 3 The fluorescence photos of Cy5-FA binding and internalization in different cells over time are shown. The complete circle is the fluorescence of the cell nucleus, and the dot-like distribution is the fluorescence of Cy5-FA.

[0089] Figure 4A The inhibitory activity of the conjugate compound CB-20BK on the proliferation of the indicated tumor cells is shown. Figure 4B The inhibitory activity of the conjugate compound CB-20B on the proliferation of the indicated tumor cells is shown. Figure 4C The inhibitory activity of the conjugate compound CB-10S on the proliferation of the indicated tumor cells is shown. Figure 4D The inhibitory activity of the conjugate compound CB-60S on the proliferation of the indicated tumor cells is shown. Figure 4E The inhibitory activity of the conjugate compound CB-60SK on the proliferation of the indicated tumor cells is shown. Figure 4F The inhibitory activity of the conjugate compound CB-18G on the proliferation of the indicated tumor cells is shown. Figure 4G The inhibitory activity of the conjugate compound CB-50S on the proliferation of the indicated tumor cells is shown.

[0090] Figures 5A-5E The results show that the coupled compound CB-20BK has an inhibitory effect on tumors in mice.

[0091] Figures 6A-6C The results show that the coupled compound CB-20B has an inhibitory effect on tumors in mice.

[0092] Figures 7A-7E The results showed that the coupled compound CB-18G has an inhibitory effect on tumors in mice.

[0093] Figures 8A-8B The effect of injected CBP-1018 on tumor volume in the LU2505 lung cancer model and the LU1206 lung cancer model is shown. DETAILED DESCRIPTION

[0094] Although the present application will disclose various aspects and embodiments, it is obvious that, without departing from the subject matter and scope of the present application, those skilled in the art may make various equivalent changes and modifications to these aspects and embodiments. The various aspects and embodiments disclosed in the present application are for illustrative purposes only and are not intended to be limiting, and the true scope is represented by the attached claims. All publications, patents or patent applications cited in the present application are incorporated herein by reference in their entirety. Unless otherwise stated, all scientific and technological terms used in the present application have the same meaning as those commonly understood by those skilled in the art to which the present application belongs.

[0095] As used herein and in the appended claims, the singular forms "a," "an," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably in this application. It should also be noted that the terms "comprising," "including," and "having" may be used interchangeably.

[0096] As used herein and in the appended claims, the term "analog" includes structural analogs and functional analogs. Structural analogs refer to a class of compounds with similar chemical structures, which may have one or more different atoms or one or more different functional groups. Functional analogs refer to a class of compounds with the same or similar chemical, biological or pharmacological effects. For example, folic acid analogs include 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate and 5,10-methylenetetrahydrofolate.

[0097] As used herein and in the appended claims, the term "derivative" refers to a class of more complex compounds derived from a parent compound molecule in which one or more atoms or groups of atoms are replaced by other atoms or groups of atoms. For example, camptothecin derivatives include irinotecan, SN-38, Dxd, topotecan, GI-147211C, topotecan, 9-aminocamptothecin, 7-hydroxymethylcamptothecin, 7-aminomethylcamptothecin, 10-hydroxycamptothecin, (20S)-camptothecin, 9-nitrocamptothecin, gimatine, karenitecin, silatecan, lortotecan, exatecan, diflutecan, belotecan, lortotecan and S39625

[0098] One aspect of the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, wherein the conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and a prostate-specific membrane antigen ligand portion, respectively.

[0099] Another aspect of the present application discloses a coupled compound or a pharmaceutically acceptable salt thereof, wherein the coupled compound or a pharmaceutically acceptable salt thereof comprises a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and a ligand portion having formula (I):

[0100]

[0101] Another aspect of the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, wherein the conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and P10, respectively, and the payload is camptothecin and any derivative thereof.

[0102] The term "payload" used in this application refers to a molecule or substance intended to be delivered to a target cell or tissue. Without limitation, a payload can be any molecule or substance intended to be used for diagnosing, treating or preventing a disease in a subject. In some embodiments, the payload has a molecular weight less than or equal to about 5 kDa. In some embodiments, the payload has a molecular weight less than or equal to about 1.5 kDa. In some embodiments, the payload is a drug or diagnostic agent that has been considered safe and effective by appropriate drug approval and registration agencies (e.g., FDA, EMEA or NMPA).

[0103] In some embodiments, the effective load of the present application is a small molecule compound, a nucleotide (e.g., DNA, plasmid DNA, RNA, siRNA, antisense oligonucleotide or nucleic acid aptamer, etc.), a peptide or a protein (e.g., an enzyme). In some embodiments, the effective load is a small molecule compound.

[0104] In some embodiments, the payload of the present application includes but is not limited to: anticancer drugs, radioactive substances, vitamins, anti-AIDS drugs, antibiotics, immunosuppressants, antiviral drugs, enzyme inhibitors, neurotoxins, opioids, regulators of cell-extracellular matrix interactions, vasodilators, antihypertensives, hypnotics, antihistamines, anticonvulsants, muscle relaxants, anti-Parkinson's substances, anti-spasmodics and muscle contractants, anti-parasitic and / or anti-protozoan drugs, analgesics, antipyretics, steroidal and non-steroidal anti-inflammatory drugs, anti-angiogenic factors, antisecretory factors, anticoagulants and / or antithrombotic agents, local anesthetics, prostaglandins, antidepressants, antipsychotics, antiemetics or imaging agents.

[0105] In some embodiments, the effective load of the present application has a free amino group or carboxyl group before being connected to the conjugate compound of the present application, and the effective load is coupled to the conjugate compound by acylation reaction of the above-mentioned amino group or carboxyl group with the corresponding part (e.g., linker) of the conjugate compound. In some embodiments, modification of the above-mentioned free amino group or carboxyl group (e.g., by conjugation to the conjugate compound of the present application) can significantly reduce the activity of the effective load (e.g., by reducing at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%).

[0106] "Small molecule compound" used in the present application refers to a compound having a molecular weight of less than or equal to about 2kDa. In some embodiments, the small molecule compound has a molecular weight of less than or equal to about 1.5kDa. In some preferred embodiments, the small molecule compound has a molecular weight of less than or equal to about 1kDa, 800Da, 700Da, 600Da or 500Da. In some embodiments, the small molecule compound of the present application is selected from the group consisting of camptothecin and any derivative thereof (e.g., SN38 or Dxd), auristatin and any derivative thereof (e.g., MMAE and MMAF), maytansine and any derivative thereof, cyclooxygenase-2 inhibitors (e.g., celecoxib), radionuclide complexes, paclitaxel and any derivative thereof, epothilone and any derivative thereof, bleomycin and any derivative thereof, dactinomycin and any derivative thereof, plicamycin and any derivative thereof, and mitomycin C. In some embodiments, the small molecule compound is camptothecin and any derivative thereof, auristatin and any derivative thereof, a radionuclide complex or a cyclooxygenase-2 inhibitor. In some embodiments, the small molecule compound described herein is a drug for alleviating or treating cancer. In some embodiments, the small molecule compound described herein is a drug for alleviating or treating autoimmune diseases.

[0107] The term "camptothecin" used in this application refers to a cytotoxic alkaloid, mainly derived from the plant Camptotheca acuminata of the Davidiaceae family, which shows strong anti-tumor activity. The camptothecin and its derivatives of the present application include camptothecin and its derivatives that are currently existing or produced later. The camptothecin and its derivatives of the present application include but are not limited to: camptothecin, irinotecan, SN-38, Dxd, topotecan, GI-147211C, topotecan, 9-aminocamptothecin, 7-hydroxymethylcamptothecin, 7-aminomethylcamptothecin, 10-hydroxycamptothecin, (20S)-camptothecin, 9-nitrocamptothecin, gimatine, karenitecin, silatecan, lertotecan, exatecan, diflutecan, belotecan, lertotecan and S39625.

[0108] The term "auritasin and any derivatives thereof" used in this application refers to the natural anti-tumor product dolastatin 10 and a series of its derivatives, which have a strong killing effect on cells by interfering with microscopic self-assembly to cause cells to arrest in mitosis. The auristatins and any derivatives thereof of this application include auristatins and any derivatives thereof that are currently existing or produced later. The auristatins and derivatives thereof of this application include, but are not limited to, auristatins, monomethyl auristatins E (MMAE), monomethyl auristatins F (MMAF), monomethyl auristatins D (MMAD), AFP and AFHPA.

[0109] The term "cyclooxygenase-2 inhibitor" used in the present application is a class of specific cyclooxygenase-2 inhibitors. Cyclooxygenase-2 participates in the development and infiltration of malignant tumors through multiple mechanisms, and cyclooxygenase-2 inhibitors can inhibit the migration and adhesion of tumor cells and intravascular infiltration, thereby inhibiting the generation and development of malignant tumors. The cyclooxygenase-2 inhibitor of the present application comprises the cyclooxygenase-2 inhibitor that currently exists or produces afterwards. Cyclooxygenase-2 inhibitors include but are not limited to celecoxib, rofecoxib, parecoxib, valdecoxib and etoxib.

[0110] The term "radionuclide complex" used in this application refers to a special type of complex containing radionuclides, in which the chelating agent in the complex can chelate with the radionuclide and provide a linking portion that more stably binds to the targeting substance. The term "radionuclide" used in this application refers to an element that can spontaneously emit radiation (such as α rays, β rays or γ rays, etc.). The radionuclides of this application include all radionuclides that are currently in existence or will be produced in the future and can be used for treatment and diagnosis. The radionuclides of this application include but are not limited to 67 Cu, 64 Cu, 90 Y. 109 Pd, 111 Ag, 149 Pm, 153 Sm, 165 Ho, 166 Ho, 177 Lu, 186 Re, 188 Re, 99m Tc, 67 Ga, 68 Ga, 111 In, 90 Y. 177 Lu, 186 Re, 188 Re, 197 Au, 198 Au, 199 Au, 105 Rh, 161 Tb, 149 Pm, 44 Sc, 47 Sc, 70 As, 71 As, 72 As, 73 As, 74 As, 76 As, 77 As, 212 Pb, 212 Bi, 213 Bi,225 Ac、 117m Sn、 67 Ga、 201 Tl、 123 I、 131 I、 160 Gd、 148 Nd、 89 Sr 211At H4octapa, H2azapa, DTPA, CHX-A”-DTPA, DTPA-bis anhydride、Maleimide-DTPA、DTPA(tBu)4、DiamSar CB-TE2A、Cyclam、DO2A、DOTA、OTA-GA(tBu)4、Maleimide-DOTA-GA、p-NCS-Bz-DOTA-GA、NH2-DOTA-GA、DOTA-GA anhydride、DOTA-tris(tBu)ester、Propargyl-DOTA-tris(tBu)ester、DO3AM-acetic acid、DO3AM-N-(2-aminoethyl)ethanamide、DO3AtBu-N-(2-aminoethyl)ethanamide、DOTA-di(tBu)ester、DOTA-tris(tBu)ester NHS ester、DOTA-NHS ester、Propargyl-DOTA-tris(tBu)ester、DOTADOTA-GA anhydride、DOTA-GA(tBu)4、p-NCS-Bz-DOTA-GA、NH2-DOTA-GA、Maleimide-DOTA-GA、AGuIX、Gado-H、CY CLEN, DO2AtBu, DO3AtBu, DO3AEt, DO3AM, DOTAEt, DOTPrEt, cis-Glyoxal-Cyclen, Mono-N-Benzyl-Cycl en、trans-N-Dibenyl-Cyclene、TriBOC-Cyclene、Mono-N-Benzyl-TACN、DiBOC-TACN、Cross-bridge-Cyc lam(CB-Cyclam), (13)aneN4, TACN, TACN·3HCl, TACD, Mono-N-benzyl-TACD, DiBOC-TACD, 1,7-Dioxa-4,10-diazacyclododecane, C-Methyl-Ester-Cyclam, C-Carboxylic-Acid-Cyclam, trans-N-Dimethyl-Cyclam, TETRAM, TETAEt, TETAMEt2, TETAMMe2, TETAM, CPTA, C B-Cyclamderivatives, CB-TE2A, Methylamino-(13)aneN4, Bis-(13)aneN4, Oxo-(13)aneN4, Mono-N-Benzyl-(13)aneN4, TriBOC-(13)aneN4, TRITRAM, TRI3AEt, TR I3AtBu, TRITAM, TRITA, Mono-N-Benzyl-Cyclam, Formaldehyde-Cyclam, cis-Glyoxal-Cyclam, Dioxocyclam, Oxocyclam, trans-N-Dibenzyl-Cyclam, TriBOC-Cycl am, DOTP, DOTMA, TETA, DOTAM, DiAmSar, CB-Cyclam, CB-TE2A, NOTA, NOTAM, NH2-NODA-GA, Iodo-NODA-GA, NCS-MP-NODA, NH2-MPAA-NODA, NODA-GA(tBu)3, NODA-GA-NHS ester, Maleimide-NODA-GA, NOTA-NHS ester, Maleimide-NOTA, Propargyl-NOTA(tBu)2, p-NCS-benzyl-NODA-GA, NOTA(tBu)2, NCS-MP-NODA, NH2-MPAA-NODA, NH2-NODA-GA, Iodo-NODA-GA and TACN. ,

[0111] In some embodiments, the conjugate compound of the present application or its pharmaceutically acceptable salt comprises a payload. In some embodiments, the conjugate compound of the present application or its pharmaceutically acceptable salt comprises two or more payloads. For example, the conjugate compound of the present application or its pharmaceutically acceptable salt comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more payloads. In a conjugate compound containing multiple payloads, each payload may be the same or different from each other. In some embodiments, at least two payloads are different from each other.

[0112] The term "targeting molecule" used in the present application refers to any molecule or part that can target the conjugate compound of the present application to a target site, target tissue, target organ, target cell or target cell intraregional area. In some embodiments, the targeting molecule makes the conjugate compound of the present application more distributed in the target site, target tissue, target organ, target cell or target cell intraregional area compared to the non-target site, non-target tissue, non-target organ, non-target cell or non-target cell intraregional area, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or higher. In some embodiments, the targeting molecule allows the conjugate compound with the targeting molecule to be distributed more at the target site, target tissue, target organ, target cell or target cell intracellular region than without the targeting molecule, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more. In some embodiments, the targeting molecule can trigger or promote the specific binding of the conjugate compound containing such targeting molecule to the target molecule, trigger or promote the endocytosis of the conjugate compound by the target cell, trigger or promote the enrichment of the conjugate compound around the target cell and / or enter the target cell.

[0113] In some embodiments, the conjugate compound of the present application includes at least two targeting molecules. In some embodiments, the two or more targeting molecules included in the conjugate compound of the present application are the same or different. In some embodiments, at least two of the two or more targeting molecules included in the conjugate compound of the present application are different. In some embodiments, the two or more targeting molecules included in the conjugate compound of the present application are different from each other. In some embodiments, at least two of the two or more targeting molecules included in the conjugate compound of the present application are capable of specifically binding to different cell surface proteins or markers. In some embodiments, the two or more targeting molecules included in the conjugate compound of the present application are capable of specifically binding to different cell surface proteins or markers.

[0114] In some embodiments, the conjugate compound of the present application comprises at least two targeting molecules, at least one of which is a synergistic molecule.

[0115] The term "synergistic molecule" used in the present application refers to any molecule or part that can synergize with other targeting molecules contained in the conjugate compound of the present application to better trigger or promote the specific binding of the conjugate compound to the target molecule, trigger or promote the endocytosis of the conjugate compound by the target cell, trigger or promote the enrichment of the conjugate compound around the target cell and / or enter the target cell, and / or cause the conjugate compound to specifically bind to and retain the target cell in other forms. In some embodiments, the synergistic molecule makes the conjugate compound of the present application more distributed in the target site, target tissue, target organ, target cell or target cell intraregional area compared to the non-target site, non-target tissue, non-target organ, non-target cell or non-target cell intraregional area, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more. In some embodiments, the synergistic molecule makes the conjugate compound with the synergistic molecule more distributed in the target site, target tissue, target organ, target cell or target cell intracellular area than without the synergistic molecule, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more. In some embodiments, the synergistic molecule makes the conjugate compound with the synergistic molecule more active on the target cell than without the synergistic molecule, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more.

[0116] In some embodiments, the synergistic molecules of the present application are cell interaction molecules.

[0117] The term "cell interaction molecule" as used in this application refers to a molecule that is capable of interacting with cell surface substances of target cells to trigger or promote the specific binding of a conjugate compound containing such a cell interaction molecule to the cells, trigger or promote endocytosis of the conjugate compound by the target cells, and / or trigger or promote the enrichment of the conjugate compound around the target cells and / or entry into the target cells.

[0118] The cell interaction molecule can be a small chemical molecule or a large biological molecule. In some embodiments, the cell interaction molecule is a small molecule compound or a polypeptide. In some embodiments, the cell interaction molecule is a small molecule compound or a polypeptide comprising 2-50, 2-40, 2-30, 2-25, 2-22, 2-20, 2-18, 2-15, 2-12, 2-10, 2-8, 4-50, 5-50, 5-40, 5-30, 5-25, 5-22, 5-20, 5-18, 5-15, 5-12, 5-10, 6, 7, 8, 9 amino acids.

[0119] In some embodiments, the targeting molecule is a ligand capable of binding to a cell surface receptor or other molecule. In some embodiments, at least one targeting molecule is a ligand capable of binding to a cell surface receptor or other molecule.

[0120] The ligands of the present application may include a variety of chemical or biological molecules that have specific binding affinity for a selected target, which may be, for example, a cell surface receptor, a cell surface antigen, a cell, a tissue, an organ, etc. In some embodiments, the ligands of the present application may specifically bind to a protein or marker expressed on the surface of a target cell. -6 ~10 -11 M(K d In some embodiments, the ligand of the present application binds to a cell surface protein or marker with an affinity of at least 10 -7 , at least 10 -8 , at least 10 -9 M(K d In some embodiments, the ligand of the present invention binds to a cell surface protein or marker with an affinity of less than 10 -6 , less than 10 -7 , less than 10 -8 M(K d In some embodiments, the ligand of the present application is combined with a cell surface protein or a marker with an affinity of at least two times, three times, four times, five times, six times, eight times, ten times, twenty times, fifty times, one hundred times or more compared with the affinity of the non-target cell surface protein or the marker. In some embodiments, the expression of the cell surface protein or the marker of the present application in target cells (e.g., cancer cells) is significantly higher than that in normal cells. The term "significant" used in the present application refers to a statistically significant difference, or a significant difference that can be recognized by those skilled in the art.

[0121] In some embodiments, the expression level of the cell surface protein or marker of the present application in target cells (e.g., cancer cells) is 2 to 1,000,000 times the expression level in normal cells, for example, the expression level in target cells (e.g., cancer cells) is 2 to 10 times, 2 to 100 times, 2 to 1,000 times, 2 to 10,000 times, 2 to 100,000 times, 2 to 1,000,000 times (can be equal to any value in the above numerical range, including the endpoints of the range). In some embodiments, the expression level of the cell surface receptor in the target cell (e.g., cancer cell) is at least 10 times, or at least 100 times, or at least 1,000 times, or at least 10,000 times, or at least 100,000 times higher than the expression level in normal cells. In some embodiments, the level of a cell surface receptor on a normal cell is reduced by at least 50%, 60%, 70%, 80%, 90%, 95% or 99% when compared to the level of a cell surface protein or marker on a target cell (e.g., a cancer cell). In some embodiments, a cell surface protein or marker described herein is not detectable in a normal cell.

[0122] In some embodiments, the cell surface protein or marker of the present application is a cell surface receptor.

[0123] In some embodiments, the cell surface receptor of the present application is selected from the following group: transferrin receptor (TFR), low-density lipoprotein receptor (LDLR), folate receptor (FR), somatostatin receptor, urate kinase receptor, tumor necrosis factor receptor (TNFR), integrin receptor (LFA-1), SST-14 receptor (SSTR2), GNRH receptor (GNRHR), TRPV6 and integrin α receptor.

[0124] In some embodiments, the cell surface protein or marker of the present application is a cell surface antigen.

[0125] In some embodiments, the cell surface antigen of the present application is selected from the group consisting of prostate specific membrane antigen, MUC1 mucin, acute lymphoblastoid common antigen, Thy-1 cell surface antigen, Melan-A protein, squamous cell carcinoma antigen, galectin 3 and human leukocyte antigen.

[0126] In some embodiments, the cell interaction molecules of the present application can bind to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), GNRHR, Her2, Trop2, Her3, NECTIN4, LRP1, GLUT1, EGFR1, AXL, CA9, CD44, Claudin18.2, APN, DLL3, CEACAM5, FZD10, TFRC, MET, IGFR1, SSTR2, CCKBR, LFA1, ICAM, GPR87, GM-CSF, GM-CSFR, TIM3, TLR family, CD40, CD40L, OX40, OX40L, GITRL, GITR, 4-BBL, 4-1BB, C D70, CD27, ICOSL, ICOS, HHLA2, CD28, CD86 / 80, CD28, MHCII antigens, TCR, CTLA-4, CD155, CD122, CD113, IGIT, PD-L1, PD1, Galectin-9, TIM-3, HVEM, BTLA, CD160, VISTA, B7-H4, B7-H3, phosphatidylserine, HHLA2, LAG3, Galectin-3, LILRB4, SIGLEC15, NKG2A, NKG2D, SLAMF7, KIR2DL1, KIR2DL2, KIR2DL3, FGFR1, FGFR2, FGFR4, NeuGcGM3 and CXCR4.

[0127] In some embodiments, the conjugate compound of the present application or a pharmaceutically acceptable salt thereof comprises a prostate-specific membrane antigen ligand portion and a synergistic molecule portion, wherein the synergistic molecule portion binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), SSTR2 and GNRHR.

[0128] In some embodiments, the conjugate compound of the present application or a pharmaceutically acceptable salt thereof comprises a ligand portion represented by formula (I) and a synergistic molecule portion, wherein the synergistic molecule portion binds to a molecule selected from the group consisting of FOLR1, TRPV6, SSTR2 and GNRHR.

[0129] In some other embodiments, the conjugate compound of the present application or a pharmaceutically acceptable salt thereof comprises P10 and a synergistic molecule portion, wherein the synergistic molecule binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA) and GNRHR.

[0130] In some embodiments, one of the synergistic molecules in the conjugate compound of the present application or its pharmaceutically acceptable salt is an endocytosis molecule portion capable of mediating endocytosis. The term "endocytosis" used in the present application refers to the ability of the conjugate compound or its pharmaceutically acceptable salt to mediate its own endocytosis, internalization or uptake into the target cell after interacting with the target cell. The term "endocytosis molecule" used in the present application refers to a molecule that, after interacting with the target cell, can mediate the endocytosis, internalization or uptake of the conjugate compound of the present application or its pharmaceutically acceptable salt into the target cell.

[0131] In some embodiments, the endocytosis molecule is selected from the group consisting of folic acid and its analogs, peptides capable of mediating endocytosis, and membrane-penetrating peptides.

[0132] In some embodiments, the endocytosis molecule of the present application is folic acid or an analog thereof.

[0133] Folic acid is conducive to forming chemical bonds with other groups due to its small molecular weight, non-immunogenicity and good stability. Folic acid can bind to the folate receptor expressed on the cell surface with high affinity to mediate the cell uptake of folic acid. Although the expression level of folate receptors in most normal cells is very low, they are expressed at high levels in a large number of cancer cells to meet the high demand for folic acid in rapidly dividing cells under low folic acid conditions (see Kelemen LE, Int J Cancer, 2006; 119: 243-50; Kane MA et al., J Clin Invest. 1988; 81: 1398-406; Matsue H et al., Proc Natl Acad Sci USA. 1992; 89: 6006-9; Zhao R et al., Annu Rev Nutr. 2011; 31: 177-201). Folic acid can specifically bind to the folate receptor on the cell surface, and it can also mediate the endocytosis of the conjugate compound or its pharmaceutically acceptable salt into the target cell.

[0134] In some embodiments, the analog of folic acid is selected from the group consisting of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate, and 5,10-methylenetetrahydrofolate.

[0135] In some embodiments, the endocytosis molecule is a peptide capable of mediating endocytosis.

[0136] In some embodiments, the peptide capable of mediating endocytosis comprises an amino acid sequence selected from the following group: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, Arg-Gly-Asp (referred to as RGD), and a homologous peptide having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence homology with any one of SEQ ID NO: 16-18, wherein the homologous peptides are functional equivalents of the peptides shown in SEQ ID NO: 16-18, respectively.

[0137] In some embodiments, the peptide capable of mediating endocytosis as described in the present application has a conservative substitution of amino acids at only one amino acid position compared to the sequences of SEQ ID NOs: 16-20 and RGD. In some embodiments, the peptide capable of mediating endocytosis as described in the present application has a conservative substitution of amino acids at 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid positions compared to the sequences of SEQ ID NOs: 16-20.

[0138] Without affecting its biological activity, the peptide capable of mediating endocytosis as described in the present application may also contain non-naturally occurring amino acids, including, for example, β-fluoroalanine, 1-methyl-histidine, γ-methylene-glutamate, α-methyl-leucine, 4,5-dehydro-lysine, hydroxyproline, 3-fluoro-phenylalanine, 3-amino-tyrosine, 4-methyl-tryptophan, etc.

[0139] Percent homology can be determined using a variety of methods well known in the art. For example, sequences can be compared using the following publicly available tools: BLASTp software (available from the website of the National Center for Biotechnology Information (NCBI): http: / / blast.ncbi.nlm.nih.gov / Blast.cgi , see also Altschul SF et al., J. Mol. Biol., 215: 403-410 (1990); Stephen F et al., Nucleic Acids Res., 25: 3389-3402 (1997)), ClustalW2 (available from the European Bioinformatics Institute website: http: / / www.ebi.ac.uk / Tools / msa / clustalw2 / , see also Higgins DG et al., Methods in Enzymology, 266: 383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23 (21): 2947-8 (2007)) and Tcoffee (available from the website of the Swedish Institute of Bioinformatics, see also Poirot O. et al., Nucleic Acids Res., 31 (13): 3503-6 (2003); Notredame C. et al., J. Mol. Boil, 302 (1): 205-17 (2000)). If software is used for sequence alignment, the default parameters provided in the software can be used, or the parameters can be customized in other ways to suit the purpose of alignment. All of these are within the knowledge of those skilled in the art.

[0140] The term "functional equivalent" used in this application refers to a derivative peptide that retains a biologically active, substantially similar biological activity to the original peptide sequence from which the derivative peptide originated. Functional equivalents can be natural derivatives or synthetic preparations. Exemplary functional equivalents include amino acid sequences with one or more amino acid substitutions, deletions or additions, provided that the biological activity of the peptide is maintained. The substituted amino acid ideally has a chemical-physical property similar to that of the substituted amino acid. Desirably similar chemical-physical properties include similarities in charge, bulkiness, hydrophobicity, hydrophilicity, etc.

[0141] In some embodiments, functional equivalents include conservative substitutions of amino acid residues. Conservative substitutions of amino acid residues refer to substitutions between amino acids with similar properties, such as substitutions between polar amino acids (e.g., substitutions between glutamine and asparagine), substitutions between hydrophobic amino acids (e.g., substitutions between leucine, isoleucine, methionine and valine), and substitutions between amino acids with the same charge (e.g., substitutions between arginine, lysine and histidine, or substitutions between glutamic acid and aspartic acid), etc.

[0142] In some embodiments, the endocytosis molecule is a cell-penetrating peptide. Cell-penetrating peptides (CPP), also known as protein transduction domains (PTDs), are short peptides (usually less than 40 amino acids) that can enter the interior of cells in a receptor-independent manner. When coupled to a payload, a cell-penetrating peptide can mediate transmembrane transport of the payload and has protein transduction activity. In some embodiments, the cell-penetrating peptide described in the present application is selected from the group consisting of tumor homing peptides, mitochondrial penetrating peptides, activatable cell-penetrating peptides, and antimicrobial peptides. In some embodiments, the cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19 (RRRRRRRRRR, referred to as R9) and SEQ ID NO: 20 (GRKKRRQRRRPPQ, which is a Tat peptide, i.e., a cell-penetrating peptide of the HIV transcription protein transactivator).

[0143] In some embodiments, one of the targeting molecules in the conjugate compound of the present application or a pharmaceutically acceptable salt thereof is a prostate-specific membrane antigen ligand portion.

[0144] The term "PSMA" used in this application refers to a type II transmembrane glycoprotein present in the membrane of prostate epithelial cells, which consists of 750 amino acids, 19 intracellular amino acids, 24 transmembrane amino acids and 707 extracellular amino acids.PSMA is expressed in normal prostate epithelial cells, but its expression level in prostate cancer cells is much higher. Compared with the traditional PSA for clinical detection, PSA is a more sensitive and specific prostate cancer tumor marker, especially in hormone-refractory prostate cancer and prostate cancer metastasis, which is highly expressed, and has high sensitivity and specificity in distinguishing prostate cancer from other types of malignant tumors. At the same time, in a variety of non-prostate-derived solid tumors (such as lung cancer, bladder cancer, gastric cancer, pancreatic cancer, kidney cancer and colorectal cancer, etc.), PSA is also highly specifically expressed on tumor vascular endothelial cells.

[0145] The term "prostate-specific membrane antigen ligand" used in this application refers to antibodies, nucleic acid aptamers and small molecules that can specifically recognize and bind to prostate-specific membrane antigen. The prostate-specific membrane antigen ligand of this application includes prostate-specific membrane antigen ligands that are currently in existence or will be produced later, and also includes fragments of the aforementioned ligands, as long as these fragments still retain the ability to bind to prostate-specific membrane antigen. Antibody ligands are the most common prostate-specific membrane antigen ligands, which include but are not limited to monoclonal antibodies J591, J533, J415 and E99 (for example, see Liu H, Rajasekaran AK, Moy P et al. Constitutive and antibody-induced internalization of prostate-specific memberane antigen [J]. Cancer Res, 1998, 58 (18): 4055-4060). Nucleic acid aptamers are single-stranded DNA or RNA that can bind to prostate-specific membrane antigen with high affinity and high specificity, obtained by technical screening of exponential enrichment ligand system. Such prostate-specific membrane antigen ligands include but are not limited to xPSM-A10 nucleic acid aptamers and their derivatives and xPSM-A9 nucleic acid aptamers and their derivatives (for example, see Lupoid SE et al., Identification and Characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen, Cncer Res, 2002, 62(14): 4029-4033). Compared with antibody and nucleic acid aptamer ligands, prostate-specific membrane antigen small molecule ligands have the advantages of small molecular weight, high permeability, low immunogenicity, and easy synthesis, and include but are not limited to glutamine urea small molecule ligands and aminophosphoryl ester small molecule ligands.

[0146] In some embodiments, the prostate-specific membrane antigen small molecule ligand of the present application can be selected from the group consisting of: 2-[[methylhydroxyphosphinyl]methyl]glutaric acid; 2-[[ethylhydroxyphosphinyl]methyl]glutaric acid; 2-[[propylhydroxyphosphinyl]methyl]glutaric acid; 2-[[butylhydroxyphosphinyl]methyl]glutaric acid; 2-[[cyclohexylhydroxyphosphinyl]methyl]glutaric acid; 2-[[phenylhydroxyphosphinyl]methyl]glutaric acid; 2-[[2-(tetrahydrofuranyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[(2-tetrahydropyranyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[((4-pyridyl)methyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[((2-pyridyl)methyl 2-[[(phenylmethyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[((2-phenylethyl)methyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[((3-phenylpropyl)methyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[((3-phenylbutyl)methyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[((2-phenylbutyl)methyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[(4-phenylbutyl)hydroxyphosphinyl]methyl]glutaric acid; and 2-[[(aminomethyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[methylhydroxyphosphinyl]oxy]glutaric acid; 2-[[ethylhydroxyphosphinyl]oxy]glutaric acid; 2-[[propyl 2-[[((4-pyridyl)methyl)hydroxyphosphinyl]oxy]glutaric acid; 2-[[((2-pyridyl)methyl)hydroxyphosphinyl]oxy]glutaric acid; 2-[[(phenylmethyl)hydroxyphosphinyl]oxy]glutaric acid; and 2[[((2-phenylethyl)methyl)hydroxyphosphinyl]oxy]glutaric acid; 2-[[(N-hydroxy)carbamoyl]methyl]glutaric acid; 2-[[(N-hydroxy-N-methyl)carbamoyl]methyl]glutaric acid; 2-[[(N-butyl-N-hydroxy)carbamoyl]methyl]glutaric acid; 2-[[(N-benzyl-N-hydroxy)carbamoyl]methyl]glutaric acid; 2-[[(N-hydroxy-N-propyl)carbamoyl]methyl]glutaric acid; 2-[[(N-hydroxy-N-phenyl)carbamoyl]methyl]glutaric acid; 2-[[(N-hydroxy-N-2-phenylethyl)carbamoyl]methyl]glutaric acid; 2-[[(N-ethyl-N-hydroxy)carbamoyl]methyl]glutaric acid; 2-[[(N-hydroxy-N-propyl)carbamoyl]methyl]glutaric acid; 2-[[(N-hydroxy-N-3-phenylpropyl)carbamoyl]methyl]glutaric acid; 2-[[(N-hydroxy-N-4-pyridyl)carbamoyl]methyl]glutaric acid; 2-[[(N-hydroxy)amido]methyl]glutaric acid; 2-[[N-hydroxy(methyl)amido]methyl]glutaric acid; 2-[[N-hydroxy(benzyl)amido]methyl]glutaric acid;2-[[N-hydroxy(phenyl)amide]methyl]glutaric acid; 2-[[N-hydroxy(2-phenylethyl)amide]methyl]glutaric acid; 2-[[N-hydroxy(ethyl)amide]methyl]glutaric acid; 2-[[N-hydroxy(propyl)amide]methyl]glutaric acid; 2-[[N-hydroxy(3-phenylpropyl)amide]methyl]glutaric acid; and 2-[[N-hydroxy(4-pyridyl)amide]methyl]glutaric acid; 2-[(sulfinyl)methyl]glutaric acid; 2-[(methylsulfinyl)methyl]glutaric acid; 2-[(ethylsulfinyl)methyl]glutaric acid; 2-[(propylsulfinyl)methyl]glutaric acid; 2-[(butylsulfinyl)methyl]glutaric acid; 2-[(phenylsulfinyl)methyl]glutaric acid ] methyl] glutaric acid; 2-[[(2-phenylethyl)sulfinyl]methyl] glutaric acid; 2-[[(3-phenylpropyl)sulfinyl]methyl] glutaric acid; 2-[[(4-pyridyl)sulfinyl]methyl] glutaric acid; 2-[(benzylsulfinyl)methyl] glutaric acid; 2-[(sulfonyl)methyl] glutaric acid; 2-[(methylsulfonyl)methyl] glutaric acid; 2-[(ethylsulfonyl)methyl] glutaric acid; 2-[(propanesulfonyl)methyl] glutaric acid; 2-[(butanesulfonyl)methyl] glutaric acid; 2-[(phenylsulfonyl]methyl] glutaric acid; 2-[[(2-phenylethyl)sulfonyl]methyl] glutaric acid; 2-[[(3-phenylpropyl)sulfonyl]methyl] glutaric acid; 2-[[ 2-[(4-pyridyl)sulfonyl]methyl]glutaric acid; 2-[(benzylsulfonyl)methyl]glutaric acid; 2-[(sulfoximinyl)methyl]glutaric acid; 2-[(methylsulfoximinyl)methyl]glutaric acid; 2-[(ethylsulfoximinyl)methyl]glutaric acid; 2-[(propylsulfoximinyl)methyl]glutaric acid; 2-[(butylsulfoximinyl)methyl]glutaric acid; 2-[(phenylsulfoximinyl]methyl]glutaric acid; 2-[[(2-phenylethyl)sulfoximinyl]methyl]glutaric acid; 2-[[(3-phenylpropyl)sulfoximinyl]methyl]glutaric acid; 2-[[(4-pyridyl)sulfoximinyl]methyl]glutaric acid; and 2-[(benzylsulfoximinyl)methyl]glutaric acid.

[0013] The prostate-specific membrane antigen ligands of the present application also include all prostate-specific membrane antigen small molecule ligands disclosed in PCT applications WO2010 / 108125 and WO2006 / 093991, and the above two patent applications are incorporated herein in their entirety. ;

[0147] In some embodiments, the small molecule ligand of prostate-specific membrane antigen of the present application is a glutaric acid derivative. In some embodiments, the small molecule ligand of prostate-specific membrane antigen of the present application is an aminocarbonyl derivative of glutaric acid.

[0148] In some embodiments, the prostate-specific membrane antigen small molecule ligand of the present application comprises the following structure:

[0149]

[0150] In some embodiments, the prostate-specific membrane antigen ligand contained in the conjugate compound or a pharmaceutically acceptable salt thereof comprises the following structure:

[0151]

[0152] In some embodiments, the prostate-specific membrane antigen ligand contained in the conjugate compound or a pharmaceutically acceptable salt thereof comprises the following structure:

[0153]

[0154] In some embodiments, the prostate-specific membrane antigen ligand contained in the conjugate compound or a pharmaceutically acceptable salt thereof comprises the following structure:

[0155]

[0156] In some embodiments, the prostate-specific membrane antigen ligand contained in the conjugate compound or a pharmaceutically acceptable salt thereof comprises the following structure:

[0157]

[0158] In some embodiments, one of the targeting molecules in the conjugate compound or a pharmaceutically acceptable salt thereof of the present application is a ligand portion having formula (I):

[0159]

[0160] Or a ligand portion having at least 70%, at least 80%, at least 85% or at least 90% amino acid sequence homology thereto or having at most 3, 2 or 1 amino acid substitutions (e.g., conservative substitutions) therewith.

[0161] In some embodiments, a targeting molecule in the conjugate compound of the present application or a pharmaceutically acceptable salt thereof is P10 or a ligand portion having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% or at least 93% amino acid sequence homology thereto or having at most 3, 2 or 1 amino acid substitutions (e.g., conservative substitutions) therewith.

[0162] The term "P10" as used in this application refers to a peptide having the amino acid sequence Cys-Lys-Glu-Phe-Leu-His-Pro-Ser-Lys-Val-Asp-Leu-Pro-Arg.

[0163] In some embodiments, the conjugate compound of the present application has a targeting molecule selected from the following combinations: (1) a folate ligand and a prostate-specific membrane antigen ligand; (2) a TRPV6 ligand and a prostate-specific membrane antigen ligand; (3) a GNRHR ligand and a prostate-specific membrane antigen ligand; (4) an SSTR2 ligand and a prostate-specific membrane antigen ligand; (5) a folate ligand and an SSTR2 ligand; or (6) a TRPV6 ligand and a folate ligand.

[0164] In some embodiments, the two targeting molecules of the conjugate compound or its pharmaceutically acceptable salt provided herein are respectively a synergistic molecule portion and a prostate-specific membrane antigen ligand portion. In some embodiments, the synergistic molecule can mediate endocytosis. In some embodiments, the two targeting molecules of the conjugate compound or its pharmaceutically acceptable salt provided herein are respectively folic acid or its analogue and a prostate-specific membrane antigen ligand portion. Without wishing to be limited by theory, selecting specific folic acid or its analogue and a prostate-specific membrane antigen ligand portion will have better stability than the ligand combination in the prior art.

[0165] In some embodiments, the two targeting molecules of the conjugate compound or its pharmaceutically acceptable salt provided herein are respectively a synergistic molecule portion and a ligand portion having formula (I). In some embodiments, the synergistic molecule can mediate endocytosis. In some embodiments, the two targeting molecules of the conjugate compound or its pharmaceutically acceptable salt provided herein are respectively folic acid or its analogue and a ligand portion having formula (I).

[0166] In some embodiments, the two targeting molecules of the conjugate compound or its pharmaceutically acceptable salt provided herein are respectively a synergistic molecule portion and P10. In some embodiments, the synergistic molecule can mediate endocytosis. In some embodiments, the two targeting molecules of the conjugate compound or its pharmaceutically acceptable salt provided herein are respectively folic acid or its analogue and P10.

[0167] In some embodiments, the conjugate compound provided herein comprises only a single effective load coupled to two targeting molecules. In some embodiments, the conjugate compound provided herein comprises multiple effective loads coupled to two targeting molecules.

[0168] The term "coupled" used in the present application refers to the connection of two chemical groups through a covalent bond, which may be a direct covalent bond between the two chemical groups or an indirect connection of the two chemical groups through a linker.

[0169] In some embodiments, the conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload (e.g., 1) and two targeting molecules, wherein the payload is directly covalently linked to at least one targeting molecule. In some embodiments, the payload is directly covalently linked to both targeting molecules.

[0170] In some embodiments, the conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload (e.g., 1) and two targeting molecules, wherein the payload is covalently linked to at least one targeting molecule via a linker. In some embodiments, the payload is covalently linked to both targeting molecules via a linker.

[0171] The term "connector" used in this application refers to a molecule or part that covalently connects a payload to a targeting molecule. The connector includes a functional group for connecting a payload to at least one targeting molecule. In some embodiments, the functional group may contain two reactive moieties, one for connecting to the payload and the other for connecting to the targeting molecule. In some embodiments, the functional groups are different from each other. In some embodiments, the functional group comprises a group containing a sulfhydryl reactive moiety and an amine reactive moiety. In some embodiments, the functional groups are identical to each other. In some embodiments, the functional group is a maleimide group. In some embodiments, the connector contains an amino acid. In some embodiments, the carboxylic acid in the amino acid contained in the connector is amidated. In some embodiments, the connector contains a short-chain polyethylene glycol (e.g., including 2-10, 2-8, 3-8, 4-8, 4-7, 4-6 or 5 repeating units).

[0172] In some embodiments, the linker of the present application is a multivalent linker capable of binding at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload and at least one targeting molecule. The payloads bound to the multivalent linker may be the same or different, and the targeting molecules bound to the multivalent linker may be the same or different.

[0173] In one aspect, the linker should be stable enough to avoid accidental release of the payload during blood circulation, to increase the effective amount of the payload to the target cell or tissue and to avoid toxicity. In another aspect, the linker should be able to release the payload around or inside the target cell to effectively kill the target cell or block the function of the target cell. In some embodiments, the linker comprises at least one cleavable functional group. Preferably, the cleavable functional group is stable enough outside the target cell, but it is cleaved after entering the target cell to release the payload. In some embodiments, the cleavage efficiency of the cleavage functional group in the target cell is at least 10, 20, 30, 50, 100 times or more higher than the cleavage efficiency in blood or serum.

[0174] Cleavable linkers can be cleaved by hydrolysis, enzymatic reaction, or reduction reaction, or by pH changes. In some embodiments, linkers are cleavable under specific physiological conditions (e.g., under a suitable pH environment). In some embodiments, linkers can be cleaved in an acidic environment of pH about 6.5 or lower, or by reagents such as enzymes. In some embodiments, linkers are sensitive to cleavage agents. For example, pH, redox potential, or the presence of degradation molecules.

[0175] In some embodiments, the linker is non-cleavable. As used herein, a non-cleavable linker refers to a linker that remains substantially intact during metabolism within a cell.

[0176] In some embodiments, the connexon is a peptide connexon, which is composed of straight or branched amino acids connected by peptide bonds. In some embodiments, the peptide connexon can be cleaved by a protease that is highly or specifically expressed around or in the target cell, such as cathepsin B in a lysosome or endosome. The length of the peptide connexon used in the present application can be various. Generally, the length of the peptide connexon of the present application is 1 to 50 amino acids. In some embodiments, the length of the peptide connexon is 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acid. In some embodiments, the length of the peptide linker is 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3 or 2 amino acids. The number of amino acids in the peptide linker described in the present application can be equal to any integer value in the above-mentioned numerical range, including the endpoints of the range. In some embodiments, the length of the peptide linker is preferably 1, 2, 3, 4 or 5 amino acids. In some embodiments, the peptide linker is cysteine, lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-lysine, cysteine-glutamic acid aspartic acid-aspartic acid and aspartic acid-aspartic acid-lysine, and optionally, the carboxylic acid in the above amino acids is amidated.

[0177] In some embodiments, the linker is a disulfide linker containing a disulfide bond. The disulfide bond can be cleaved under the reducing environment in the cell and remain stable in the circulatory system. The disulfide linker of the present application can be DSDM, DMDS, MDS or NDMDS. The structures of these disulfide linkers are shown in Table 1 below.

[0178] Table 1: Structure of DSDM, DMDS, MDS and NDMDS

[0179]

[0180]

[0181] In some embodiments, the linker is a pH-dependent linker. The pH-dependent linkers described herein can be cleaved under a specific pH environment. In some embodiments, the pH-dependent linker can be stable under alkaline conditions, but cleaved under acidic conditions (e.g., at a pH of 6.5 or less). In some embodiments, the pH-dependent linker is cis-aconitic anhydride.

[0182] In some embodiments, the linker of the conjugate compound or a pharmaceutically acceptable salt thereof is

[0183]

[0184] Or it may be a combination of the above structure and a peptide linker (for example, connected to the targeting molecule via a peptide linker containing 1-3 amino acids).

[0185] In some embodiments, the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

[0186]

[0187] Or it may be a combination of the above structure and a peptide linker (for example, connected to the targeting molecule via a peptide linker containing 1-3 amino acids).

[0188] In some embodiments, the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

[0189]

[0190] Or it may be a combination of the above structure and a peptide linker (for example, connected to the targeting molecule via a peptide linker containing 1-3 amino acids).

[0191] In some embodiments, the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

[0192]

[0193] Or it may be a combination of the above structure and a peptide linker (for example, connected to the targeting molecule via a peptide linker containing 1-3 amino acids).

[0194] In some embodiments, the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

[0195]

[0196] Or it may be a combination of the above structure and a peptide linker (for example, connected to the targeting molecule via a peptide linker containing 1-3 amino acids).

[0197] In some embodiments, the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

[0198]

[0199] Or it may be a combination of the above structure and a peptide linker (for example, connected to the targeting molecule via a peptide linker containing 1-3 amino acids).

[0200] In some embodiments, the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

[0201]

[0202] Or it may be a combination of the above structure and a peptide linker (for example, connected to the targeting molecule via a peptide linker containing 1-3 amino acids).

[0203] In some embodiments, the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

[0204]

[0205] Or it may be a combination of the above structure and a peptide linker (for example, connected to the targeting molecule via a peptide linker containing 1-3 amino acids).

[0206] In some embodiments, the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

[0207]

[0208] Or it may be a combination of the above structure and a peptide linker (for example, connected to the targeting molecule via a peptide linker containing 1-3 amino acids).

[0209] In some embodiments, the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

[0210]

[0211] Or it may be a combination of the above structure and a peptide linker (for example, connected to the targeting molecule via a peptide linker containing 1-3 amino acids).

[0212] In some embodiments, the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

[0213]

[0214] Or it may be a combination of the above structure and a peptide linker (for example, connected to the targeting molecule via a peptide linker containing 1-3 amino acids).

[0215] In some embodiments, the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

[0216]

[0217] Or it may be a combination of the above structure and a peptide linker (for example, connected to the targeting molecule via a peptide linker containing 1-3 amino acids).

[0218] In some embodiments, the linker of the present application may include any one of the linkers described above or a combination thereof.

[0219] In some embodiments, the payload is directly or indirectly coupled to the first targeting molecule, and the first targeting molecule is directly or indirectly coupled to the second targeting molecule. In some embodiments, the payload is directly coupled to the first targeting molecule and the second targeting molecule. In some embodiments, the payload is indirectly coupled to the first targeting molecule and the second targeting molecule. In some embodiments, the payload is indirectly (e.g., through a connexon) coupled to the first targeting molecule, and the first targeting molecule is directly or indirectly coupled to the second targeting molecule. In some embodiments, the payload is coupled to the first targeting molecule through the first connexon, and the payload is coupled to the second targeting molecule through the second connexon. In some embodiments, the connexon is a multivalent connexon that is combined with at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload and two targeting molecules.

[0220] In some embodiments, two targeting molecules are connected to each other by a spacer. In some embodiments, the spacer can be cleaved by a protease specifically expressed by the target cell or expressed by the target cell. Such proteases include, for example, the proteases listed in Table 2 below. In some embodiments, the spacer comprises an amino acid sequence selected from any one of the amino acid sequences listed in Table 2 below.

[0221] Table 2: List of enzymatically cleavable sequences

[0222] Protease Amino acid sequence of the recognition site SEQ ID NO. Cathepsin B RR - Pea Protein ASN - Matripase KSRAEDE SEQ ID NO: 1 MMP-2 PLGLAG SEQ ID NO: 2 Prostate-specific antigen SSLY SEQ ID NO: 3 stromelysin-3 AAA - TMPRSS2 LLRSLIG SEQ ID NO: 4 Urokinase-type plasminogen activator SSR - Activated Protein C LVKR SEQ ID NO: 5 Factor Ixa LVVR SEQ ID NO: 6 Factor VIIa QLTR SEQ ID NO: 7 Factor Xa LEGR SEQ ID NO: 8 Thrombin PR - Calpain-a PLFAEP SEQ ID NO: 9 Calpain-2 GLGSEP SEQ ID NO: 10 Enteropeptidase DDDDK SEQ ID NO: 11 MMP-8 GPSG SEQ ID NO: 12 Cathepsin L PLG - Proprotein convertase 5 RSKR SEQ ID NO: 13 Calpain-3 VGV SEQ ID NO: 14

[0223] The term "cleavable" or "cleaved" used in the present application refers to a metabolic process or reaction process performed on the conjugate compound provided in the present application, thereby destroying the linker between the payload and the targeting molecule, or the spacer between the targeting molecules to release the free payload or targeting molecule. The linker or spacer is cleaved by a protease or cleaved under a specific physiological environment (e.g., pH environment).

[0224] In some embodiments, the conjugate compound has a structure as shown in the following formula I, II, III or IV, wherein n, m, p and q are independently 0 or 1, which represents the independent presence or absence of a linker or spacer. The "molecule" in the following formula is an abbreviation for "targeting molecule".

[0225]

[0226]

[0227] In some embodiments, the conjugate compound provided herein or its pharmaceutically acceptable salt comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload as provided herein, two targeting molecules as provided herein, and optionally a linker or spacer as provided herein. In some embodiments, the conjugate compound provided herein or its pharmaceutically acceptable salt comprises a payload as provided herein, a ligand specifically bound to a cell surface protein or marker as provided herein, a synergistic molecule as provided herein, and a linker or spacer as provided herein.

[0228] In some embodiments, the conjugate compound has a structure of Formula V, VI, VII or VIII as shown below, wherein n, m, p, q and s are independently 0 or 1, which independently represent the presence or absence of a linker, a multivalent linker and a spacer.

[0229]

[0230] In some embodiments, the conjugate compound provided herein or a pharmaceutically acceptable salt thereof comprises a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and a prostate-specific membrane antigen ligand portion, such as CB-20B, CB-20BK, CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB - 1820, CR19428, 20R-SM09 and CB-20R.

[0231] In some embodiments, the conjugate compound provided herein or a pharmaceutically acceptable salt thereof comprises one or more payloads and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and a ligand portion represented by formula (I), such as CB-18G, CB-1820 and CR19426.

[0232] In some embodiments, the conjugate compound provided herein or a pharmaceutically acceptable salt thereof comprises a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and P10, respectively, and the payload is camptothecin and any derivative thereof, such as CB-10S, CR19425 and CB-50S.

[0233] In some embodiments, the conjugate compound of the present application is selected from the group consisting of the following compounds: CB-20B, CB-20BK, CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB-1820, CR19428, 20R-SM09, CB-20R, CB-18G, CR19426, CB-10S, CR19425 and CB-50S (the specific structure of each conjugate compound is as shown in Figure 1 As shown). In some embodiments, the conjugate compound of the present application is formed by connecting the linker-drug part and the ligand part through a covalent bond. The linker-drug part of the present application includes a payload and a linker, and the ligand part of the present application includes two targeting molecules and an optional spacer or linker. The two parts react to form a covalent bond to form the conjugate compound of the present application. The covalent bond can be formed between the linker in the linker-drug part and the ligand molecule of the ligand part, or between the linker in the linker-drug part and the spacer or linker of the ligand part.

[0234] The coupled compound CB-20B of the present application is formed by connecting the linker-drug part LT1002 and the ligand part 20B-SM09 through a covalent bond. The coupled compound CB-20BK of the present application is formed by connecting the linker-drug part LT1002 and the ligand part 20BK-SM09 through a covalent bond. The coupled compound CB-60S of the present application is formed by connecting the linker-drug part LT2000C and the ligand part 60S-SM09 through a covalent bond. The coupled compound CB-60SK of the present application is formed by connecting the linker-drug part LT2000C and the ligand part 60SK-SM09 through a covalent bond. The coupled compound CB-20C of the present application is formed by connecting the linker-drug part LD1001 and the ligand part 20BK-SM09 through a covalent bond. The coupled compound CB-1020 of the present application is formed by connecting the linker-drug part LT1002 and the ligand part 1020BK-SM09 through a covalent bond. The coupled compound CB-1320 of the present application is formed by connecting the linker-drug part LT1002 and the ligand part 1320BK-SM09 through a covalent bond. The coupled compound CB-1820 of the present application is formed by connecting the linker-drug part LT1002 and the ligand part 1820BK-SM09 through a covalent bond. The coupled compound CR19428 of the present application is formed by connecting the linker-drug part CR19423 and the ligand part 20BK-SM09 through a covalent bond. The coupled compound CB-20R of the present application is complexed by 20R-SM09 and the radionuclide ion M. The conjugate compound CB-18G of the present application is formed by connecting the linker-drug part LT1002 and the ligand part 18G-SM09 through a covalent bond. The conjugate compound CR19426 of the present application is formed by connecting the linker-drug part CR19423 and the ligand part 18G-SM09 through a covalent bond. The conjugate compound CB-10S of the present application is formed by connecting the linker-drug part LT1000 and the ligand part CBSM09 through a covalent bond. The conjugate compound CR19425 of the present application is formed by connecting the linker-drug part CR19423 and the ligand part CBSM09 through a covalent bond. The conjugate compound CB-50S of the present application is formed by connecting the linker-drug part LT1000N3 and the ligand part 50S-SM09 through a covalent bond. Each structure is shown in Table 3 below.

[0235] Table 3: Structures of Linker-Drug and Ligand Moieties

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243] In some embodiments, the conjugate compound provided herein or a pharmaceutically acceptable salt thereof enters the blood circulation and the outside of the cell (the extracellular matrix), and since the linker is very stable in the extracellular environment and cannot release the drug molecule, the toxicity of the drug molecule is blocked. The conjugate is a non-cytotoxic or low-toxic drug that does not produce toxic effects on normal cells.

[0244] In some embodiments, the conjugate compound or its pharmaceutically acceptable salt provided by the present application binds to multiple receptors or antigens and other molecules that are highly expressed on diseased cells at the same time, and the synergistic effect greatly increases the affinity of the conjugate compound to the target cells and reduces the possibility of binding to normal cells. Thus, it can carry highly effective toxin drugs such as MMAE / Dxd / SN38 / radionuclide complexes, enhance drug efficacy, broaden the treatment window and avoid drug side effects.

[0245] In some embodiments, after the conjugate compound provided in the present application or a pharmaceutically acceptable salt thereof enters the interior of the targeted cell, the linker can be cleaved to release the drug molecule (equivalent to removing the modifying group of the drug molecule) through changes in the internal environment of the cell (specific enzymatic cleavage, pH change, disulfide bond reduction, etc.), thereby producing a therapeutic effect on the tumor cell.

[0246] In some embodiments, the conjugate compound of the present application or its pharmaceutically acceptable salt can be used to deliver the payload specifically to the target cell in the target tissue environment. Under normal circumstances, the two targeting molecules of the conjugate compound or its pharmaceutically acceptable salt have three advantages. First, the two targeting molecules can act in a variety of ways (usually synergistically), thereby improving the therapeutic effect while reducing side effects. Secondly, the combination of the two targeting molecules increases the affinity or affinity of the conjugate compound or its pharmaceutically acceptable salt to the target receptor or target cell, thereby enhancing its specificity and avoiding off-target toxicity. Finally, when the design is reasonable, the combination of two targeting molecules can meet the multifunctional requirements usually required for drug conjugates.

[0247] The conjugate compound or its pharmaceutically acceptable salt of the present application has achieved unexpected technical effects, including but not limited to: (1) The combination of a ligand capable of binding to a cell surface receptor and a synergistic molecule capable of mediating endocytosis enables the conjugate compound to specifically enter the target cell; (2) The conjugate compound or its pharmaceutically acceptable salt enhances the affinity and targeting specificity of the drug compound, thereby delivering highly effective chemotherapeutic agents (such as MMAE) to patients, broadening the therapeutic window of such agents and avoiding side effects; (3) The linker can prevent the release of the payload outside the target cell (for example, the blood circulation system, intercellular matrix, etc.), ensuring the stability of the conjugate compound in the blood circulation and reducing the toxicity of the drug. After entering the target cell, the linker is cleaved and the payload is released, thereby exerting the effect of the drug. At the same time, multidrug resistance (MDR) can be avoided; (4) A variety of drugs can be delivered in the form of the conjugate compound of the present application, thereby expanding the application range of the relevant drugs. Therefore, the conjugate compound or a pharmaceutically acceptable salt thereof of the present application not only broadens the target range and therapeutic window of LDC drugs, but also reduces the toxicity and side effects of some drugs.

[0248] The terms "polypeptide", "protein" and "peptide" as used in this application may be a single amino acid or a polymer of amino acids. The polypeptides, proteins or peptides as described herein may contain naturally occurring amino acids, as well as non-naturally occurring amino acids, or analogs and mimetics of amino acids. The polypeptides, proteins or peptides may be obtained by any method well known in the art, such as, but not limited to, separation and purification from natural materials, recombinant expression, chemical synthesis, etc.

[0249] Another aspect of the present application discloses a pharmaceutical composition, which contains the conjugate compound provided by the present application or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0250] The term "pharmaceutically acceptable" as used in this application means that it is suitable for contact with cells of humans and other animals without undue toxicity, irritation, allergic response, etc., within the scope of reasonable medical judgment, and is commensurate with a reasonable benefit / risk ratio.

[0251] The term "pharmaceutically acceptable salt" used in the present application refers to relatively nontoxic, inorganic and organic acid addition salts and base addition salts of the couplet compound of the present application. Representative acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, mesylate, glucoheptonate, lactobionate, sulfamate, malonate, salicylate, propionate, methylene-bis-b-hydroxynaphthoate, gentisate, isethionate, di-p-toluoyl tartrate, mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylaminosulfonate and quinic acid lauryl sulfonate etc. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium and aluminum salts. In some embodiments, sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases, including, for example, sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide and zinc hydroxide. Suitable amine base addition salts are prepared from amines having sufficient basicity to form stable salts, and preferably include the following amines commonly used in pharmaceutical chemistry because of their low toxicity and acceptable medical use: ammonia, ethylenediamine, N-methylglucamine, lysine, arginine, ornithine, choline, N,N′-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, trishydroxymethylaminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, diphenylhydroxymethylamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids (e.g., lysine and arginine) and dicyclohexylamine and the like.

[0252] The term "pharmaceutically acceptable carrier" as used in the present application refers to a pharmaceutically acceptable solvent, suspension or any other pharmaceutically inert carrier for delivering the conjugate compound provided herein to a subject, which does not interfere with the structure and properties of the conjugate compound. Some such carriers can formulate the conjugate compound into, for example, tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and pastilles for oral ingestion by a subject. Some such carriers can formulate the conjugate compound into a formulation for injection, infusion or topical administration.

[0253] Pharmaceutically acceptable carriers used in the pharmaceutical compositions provided herein include, but are not limited to, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous carriers (e.g., sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection), non-aqueous carriers (e.g., fixed oils of plant origin, cottonseed oil, corn oil, sesame oil or peanut oil), antimicrobial agents, isotonic agents (e.g., sodium chloride or dextrose), buffers (e.g., phosphate or citric acid buffers), antioxidants (e.g., sodium bisulfate), anesthetics (e.g., procaine hydrochloride), suspending / dispersing agents (e.g., sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifiers (e.g., polysorbate 80 (Tween-80)), diluents, adjuvants, excipients, or non-toxic auxiliary substances, other ingredients known in the art, or various combinations thereof. Suitable ingredients may include, for example, fillers, binders, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants or emulsifiers.

[0254] In some embodiments, the pharmaceutical composition is an injection. The injection includes a sterile aqueous solution or a dispersant, a suspension or an emulsion. In all cases, the injection should be sterile and should be fluid for injection. The injection should remain stable under production and storage conditions, and must prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures and / or vegetable oils. The injection should maintain appropriate fluidity. For example, appropriate fluidity can be maintained by using a coating such as lecithin, by using a surfactant, etc. The effect of preventing microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.

[0255] In some embodiments, the pharmaceutical composition is an oral formulation. Oral formulations include, but are not limited to, capsules, cachets, pills, tablets, lozenges (using a flavored base, typically sucrose and gum arabic or tragacanth), powders, granules, or solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and gum arabic), and / or as mouthwashes, etc.

[0256] In a solid dosage form for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules, etc.), the conjugate compound is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) a filler or extender, such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) a binder, such as carboxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and / or gum arabic; (3) a humectant. , for example, glycerol; (4) disintegrating agents, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retarding agents, for example, paraffin; (6) absorption accelerators, for example, quaternary ammonium compounds; (7) wetting agents, for example, acetyl alcohol and glyceryl monostearate; (8) absorbents, for example, kaolin and bentonite; (9) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and (10) coloring agents.

[0257] In liquid dosage forms for oral administration, the conjugate compound is mixed with any of the following: pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the conjugate compound, the liquid dosage form may contain inert diluents commonly used in the art, for example, water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, isopropanol, 1,3-butylene glycol, oils (particularly, cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan fatty acid esters and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants, for example, wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, coloring agents, flavoring agents and preservatives.

[0258] In some embodiments, the pharmaceutical composition is an oral spray preparation or a nasal spray preparation. Spray preparations include but are not limited to aqueous aerosols, non-aqueous suspensions, liposome preparations or solid particle preparations, etc. Aqueous aerosols are prepared by mixing an aqueous solution or suspension of a medicament with a conventional pharmaceutically acceptable carrier and stabilizer. Carriers and stabilizers are changed according to the requirements of specific compounds, but in general, they include nonionic surfactants (tween or polyethylene glycol), oleic acid, lecithin, amino acids, for example, glycine, buffer solutions, salts, sugars or sugar alcohols. Aerosols are usually prepared by isotonic solutions and can be delivered by spraying.

[0259] In some embodiments, the pharmaceutical composition can be used by mixing with one or more other drugs. In some embodiments, the pharmaceutical composition comprises at least one other drug. In some embodiments, the other drug is an anti-tumor drug, a cardiovascular drug, an anti-inflammatory drug, an antiviral drug, a digestive system drug, a nervous system drug, a respiratory system drug, an immune system drug, a dermatological drug, a metabolic drug, etc.

[0260] In some embodiments, the pharmaceutical composition can be administered to a subject in need by a suitable route, including but not limited to oral, injection (e.g., intravenous, intramuscular, subcutaneous, intradermal, intracardiac, intrathecal, intrapleural, intraperitoneal injection, etc.), mucosal (e.g., intranasal, oral administration, etc.), sublingual, rectal, transdermal, intraocular and pulmonary administration. In some embodiments, the pharmaceutical composition can be administered intravenously, subcutaneously, orally, intramuscularly or intraventricularly.

[0261] Due to the properties of some payloads, such as high toxicity and high hydrophilicity, it is desirable to deliver the payload more specifically and more effectively to an object in need. For example, in cancer treatment, it is desirable to deliver chemotherapeutic agents specifically to cancer cells without toxicity to normal cells. Thus, another aspect of the present application discloses a method for delivering a payload to an object in need, the method comprising administering to the object a therapeutically effective amount of a conjugate compound provided herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition provided herein. The payload described herein can be any agent that a researcher, veterinarian, doctor or other physician is looking for to cause a biological or medical response in a tissue, system, individual animal or human to prevent, inhibit, improve or treat a disease.

[0262] The term "subject" as used in this application refers to humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals. The subject can also be livestock, such as cattle, pigs, sheep, poultry and horses, or domestic animals, such as dogs and cats. The subject can be male (e.g., male) or female (e.g., female), and can be elderly, adult, adolescent, child or infant. A person can be Caucasian, African, Asian, Semitic or other ethnic background, or a mixture of these ethnic backgrounds.

[0263] The term "therapeutically effective amount" as used in this application refers to the amount of the conjugate compound or its pharmaceutically acceptable salt or pharmaceutical composition to alleviate one or more symptoms of a disease or condition in a subject to a certain extent; the amount that partially or completely restores one or more physiological or biochemical parameters associated with or causing the disease or condition to normal; and / or the amount that reduces the likelihood of the onset of the disease or condition. This amount usually varies according to a variety of factors, and a person of ordinary skill in the art can determine and describe it according to the scope of the specification provided in this application. These include, but are not limited to: a specific subject and its age, weight, height, general physical condition and medical history, the specific compound used, and the carrier and selected route of administration of its preparation; and the nature and severity of the condition being treated.

[0264] In some embodiments, the amount of the conjugate compound or its pharmaceutically acceptable salt, or the pharmaceutical composition is sufficient to suppress the disease or condition in the subject, or preventively suppress or prevent the onset of the disease or condition. Although the therapeutically effective amount can be changed in different subjects, it generally ranges from 0.01 to 100 mg / kg, such as 0.01 to 90 mg / kg, 0.01 to 80 mg / kg, 0.01 to 70 mg / kg, 0.01 to 60 mg / kg, 0.01 to 50 mg / kg, 0.01 to 40 mg / kg, 0.01 to 30 mg / kg, 0.01 to 20 mg / kg, 0.01 to 10 mg / kg, 0.01 to 5 mg / kg, 0.01 to 4 mg / kg, 0.01 to 3 mg / kg, 0.01 to 2 mg / kg, 0.01 to 1 mg / kg, 0.01 to 0.1 mg / kg. The therapeutically effective amount described herein can be equal to any value within the above numerical range, including the endpoint of the range.

[0265] Another aspect of the present application discloses a method for delivering a payload to a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the conjugate compound provided herein or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition provided herein.

[0266] Another aspect of the present application discloses a method for treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the conjugate compound provided herein or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition provided herein.

[0267] In some embodiments, the disease is cancer, including but not limited to prostate cancer, breast cancer, lung cancer, kidney cancer, leukemia, ovarian cancer, stomach cancer, uterine cancer, endometrial cancer, liver cancer, thyroid cancer, pancreatic cancer, colon cancer, colorectal cancer, esophageal cancer, skin cancer, lymphoma, and multiple myeloma.

[0268] In some embodiments, the cancer cells of the cancer have expression of the cell surface receptors or antigens mentioned in the present application. In some embodiments, the cancer cells of the cancer have high expression of the cell surface receptors or antigens mentioned in the present application (e.g., according to Depmap data (see https: / / depmap.org / portal / ), the corresponding gene expression is at least 0, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10). In some embodiments, the cancer cells of the cancer have high expression of FOLR1 and FOLH1, TRPV6 and FOLH1, GNRHR and FOLH1, SSTR2 and FOLH1, FOLR1 and SSTR2, or TRPV6 and FOLR1. In some embodiments, the disease is an immune disease, for example, an autoimmune disease, including but not limited to connective tissue disease, systemic sclerosis, rheumatoid arthritis and systemic lupus erythematosus.

[0269] In some embodiments, the disease is a cardiovascular disease, including but not limited to angina, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmias, and congenital heart disease.

[0270] In some embodiments, the disease is a metabolic disease, including but not limited to diabetes, gout, obesity, hypoglycemia, hyperglycemia, and dyslipidemia.

[0271] In some embodiments, the disease is a neurological disease, including but not limited to Alzheimer's disease, Parkinson's disease, Huntington's disease, head injury, multiple sclerosis, vertigo, coma, and epilepsy.

[0272] In some embodiments, the method provided herein further comprises administering one or more therapeutic agents in combination with the conjugate compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition. In some embodiments, the therapeutic agent targets an anti-cancer therapeutic target, induces or enhances an immune response against cancer, or is a chemotherapeutic agent.

[0273] The application will be described in more detail below by specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the present invention in any way. Those skilled in the art will readily appreciate that multiple non-critical parameters can be changed or modified to produce substantially the same results.

[0274] Example

[0275] The following examples are intended to further illustrate the application. Through description, the advantages and features of the application will become clear. However, these descriptions are merely exemplary and should not be construed as limiting the scope of the application.

[0276] Example 1: Preparation of coupled compounds

[0277] Coupled compounds CB-20BK, CB-18G, CB-20B, CB-10S, CB-20C, FA-MMAE, CB-20AK, CB- Synthesis of 1020, CB-1320 and CB-1820

[0278] 1. Weigh 10g of Rink amide-am resin (hereinafter referred to as "Rink Resin", Xi'an Lanxiao Technology New Materials Co., Ltd., item number 183599-10-2) with a substitution degree of 0.45mmol / g, load it into a solid phase reaction column, add DCM, bubble nitrogen into the solvent, and swell the resin for 30 minutes; remove the solvent, remove the Fmoc protecting group on the resin with DBLK, and then wash it with DMF 5 times. Weigh 4.79g (9mmol) of Fmoc-Lys(Dde)-OH and 1.47g (10.8mmol) of HOBt, dissolve them with DMF, add 1.67ml (10.8mmol) of DIC to the above solution under a 0℃ ice water bath, mix and activate it for 5 minutes, add the solution to the above reaction column, react for 3 hours, drain the solvent, and wash the resin in the reaction column 3 times. Then remove the Fmoc protecting group with DBLK.

[0279] 2. Repeat the above operation and couple Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH and intermediate 108 in sequence according to the structure.

[0280]

[0281] 3. Remove the Dde protecting group twice with 2% hydrazine hydrate / DMF, 10 minutes each time, and then wash the resin 5 times with DMF. Sequentially couple Fmoc-Glu-OtBu and pteroic acid. Finally, shrink the resin twice with methanol, drain the solvent, and obtain 17.4g of protected peptide resin.

[0282] 4. Add 17.4 g of the peptide resin obtained in the previous step to a 250 ml single-necked flask, prepare 139 ml of lysate in advance, TFA: H2O: TIS = 95: 3: 2 (volume ratio), and weigh 2.1 g of DTT to add to the lysate. Add the lysate to the above flask, react at room temperature for 2.5 hours, filter, and continue to wash the resin with 30 ml of TFA. Combine the above filtrate and add it to 834 ml of anhydrous ether. At this time, a yellow solid precipitates. Centrifuge to obtain a solid, wash the solid with anhydrous ether, and vacuum dry to obtain 6.4 g of a yellow solid. The crude product yield is 93.4%. HPLC purity is 82.3%. The product was separated by HPLC (preparative conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid aqueous solution, B: acetonitrile, elution gradient (15-25)% B, time 60 minutes, fraction collection), and the fraction containing qualified product was lyophilized to obtain 20BK-SM09 4.73 g with a purity of 98.8%.

[0283] 5. Weigh 4.09g (3.11mmol) of Mc-Val-Cit-PAB-MMAE (LT1002) into a 1000ml single-necked flask, add 500ml of phosphate buffer and 100ml of acetonitrile, stir, keep pH=7.2 until clear, add 4.73g (3.11mmol) of intermediate 20BK-SM09, react at room temperature for 2 hours, and monitor the reaction by HPLC. After the reaction is complete, filter, and separate the filtrate by HPLC preparation (preparation conditions: C18 column, mobile phase A: ammonium bicarbonate solution (pH=7.2), B: acetonitrile, elution gradient (25-35)% B, time 60 minutes, collect fractions), freeze-dry the fraction containing qualified products to obtain CB-20BK 6.96g product, purity 98.8%, yield 78.8%.

[0284] Similarly, the coupled compounds CB-18G, CB-20B, CB-10S, CB-20C, FA-MMAE (structure shown below), CB-20AK (structure shown below), CB-1020, CB-1320 and CB-1820 can be obtained by steps similar to the above method.

[0285]

[0286] Synthesis of coupled compounds CB-50S, CB-60S and CB-60SK

[0287] 1. Weigh 10 g of Wang resin (hereinafter referred to as "Wang Resin", Xi'an Lanxiao Technology New Materials Co., Ltd., item number 1365700-43-1) with a substitution degree of 1.1 mmol / g, load it into a solid phase reaction column, add DMF, bubble nitrogen into the solvent, and swell for 30 minutes; weigh 14.3 g (22 mmol) of Fmoc-Arg(pbf)-OH, 3.56 g (26.4 mmol) of HOBt, and 0.27 g (2.2 mmol) of DMAP, dissolve them in DMF, add 4.1 ml of DIC (26.4 mmol) in a 0°C ice-water bath, mix to activate it for 5 minutes, add the solution to the reaction column, react for 3 hours, drain the solvent, and wash 3 times.

[0288] 2. Dissolve 10.4 ml of acetic anhydride and 8.9 ml of pyridine in 50 ml of DMF, add the mixed solution to the washed resin, block at room temperature for 5 hours, wash three times with DMF, shrink with methanol and then drain the resin to obtain Fmoc-Arg(pbf)-Wang Resin with a detection substitution degree of 0.53 mmol / g.

[0289] 3. Weigh 3.8 g (2 mmol) of Fmoc-Arg(pbf)-Wang Resin (Sub = 0.53 mmol / g) into a reaction column, wash it with DMF 3 times, and then add DMF to swell the resin for 30 minutes. Then remove the Fmoc protecting group with DBLK, and then wash it with DMF 6 times. Weigh 2.0 g (6 mmol) of Fmoc-Pro-OH and 0.97 g (7.2 mmol) of HOBt, dissolve them with DMF, add 1.1 ml of DIC (7.2 mmol) in a 0°C ice-water bath, mix to activate it for 5 minutes, add it to the reaction column, react for 2 hours, and then remove the Fmoc protecting group with DBLK.

[0290] 4. Repeat the above operation, and sequentially couple Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-propargyl-Gly-OH, Fmoc-Glu-OtBu and pteroic acid according to the structure. Shrink with methanol twice, drain the solvent, and obtain 8.4g of peptide resin.

[0291] 5. Add 8.4 g of the peptide resin obtained in the previous step to a 250 ml single-necked flask, prepare 67 ml of lysis solution in advance, TFA: H2O: TIS = 95: 3: 2 (volume ratio), and weigh 0.92 g of DTT to add to the lysis solution. Add the lysis solution to the flask, react at room temperature for 2.5 hours, filter out the resin, wash the resin with 20 ml of TFA, combine the filtrate, add it to 402 ml of anhydrous ether to precipitate a yellow solid, centrifuge to obtain a solid, wash the solid with anhydrous ether, and vacuum dry to obtain 4.06 g of a yellow solid, with a crude product yield of 97.3%. HPLC purity 84.6%. HPLC preparative separation (preparative conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid aqueous solution, B: acetonitrile, elution gradient (20-29)% B, time 60 minutes, collect fractions), freeze-dry the fraction containing qualified products to obtain 50S-SM09 2.86 g, with a purity of 97.6%.

[0292] 6. Weigh 1.29g (1.37mmol) of LT1000N3 and add it to a 500ml single-necked flask, and add 270ml of mixed solvent (ACN: H2O = 1: 4), 393mg (2.74mmol) of CuBr, and stir. Add 2.86g (1.37mmol) of intermediate 50S-SM09, react at room temperature for 2-3 hours, and monitor the reaction by HPLC. After the reaction is complete, filter and separate by HPLC preparation (preparation conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid aqueous solution, B: acetonitrile, elution gradient (22-40)% B, time 60 minutes, collect fractions), freeze-dry the fraction containing qualified products to obtain 3.17g of CB-50S, with a purity of 98.6% and a yield of 76.4%.

[0293] Similarly, the coupled compounds CB-60S and CB-60SK can be obtained by steps similar to the above method.

[0294] Synthesis of CB-20R

[0295] 1. Weigh 5g of Rink Resin with a substitution degree of 0.45mmol / g, load it into a solid phase reaction column, add DCM, bubble nitrogen into the solvent, and swell the resin for 30 minutes; remove the solvent, remove the Fmoc protecting group on the resin with DBLK, and then wash it with DMF 5 times. Weigh 2.4g (4.5mmol) of Fmoc-Lys(Dde)-OH and 0.74g (5.4mmol) of HOBt, dissolve them with DMF, add 0.84ml (5.4mmol) of DIC in a 0℃ ice water bath, mix to activate it for 5 minutes, add it to the reaction column, react for 3 hours, drain it, and wash it 3 times. Then remove the Fmoc protecting group with DBLK.

[0296] 2. Repeat the above operation and couple Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH and intermediate 108 in sequence according to the structure.

[0297] 3. Remove the Dde protecting group twice with 2% hydrazine hydrate / DMF, 10 minutes each time, and then wash with DMF 5 times. Coupling DOTA-tris(tBu)ester. Remove the Dde protecting group twice with 2% hydrazine hydrate / DMF, 10 minutes each time, and then wash with DMF 5 times. Sequentially couple Fmoc-Glu-OtBu and pteroic acid, and finally shrink with methanol twice, drain and obtain 9.2g of protected peptide resin.

[0298] 4. Add 9.2 g of the peptide resin obtained in the previous step to a 250 ml single-necked flask, prepare 74 ml of lysis solution in advance, TFA: H2O: TIS = 95: 3: 2 (volume ratio), and weigh 1.05 g of DTT to add to the lysis solution. Add the lysis solution to the flask, react at room temperature for 2.5 hours, filter out the resin, wash the resin with 20 ml of TFA, combine the filtrate, add it to 560 ml of anhydrous ether to precipitate a yellow solid, centrifuge, wash the solid with anhydrous ether, and vacuum dry to obtain 3.8 g of yellow solid, with a crude product yield of 87.4%. HPLC purity is 81.2%. Prepare and separate by HPLC (preparation conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid aqueous solution, B: acetonitrile, elution gradient (15-25)% B, time 60 minutes, collect fractions), freeze-dry the fraction containing the synthetic product to obtain 20R-SM09 2.9 g, with a purity of 97.8%.

[0299] 5. 20R-SM09 is complexed with the radioactive nuclide ion M to obtain CB-20R. Specifically, the radioactive marker 177 Lu (about 50 MBq) was mixed with 100 μl of 0.5 M sodium acetate buffer (pH=5). 40 μl of 1 mM CB-20R solution dissolved in 10% DMSO aqueous solution, 2 μl of saturated ascorbic acid solution and 100 μl of 177 The Lu solution was mixed and heated to 95°C for 10 minutes. The labeling was verified by radio-HPLC (0-100% ACN in water within 5 minutes, C18 column).

[0300] Synthesis of compound CR19425

[0301]

[0302]

[0303] 1. Under N2 protection, add 441.4 mg CR19420 (structure as shown in the above reaction step) and 8.0 mL DMF to the reaction bottle, stir to dissolve, cool in an ice bath, add 459.2 mg HATU, add 380 μL DIPEA, stir for half an hour; add 500.0 mg CR19419 (structure as shown in the above reaction step), add 190 μL DIPEA, and react at room temperature until completion. After the reaction is completed, pour the reaction solution into acetic acid water, precipitate solid, filter, wash the filter cake with acetic acid water, wash with water, and vacuum dry to obtain 835.7 mg CR19421 (structure as shown in the above reaction step), brown powder, HPLC purity: 90.0%, yield: 90.6%.

[0304] 2. Under N2 protection, add 835.7 mg CR19421 and 16 mL 10% piperidine DMF solution to the reaction bottle and react at room temperature for half an hour. After the reaction is completed, pour the reaction solution into TFA / MTBE, precipitate solid, filter, wash the filter cake with MTBE, and vacuum dry to obtain 599.7 mg CR19422 (structure as shown in the above reaction step), earthy gray powder, HPLC purity: 84.7%, yield: 83.0%.

[0305] 3. Under N2 protection, add 599.7 mg CR19422, 586.7 mg CR19424 (structure as shown in the above reaction step), 15 mL DMF to the reaction bottle, stir to dissolve, cool in an ice bath, add 495.7 mg HATU, add 410 μL DIPEA, and react at room temperature. After the reaction is completed, the reaction solution is purified, the acetonitrile is removed under reduced pressure, and the pure product solution is extracted with a mixed solvent of dichloromethane and methanol, concentrated and dried to obtain 674.9 mg CR19423 (structure as shown in the above reaction step), yellow powder, HPLC purity: 88.4%, yield: 63.1%.

[0306] 4. Under N2 protection, add 14.8 mg CR19423, 3.0 mL PBS buffer with pH = 6.6, 3.0 mL acetonitrile to the reaction bottle, stir to dissolve, add 32.9 mg CBSM09, adjust pH to 6.6-6.8 with Na2HPO4, and react for half an hour. After the reaction is completed, the reaction solution is purified, and the pure product is freeze-dried to obtain 21.8 mg CR19425, yellow powder, HPLC purity: 95.6%, yield: 48.8%.

[0307] Synthesis of compound CR19426

[0308]

[0309] 1. Under N2 protection, add 25.2 mg CR19423, 3.0 mL PBS buffer solution with pH = 6.6, and 3.0 mL acetonitrile to the reaction bottle, stir to dissolve, add 55.5 mg 18G-SM09, adjust the pH to 6.6-6.8 with Na2HPO4, and react for half an hour. After the reaction is completed, the reaction solution is purified, and the pure product is freeze-dried to obtain 44.6 mg CR19426, yellow powder, HPLC purity: 95.4%, yield: 55.2%.

[0310] Synthesis of compound CR19428

[0311]

[0312] 1. Under N2 protection, add 486 mg CR19423, 3.0 mL PBS buffer solution with pH=6.6, 3.0 mL acetonitrile to the reaction bottle, stir to dissolve, add 712 mg 20BK-SM09, adjust pH to 6.6-6.8 with Na2HPO4, and react for half an hour. After the reaction is completed, the reaction solution is purified, and the pure product is freeze-dried to obtain 508 mg CR19428, yellow powder, HPLC purity: 96.7%, yield: 42.3%.

[0313] Example 2: Determination of affinity between conjugate compounds and target proteins

[0314] 1. Affinity determination of CB-20BK binding to protein FOLR1

[0315] Experimental instruments, materials and reagents

[0316] BIAcore T200(GE)

[0317] CM5 chip (GE, part number: 29104988)

[0318] Buffer: HBS-EP+buffer 10X (GE, Catalog No.: BR100669), diluted 10 times with deionized water before use.

[0319] Amine coupling kit (GE, catalog number: BR100050)

[0320] Regeneration reagent: 10 mM Glycine 2.0 (GE, Cat. No. BR100355)

[0321] 10mM Glycine 3.0 (GE, Cat. No. BR100357)

[0322] Experimental procedures

[0323] The experiment was conducted according to the BIAcore T200 (GE) manual to determine the affinity of the analytes CB-20BK, CB-20AK, folic acid (FA) and FA-MMAE to FOLR1. The CM5 chip was coupled to the ligand FOLR1 (R&D System, Catalog No. 5646-FR). The experimental results are shown in Table 4.

[0324] Table 4. Binding affinity of CB-20BK and related compounds to FOLR1

[0325] ka kd KD FA (Folic Acid) <![CDATA[3.34×10 6 M -1 s -1 ]]> <![CDATA[2.26×10 -4 s -1 ]]> <![CDATA[6.77×10 -11 M]]> FA-MMAE <![CDATA[1.79×10 6 M -1 s -1 ]]> <![CDATA[1.15×10 -4 s -1 ]]> <![CDATA[6.43×10 -11 M]]> CB-20AK N / D N / D N / D <![CDATA[CB - 20BK]]> <![CDATA[1.03×10 6 M -1 s -1 ]]> <![CDATA[1.31×10 -4 s -1 ]]> <![CDATA[1.27×10 -10 M]]>

[0326] "N / D" means no specific binding was detected.

[0327] Table 4 shows that CB-20BK binds specifically to FOLR1 with good affinity. The affinity of CB-20BK binding to FOLR1 is slightly weaker than the affinity of FA or FA-MMAE binding to FOLR1. CB-20AK does not have the folic acid part of CB-20BK, and its specific binding to FOLR1 was not detected in the experiment.

[0328] 2. Affinity determination of CB-20BK binding to FOLH1 protein

[0329] Experimental instruments, materials and reagents

[0330] Gator TM (Probe Life)

[0331] SA probe (Probe Life, Cat. No. 1906018)

[0332] Buffer: Q buffer (Probe Life), 10 mM, pH = 7.4

[0333] Experimental procedures

[0334] (1) Synthesis steps of analyte Biotin-CB-20BK

[0335] 1) Weigh 5.1g of Rink Resin with a substitution degree of 0.45mmol / g, load it into a solid phase reaction column, add DCM, swell with nitrogen bubbling for 30 minutes; remove the solvent, remove the Fmoc protecting group with DBLK, and then wash with DMF 5 times. Weigh 2.45g of Fmoc-Lys(Dde)-OH and 0.75g of HOBt, dissolve them with DMF, add 0.83ml of DIC in a 0℃ ice water bath, activate for 5 minutes, add to the reaction column, react for 3 hours, drain, and wash 3 times. Then remove the Fmoc protecting group with DBLK.

[0336] 2) Repeat the above operation and couple Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH and intermediate 108 in sequence according to the structure.

[0337] 3) Remove the Dde protecting group with 2% hydrazine hydrate / DMF twice, 10 minutes each time, and then wash with DMF 5 times. Sequentially couple Fmoc-Lys(Biotin)-OH, Fmoc-Glu-OtBu and pteroic acid. After coupling pteroic acid, wash with DMF twice, and finally shrink with methanol twice, drain and obtain 9.7g of protected peptide resin.

[0338] 4) Add 9.7g of the peptide resin obtained in the previous step to a 250ml single-necked flask, pre-prepare 77.6ml of lysate, TFA:H2O:TIS=95:3:2 (volume ratio), and weigh 1.1g of DTT to add to the lysate. Add the lysate to the flask, react at room temperature for 2.5 hours, filter out the resin, wash the resin with 20ml of TFA, combine the filtrate, add it to 466ml of tertiary methyl ether to precipitate a yellow solid, centrifuge, wash the solid with tertiary methyl ether, and vacuum dry to obtain 4.6g of a yellow solid with an HPLC purity of 83.2%. After HPLC preparative separation and freeze-drying, 2.1g of Biotin-20BK-SM09 with a purity of more than 95% was obtained.

[0339] 5) Weigh 500 mg of Mc-Val-Cit-PAB-MMAE (LT1002) and add it to a 250 ml single-necked flask, add 65 ml of phosphate buffer and 20 ml of acetonitrile, stir, keep pH = 7.2 until clear, add 785 mg of intermediate Biotin-20BK-SM09, react at room temperature for 2 hours, and monitor the reaction by HPLC. After the reaction is complete, filter, separate by HPLC, and freeze-dry to obtain 723 mg of Biotin-CB-20BK, with a purity of 96.8% and a yield of 59.4%.

[0340] (2) According to Gator TM The experiment was conducted according to the Probe Life manual to determine the affinity of Biotin-CB-20BK binding to FOLH1. The SA probe was bound to the ligand Biotin-CB-20BK and the analyte was FOLH1 (Sino Biologicals, catalog number, 15877-H07H). The experimental results are shown in Table 5.

[0341] Table 5. Affinity of CB-20BK binding to FOLH1

[0342] ka kd KD FOLH1 <![CDATA[1.19×10 4 M -1 s -1 ]]> <![CDATA[1.70×10 -4 s -1 ]]> <![CDATA[6.98×10 -9 M]]>

[0343] Table 5 shows that CB-20BK binds to FOLH1 with good affinity.

[0344] 3. FOLH1 binds to the CB-20BK-FOLR1 complex

[0345] Experimental materials and reagents

[0346] BIAcore T200(GE)

[0347] CM5 chip (GE, part number: 29104988)

[0348] Buffer: HBS-EP+buffer 10X (GE, Catalog No.: BR100669), diluted 10 times with deionized water before use.

[0349] Amine coupling kit (GE, catalog number: BR100050)

[0350] Regeneration reagent: 10 mM Glycine 2.0 (GE, Cat. No. BR100355)

[0351] 10mM Glycine 3.0 (GE, Cat. No. BR100357)

[0352] Experimental procedures

[0353] CM5 chip coupled ligand: FOLR1 (R&D System, Cat. No. 5646-FR)

[0354] Analytes: CB-20BK, FA-MMAE and FOLH1 (Sino Biologicals, Cat. No. 15877-H07H)

[0355] According to the BIAcore T200 (GE) operating manual, FOLR1 was coupled to a CM5 chip, CB-20BK or FA-MMAE was first sampled, and then FOLH1 was sampled to detect the binding of FOLH1 to the CB-20BK-FOLR1 complex. The experimental results are shown in Table 6.

[0356] Table 6. Binding of different concentrations of FOLH1 solutions to the CB-20BK-FOLR1 complex

[0357] Concentration (μM) FA-MMAE(RU) CB-20BK(RU) 0.5 9.8 23.9 0.25 8.4 14.1 0.125 4.2 4.6 0.0625 -1 -0.9

[0358] Table 6 shows that the amount of high-concentration FOLH1 solution binding to the CB-20BK-FOLR1 complex is significantly higher than the amount of FOLH1 binding to the FA-MMAE-FOLR1 complex, and it continues to rise. The binding of FOLH1 to the FA-MMAE-FOLR1 complex tends to be saturated at high concentrations. This experiment proves that CB-20BK can bind well to both FOLR1 and FOLH1 receptors at the same time.

[0359] Example 3: Binding and endocytosis studies of ligand conjugates to target cells

[0360] 1. Cell binding and endocytosis experiments of the conjugate compound CB-20BK

[0361] Synthesis of labeled samples Cy5-pep-20BK, Cy5-FA, and Cy5-pep-20AK

[0362] (1) Weigh 2 g of Rink Resin with a substitution degree of 0.45 mmol / g, load it into a solid phase reaction column, add DCM, bubble nitrogen into the solvent, and swell the resin for 30 minutes; remove the solvent, remove the Fmoc protecting group with DBLK, and then wash it with DMF 5 times. Weigh 0.96 g (1.8 mmol) of Fmoc-Lys(Dde)-OH and 0.3 g (2.2 mmol) of HOBt, dissolve them in DMF, add 0.33 ml (2.2 mmol) of DIC in a 0°C ice water bath, mix to activate it for 5 minutes, add it to the reaction column, react for 3 hours, drain it, and wash it 3 times. Then remove the Fmoc protecting group with DBLK.

[0363] (2) Repeat the above operation to sequentially couple Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH and intermediate 108 according to the structure:

[0364] (3) Remove the Dde protecting group twice with 2% hydrazine hydrate / DMF, 10 minutes each time, and then wash with DMF 5 times. Sequentially couple Fmoc-Lys(Dde)-OH, Fmoc-Glu-OtBu and pteroic acid.

[0365] (4) The Dde protecting group was then removed with 2% hydrazine hydrate / DMF twice, each time for 10 minutes, and then washed with DMF 5 times. After coupling with Cy5-COOH, the mixture was washed with DMF twice and finally shrunk with methanol twice and dried to obtain 3.6 g of protected peptide resin.

[0366] (5) 3.6 g of the peptide resin obtained in the previous step was added to a 50 ml single-necked flask. 29 ml of lysate was prepared in advance, TFA: H2O: TIS = 95: 3: 2 (volume ratio), and 0.4 g of DTT was weighed and added to the lysate. The lysate was added to the flask and reacted at room temperature for 2.5 hours. The resin was filtered off and washed with 8 ml of TFA. The filtrate was combined and added to 173 ml of anhydrous ether to precipitate a yellow solid. The solid was centrifuged and washed with anhydrous ether. The solid was dried in vacuo to obtain 1.9 g of a blue solid. The crude product yield was 89.6%. The HPLC purity was 76.3%. The product was separated by HPLC preparation (preparation conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid aqueous solution, B: acetonitrile, elution gradient (20-28)% B, time 50 minutes, and fractions were collected). The fraction containing the qualified product was lyophilized to obtain 736 mg of Cy5-pep-20BK with a purity of 93.4%.

[0367] Similarly, compounds Cy5-FA and Cy5-pep-20AK can be obtained by steps similar to the above method.

[0368]

[0369]

[0370] Flow cytometry to detect cell binding and internalization of Cy5-pep-20AK / Cy5-pep-20BK samples

[0371] Sample information: Cy5-pep-20AK, Cy5-pep-20BK

[0372] Cell lines: LNCaP human prostate cancer cells, Du145 human prostate cancer cells, SKOV3 human ovarian cancer cells, NCI-H460 human lung cancer cells

[0373] Main reagents: IMDM culture medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, PBS

[0374] Experimental operation:

[0375] (1) Cell culture: Prepare cells, digest with Trypsin, collect and count, add cells to several culture bottles containing complete medium, and culture in a 37°C, 5% CO2 incubator. Finally, collect about 5×10 6 cells into a centrifuge tube.

[0376] (2) Sample incubation: Use PBS to dilute the Cy5-pep-20AK / Cy5-pep-20BK sample to 8 nmol / L. Centrifuge the cell suspension at 1000 rpm for 5 minutes, remove the supernatant, wash once with PBS, evenly suspend the cells, and divide them into different centrifuge tubes according to the experimental plan. Centrifuge to remove PBS, add 200 μl sample working solution to each tube, and incubate at 37°C for 15, 30, 60 and 90 minutes respectively. Keep one tube with only PBS as a blank control. Keep the operation away from light.

[0377] (3) Washing and loading: Centrifuge at 1000 rpm for 5 minutes to remove the working solution, wash the cells three times with PBS, and add an appropriate amount of PBS to suspend the cells to 1×10 6 Cells / ml. Turn on the Beckman CytoFLEX flow cytometer in advance, complete the startup and cleaning process, load the cell samples onto the instrument one by one, and read the fluorescence of 10,000 live cells under the APC channel. Keep the whole process away from light.

[0378] (4) Data analysis: Obtain the absolute MFI (mean fluorescence intensity) of APC fluorescence of each cell sample and the relative MFI to the blank control, and complete the data line graph based on the relative MFI.

[0379] Results and Analysis

[0380] Table 7. Expression levels of FOLR1 and FOLH1 in different cell lines (refer to Depmap data, source: https: / / depmap.org / portal / )

[0381] RNA-Seq LqCy SKOV3 Du145 NCI-H460 FOLR1 + / - 6+ + / - + / - FOLH1 10+ + + / - + / -

[0382] According to the Depmap data, data of 0 or negative numbers are represented as "-", 0.001-0.499 is represented as "+ / -", 0.500-1.499 is represented as "+", 1.500-2.499 is represented as "2+", and so on.

[0383] The fluorescence intensity of the cells was detected at 15 minutes, 30 minutes, 60 minutes and 90 minutes of incubation, and the results were plotted on the graph. Figure 2A and 2B. It can be seen that the sample Cy5-pep-20BK binds to and internalizes cells. In the LNCaP cell line with relatively high expression of FOLR1 and the SKOV3 cell line with relatively high expression of FOLH1, the fluorescence intensity of the bound and internalized CB-20BK is significantly higher than that in the DU145 cell line and NCI-H460 cell line with relatively low expression of FOLR1 and FOLH1. Cy5-pep-20AK only has the FOLH1 ligand, but not the FA ligand of FOLR1. Therefore, Cy5-pep-20AK shows high levels of binding and internalization in LNCaP cells with high expression of FOLH1. In SKOV3 cells with moderate expression of FOLH1, it shows moderate levels of binding and internalization; in both cell lines, the binding and internalization levels of Cy5-pep-20AK are significantly lower than those of Cy5-pep-CB-20BK. The degree of binding and internalization of the ligand conjugate to the cell is positively correlated with the expression level of cell-related receptors.

[0384] 2. Binding and endocytosis experiments of single ligand conjugate Cy5-FA to cells

[0385] Sample information: Cy5-FA

[0386] Cell lines: Hela cervical cancer cells, SKOV3 human ovarian cancer cells, and A549 human lung cancer cells

[0387] Main reagents: IMDM culture medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, PBS, anti-fluorescence quenching sealing medium containing DAPI

[0388] Experimental operation:

[0389] (1) Cell culture: Place the culture slide in a 24-well plate. Prepare the cells, digest with Trypsin, collect and count them, and dilute the cells to about 2×10 5 cells / ml, 500 μl of diluted cell solution was added to each well of a 24-well plate and cultured in a 37°C, 5% CO2 incubator for 48 hours.

[0390] (2) Sample incubation: Dilute the Cy5-FA sample to 73 nmol / L using IMDM medium. Shake off the medium in the 24-well plate, add 200 μl of sample working solution to each well of the cell slide, and incubate at 37°C for 15, 30, and 60 minutes respectively. Keep one well with only IMDM medium as a blank control. Keep the whole process away from light.

[0391] (3) Washing the sealing slide: shake off the working solution in the 24-well plate, wash three times with PBS preheated at 37°C, remove the cell slide, add 5 μl of anti-fluorescence quenching sealing medium containing DAPI on the slide, cover the cell slide with the sealing medium, and complete the sealing. Keep the operation away from light throughout the process.

[0392] (4) Sample reading: Turn on the Leica DM2500 fluorescence microscope and preheat the fluorescence exciter for 15 minutes. Take pictures of the cell nucleus and Cy5 fluorescent pigment at the same position under the corresponding fluorescence channel, and complete the image fusion in the software.

[0393] according to Figure 3 It can be seen that the fluorescence intensity of the sample Cy5-FA binding and internalization to the cells at 15 minutes and 30 minutes in the cell lines Hela and SKOV-3 with high expression of FOLR1 was significantly higher than that in the cell line A549 with relatively low expression of FOLR1. The degree of binding and internalization of the ligand conjugate to the cell is positively correlated with the expression level of the cell-related receptor.

[0394] Example 4: Cell proliferation inhibition experiment of coupled compounds

[0395] 1. CB-20BK tumor cell proliferation inhibition experiment

[0396] Sample information: CB-20BK

[0397] Cell lines: KB human oral epidermoid carcinoma cells, T-47D human breast cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung adenocarcinoma cells, HuH-7 human hepatoma cells, and LNCaP human prostate cancer cells

[0398] Main reagents: IMDM culture medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, CCK8

[0399] Experimental operation:

[0400] 1) Cell plating

[0401] Prepare cells in advance, digest with Trypsin, collect and count, and dilute KB cells to 2.5×10 4 T-47D cells were diluted to 1.3×10 4 cells / ml, and NCI-H460 cells were diluted to 3.5×10 4 cells / ml, CALU-3 cells were diluted to 4.0×10 4 cells / ml, HuH-7 cells were diluted to 3.0×10 4 cells / ml, LNCaP cells were diluted to 8.0×10 4 100 μl of diluted cell solution was added to each well of a 96-well plate. Negative and blank control wells were set up on each plate. The 96-well plate with cells was placed in a 37°C, 5% CO2 incubator for overnight culture.

[0402] 2) Dilution and sample addition

[0403] The sample was diluted to the required concentration with culture medium (see Table 8). 50 μl was added to each well of a 96-well plate after overnight culture, and three replicates were made. Negative control and blank control were set up and cultured in a 37°C, 5% CO2 incubator for 72 hours.

[0404] Table 8: Sample (CB-20BK) concentration after dilution

[0405] Tube No. Concentration after dilution μmol / L 1 201 2 50 3 13 4 3.1 5 0.8 6 0.2 7 0.05 8 0.01 9 0.003 10 0.0008

[0406] 3) Chromogenic plate reader

[0407] Take CCK-8 colorimetric solution, add 15 μl (10% of the liquid volume in the well) to each well, incubate at 37°C for an appropriate time (try to keep the OD value within the range of 1.0-2.0), remove the culture plate cover of the 96-well plate, place it on a microplate reader (Molecular Devices Spectra MAX Plus), and read the value at 450 nm.

[0408] 4) Data processing

[0409] SoftMax Pro was used to edit the data and draw a four-parameter fitting curve.

[0410] 5) Experimental results and analysis

[0411] According to the four-parameter fitting curve ( Figure 4A , where the C value corresponds to IC 50 ), CB-20BK has an inhibitory effect on the growth and proliferation of KB, T-47D, NCI-H460, CALU-3, HuH-7 and LNCaP tumor cell lines. Since the corresponding receptor expression levels of the coupled compounds in the cells are different, the inhibition levels are different. The IC 50 It was significantly lower than that of the cell line NCI-H460, which has relatively low receptor expression. CB-20BK showed a correlation between the anti-tumor effect and the expression level of cell-related receptors.

[0412] 2. Inhibition experiment of the coupled compound CB-20BK and related compounds on the proliferation of tumor cell lines LNCaP (human prostate cancer cells) and 22RV1 (human prostate cancer cells)

[0413] Different cell lines may have different sensitivity to the cytostatic toxicity of the payload in the ligand conjugate, which occasionally interferes with quantitative analysis. We selected a series of cell lines with known receptor expression levels for different ligands and tested their responses to the cytostatic effects of the ligand conjugate and the payload alone. The IC values ​​of the payload alone and the ligand conjugate in a cell line were taken as50 ratio, thereby removing this interference.

[0414] Related samples: MMAE, CB-20BK, CB-20AK and FA-MMAE

[0415] Experimental operation: Count the LNCaP and 22RV1 prepared in advance at 4×10 4 cells / ml and 2.0×10 4 Cells / ml cell density was spread on a 96-well cell culture plate, 100 μl / well, and each plate was set with corresponding negative and blank control wells. After overnight adherence, the diluted sample to be tested was added at 50 μl / well, placed in a 37°C, 5% CO2 incubator, cultured for 68-72 hours, CCK8 colorimetric solution was added, 15 μl (10% of the liquid volume in the well) was added to each well, incubated at 37°C for 45-70 minutes, and placed in an enzyme reader at 450nm to read the value. Data editing was performed using SoftMax Pro, and a 4-P fitting curve was drawn. The calculated data are shown in Table 9.

[0416] Table 9. Inhibitory effects of MMAE, FA-MMAE, CB-20BK and CB-20AK on cell line proliferation and expression levels of receptor genes in cell lines (refer to Depmap data)

[0417]

[0418]

[0419] As can be seen from the table above, the difference in FOLR1 expression levels between LNCaP and 22RV1 is not large, and the FOLH1 expression level of LNCaP is significantly higher than that of 22RV1. CB-20BK (with FOLH1 ligand and FOLR1 ligand) and CB-20AK (with only FOLH1 ligand) have inhibitory effects on the proliferation of LNCaP and 22RV1 tumor cell lines. CB-20BK and CB-20AK have different degrees of cell inhibition for different levels of receptor FOLH1 expression. In the cell line LNCaP with relatively high expression of receptor FOLH1, IC 50 The ratio is higher than the IC of 22RV1, a cell line with relatively low receptor expression. 50 FA-MMAE also has an inhibitory effect on the proliferation of LNCaP and 22RV1 tumor cell lines. Since the FOLR1 expression levels of LNCaP and 22RV1 are very low, the expression in LNCaP cells (FOLR1 gene expression level is 0.3219, refer to Depmap data) is slightly higher than that in 22RV1 cells (FOLR1 gene expression level is 0.0704, refer to Depmap data). The IC of FA-MMAE in LNCaP and 22RV1 is50 The difference in the ratio is only 1.5 times. The above data show that the cell proliferation inhibition effect is positively correlated with the expression levels of cell-expressed receptors FOLH1 and FOLR1.

[0420] 3. Inhibitory effect of the coupled compound CB-20BK and related compounds on the proliferation of tumor cell lines PANC-1 (human pancreatic cancer cells) and CFPAC-1 (human pancreatic cancer cells)

[0421] Related samples: MMAE and CB-20BK

[0422] Experimental operation: Prepare the cell count in advance, PANC-1 and CFPAC-1 at 4×10 4 Cells / ml were plated in a 96-well plate at a cell density of 100ul / well. Negative and blank control wells were set up on each plate. After overnight attachment, the diluted sample to be tested was added at 50μl / well and placed in a 37°C, 5% CO2 incubator for 68-72 hours. CCK8 colorimetric solution was added, 15μl (10% of the volume of the liquid in the well) was added to each well, incubated at 37°C for 45-70 minutes, and placed in an ELISA reader at 450nm to read the value. Data were edited using SoftMax Pro and a 4-P fitting curve was drawn. The calculated data are shown in the following table:

[0423] Table 10. Inhibitory effect of CB-20BK on cell line proliferation and expression levels of cell line receptor genes (refer to Depmap data)

[0424]

[0425] As shown in Table 10, the expression level of FOLH1 in CFPAC-1 is very low, and the expression level of FOLR1 in CFPAC-1 is significantly higher than that in the PANC1 cell line. CB-20BK has an inhibitory effect on the proliferation of both PANC-1 and CFPAC-1 tumor cell lines. The degree of inhibition varies with the expression level of receptor FOLR1. In the CFPAC-1 cell line with relatively high expression of receptor FOLR1, IC 50 The ratio was significantly higher than the IC of PANC-1, which has a relatively low expression of the receptor FOLR1. 50 The ratio showed a positive correlation between the cell proliferation inhibition effect and the expression level of cell-related receptor FOLR1.

[0426] Experiments 2 and 3 demonstrated that the two ligands in CB-20BK and their receptors (FOLR1 and FOLH1) expressed on the cell surface play an important role in the process of the compound inhibiting tumor cell proliferation.

[0427] 4. CB-20B tumor cell proliferation inhibition experiment

[0428] Sample information: CB-20B

[0429] Cell lines: A549 human lung cancer cells, HuH-7 human hepatoma cells, KB human oral epidermoid carcinoma cells, LNCaP human prostate cancer cells, DU145 human prostate cancer cells, and T-47D human breast cancer cells

[0430] Main reagents: IMDM culture medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, CCK8

[0431] Experimental operation:

[0432] 1) Cell plating

[0433] Prepare cells in advance, digest with Trypsin, collect and count, use complete cell culture medium, and culture A549 cells, HuH-7 cells, KB cells and DU145 cells at 2×10 4 T-47D cells were plated at a density of 1×10 5 LNCaP cells were plated at a density of 6×10 4 96-well plates were plated at a density of 100 cells / ml, 100 μl of diluted cell solution was added to each well, negative and blank control wells were set on each plate, and the 96-well plates with cells were placed in a 37°C, 5% CO2 incubator for overnight culture.

[0434] 2) Dilution and sample addition

[0435] The sample was diluted to the required concentration with culture medium (see Table 11). 50 μl was added to each well of a 96-well plate after overnight culture, and three replicates were made. Negative control and blank control were also set up. The plate was placed in a 37°C, 5% CO2 incubator for 72 hours.

[0436] Table 11: Sample (CB-20B) concentration after dilution

[0437] Tube No. Concentration after dilution μmol / L 1 200 2 66.7 3 22.2 4 7.4 5 2.5 6 0.8 7 0.3 8 0.09 9 0.03 10 0.01

[0438] 3) Chromogenic plate reader

[0439] Take CCK-8 colorimetric solution, add 15 μl (10% of the liquid volume in the well) to each well, incubate at 37°C for an appropriate time (try to keep the OD value within the range of 1.0-2.0), remove the culture plate cover of the 96-well plate, place it on a microplate reader (Molecular Devices Spectra MAX Plus), and read the value at 450 nm.

[0440] 4) Data processing

[0441] SoftMax Pro was used to edit the data and draw a four-parameter fitting curve.

[0442] 5) Experimental results and analysis

[0443] According to the four-parameter fitting curve ( Figure 4B , where the C value corresponds to IC 50 ), CB-20B has an inhibitory effect on the growth and proliferation of A549, HuH-7, KB, LNcap, DU145 and T-47D tumor cell lines. Since the corresponding receptor expression levels of the coupled compounds in the cells are different, the inhibition levels are different. The IC values ​​for the cell lines T-47D, LNcap, KB, HuH-7 and DU145 with relatively high receptor expression are 50 It was significantly lower than that of the cell line A549 with relatively low receptor expression. CB-20B showed a correlation between the anti-tumor effect and the expression level of cell-related receptors.

[0444] 5. CB-10S tumor cell proliferation inhibition experiment

[0445] Sample information: CB-10S

[0446] Cell lines: KB human oral epidermoid carcinoma cells, NCI-H460 human lung cancer cells, RT4 human bladder cancer cells, T-47D human breast cancer cells, and LNcap human prostate cancer cells

[0447] Main reagents: IMDM culture medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, CCK8

[0448] Experimental operation:

[0449] 1) Cell plating

[0450] Prepare cells in advance, digest with Trypsin, collect and count, and dilute KB cells to 3.5×10 4 cells / ml, LNCaP cells were diluted to 8.0×10 4 T-47D cells were diluted to 1.2×10 4 cells / ml, and NCI-H460 cells were diluted to 2.5×10 4 cells / ml, RT4 cells were diluted to 1.2×10 4 cells / ml, take a 96-well plate, add 100 μl of diluted cell solution to each well, set up negative and blank control wells on each plate, and place the 96-well plate with cells in a 37°C, 5% CO2 incubator for overnight culture.

[0451] 2) Dilution and sample addition

[0452] The sample was diluted to the required concentration with culture medium (see Table 12). 50 μl was added to each well of a 96-well plate after overnight culture, and three replicates were made. Negative control and blank control were set up and cultured in a 37°C, 5% CO2 incubator for 72 hours.

[0453] Table 12: Sample (CB-10S) concentration after dilution

[0454]

[0455]

[0456] 3) Chromogenic plate reader

[0457] Take CCK-8 colorimetric solution, add 15 μl (10% of the liquid volume in the well) to each well, incubate at 37°C for an appropriate time (try to keep the OD value within the range of 1.0-2.0), remove the culture plate cover of the 96-well plate, place it on a microplate reader (Molecular Devices Spectra MAX Plus), and read the value at 450 nm.

[0458] 4) Data processing

[0459] SoftMax Pro was used to edit the data and draw a four-parameter fitting curve.

[0460] 5) Experimental results and analysis

[0461] According to the four-parameter fitting curve ( Figure 4C , where the C value corresponds to IC 50 ), CB-10S has an inhibitory effect on the growth and proliferation of KB, LNCaP, T-47D, RT4 and NCI-H460 tumor cell lines. Due to the different expression levels of the corresponding receptors of the coupled compounds in the cells, the inhibition levels are different. For the cell lines KB, LNCaP, T-47D and RT4 with relatively high expression of receptors, the IC 50 It was significantly lower than the cell line NCI-H460 with relatively low receptor expression. CB-10S showed a correlation between the anti-tumor effect and the expression level of cell-related receptors.

[0462] 6. CB-60S tumor cell proliferation inhibition experiment

[0463] Sample information: CB-60S

[0464] Cell lines: KB human oral epidermoid carcinoma cells, T-47D human breast cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung adenocarcinoma cells, HuH-7 human hepatoma cells, and LNCaP human prostate cancer cells

[0465] Main reagents: IMDM culture medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, CCK8

[0466] Experimental operation:

[0467] 1) Cell plating

[0468] Prepare cells in advance, digest with Trypsin, collect and count, and dilute KB cells to 2.0×10 4 T-47D cells were diluted to 1.5×10 4 cells / ml, NCI-H460 cells were diluted to 2.0×10 4 cells / ml, and CALU-3 cells were diluted to 3.0×10 4 cells / ml, HuH-7 cells were diluted to 4.0×10 4 cells / ml, LNCaP cells were diluted to 8.0×10 4 cells / ml, take a 96-well plate, add 100 μl of diluted cell solution to each well, set up negative and blank control wells on each plate, and place the 96-well plate with cells in a 37°C, 5% CO2 incubator for overnight culture.

[0469] 2) Dilution and sample addition

[0470] The sample was diluted to the required concentration with culture medium (see Table 13). 50 μl was added to each well of a 96-well plate after overnight culture, and three replicates were made. Negative control and blank control were set up and cultured in a 37°C, 5% CO2 incubator for 72 hours.

[0471] Table 13: Sample (CB-60S) concentration after dilution

[0472] Tube No. Concentration after dilution μmol / L 1 320 2 80 3 20 4 5 5 1.3 6 0.3 7 0.08 8 0.02 9 0.005 10 0.001

[0473] 3) Chromogenic plate reader

[0474] Take CCK-8 colorimetric solution, add 15 μl (10% of the liquid volume in the well) to each well, incubate at 37°C for an appropriate time (try to keep the OD value within the range of 1.0-2.0), remove the culture plate cover of the 96-well plate, place it on a microplate reader (Molecular Devices Spectra MAX Plus), and read the value at 450 nm.

[0475] 4) Data processing

[0476] SoftMax Pro was used to edit the data and draw a four-parameter fitting curve.

[0477] 5) Experimental results and analysis

[0478] Four parameter fitting curve diagram ( Figure 4D , where the C value corresponds to IC 50 ), CB-60S has an inhibitory effect on the growth and proliferation of KB, T-47D, NCI-H460, CALU-3, HuH-7 and LNCaP tumor cell lines. Due to the different expression levels of the corresponding receptors of the coupled compounds in the cells, the inhibition levels vary. For the cell lines LNCaP and HuH-7 with relatively high expression of the receptors, the IC 50 The value was significantly lower than that of NCI-H460, KB, T-47D and CALU-3 cell lines with relatively low receptor expression. CB-60S showed a correlation between the anti-tumor effect and the expression level of cell-related receptors.

[0479] 7. CB-60SK tumor cell proliferation inhibition experiment

[0480] Sample information: CB-60SK

[0481] Cell lines: KB human oral epidermoid carcinoma cells, T-47D human breast cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung adenocarcinoma cells, HuH-7 human hepatoma cells, and LNCaP human prostate cancer cells

[0482] Main reagents: IMDM culture medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, CCK8

[0483] Experimental operation:

[0484] 1) Cell plating

[0485] Prepare cells in advance, digest with Trypsin, collect and count, and dilute KB cells to 2.5×10 4 T-47D cells were diluted to 1.3×10 4 cells / ml, and NCI-H460 cells were diluted to 3.5×10 4 cells / ml, CALU-3 cells were diluted to 4.0×10 4 cells / ml, HuH-7 cells were diluted to 3.0×10 4 cells / ml, LNCaP cells were diluted to 8.0×10 4 cells / ml, add 100 μl of diluted cell solution to each well, set up negative and blank control wells on each plate, and place the 96-well plate with cells in a 37°C, 5% CO2 incubator for overnight culture.

[0486] 2) Dilution and sample addition

[0487] The sample was diluted to the required concentration with culture medium (see Table 14). 50 μl was added to each well of a 96-well plate after overnight culture, and three replicates were made. Negative control and blank control were set up and cultured in a 37°C, 5% CO2 incubator for 72 hours.

[0488] Table 14: Sample (CB-60SK) concentration after dilution

[0489] Tube No. Concentration after dilution μmol / L 1 289 2 72 3 18 4 4.5 5 1.1 6 0.3 7 0.07 8 0.02 9 0.004 10 0.001

[0490] 3) Chromogenic plate reader

[0491] Take CCK-8 colorimetric solution, add 15 μl (10% of the liquid volume in the well) to each well, incubate at 37°C for an appropriate time (try to keep the OD value within the range of 1.0-2.0), remove the culture plate cover of the 96-well plate, place it on a microplate reader (Molecular Devices Spectra MAX Plus), and read the value at 450 nm.

[0492] 4) Data processing

[0493] SoftMax Pro was used to edit the data and draw a four-parameter fitting curve.

[0494] 5) Experimental results and analysis

[0495] According to the four-parameter fitting curve ( Figure 4E , where the C value corresponds to IC 50 ), CB-60SK has an inhibitory effect on the growth and proliferation of KB, T-47D, NCI-H460, CALU-3, HuH-7 and LNCaP tumor cell lines. Due to the different expression levels of the corresponding receptors of the coupled compounds in the cells, the inhibition levels vary. For the cell lines LNCaP and HuH-7 with relatively high expression of the receptors, the IC 50 The value was significantly lower than that of T-47D, NCI-H460, CALU-3 and KB cell lines with relatively low receptor expression. CB-60SK showed a correlation between the anti-tumor effect and the expression level of cell-related receptors.

[0496] 8. CB-18G tumor cell proliferation inhibition experiment

[0497] Sample information: CB-18G

[0498] Cell lines: A549 human lung cancer cells, Hela human cervical cancer cells, SCLC-21H small cell lung cancer cells, U-2 OS human osteosarcoma cells, T-47D human breast cancer cells, and NCI-H460 human lung cancer cells

[0499] Main reagents: IMDM culture medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, CCK8

[0500] Experimental operation:

[0501] 1) Cell plating

[0502] Prepare cells in advance, digest with Trypsin, collect and count, use complete cell culture medium, and culture A549 cells, Hela cells, SCLC-21H cells and U-2OS cells at 2×10 4 T-47D cells and NCI-H460 cells were plated at a density of 3×10 4 96-well plates were plated at a density of 10 cells / ml, 100 μl of diluted cell solution was added to each well of the 96-well plate, and negative and blank control wells were set up on each plate. The 96-well plates with cells were placed in a 37°C, 5% CO2 incubator for overnight culture.

[0503] 2) Dilution and sample addition

[0504] The sample was diluted to the required concentration with culture medium (see Table 15). 50 μl was added to each well of a 96-well plate after overnight culture, and three replicates were made. Negative control and blank control were also set up. The plate was placed in a 37°C, 5% CO2 incubator for 72 hours.

[0505] Table 15: Sample (CB-18G) concentration after dilution

[0506] Tube No. Concentration after dilution μmol / L 1 100 2 25 3 6.25 4 1.56 5 0.39 6 0.10 7 0.02 8 0.006 9 0.002 10 0.0004

[0507] 3) Chromogenic plate reader

[0508] Take CCK-8 colorimetric solution, add 15 μl (10% of the liquid volume in the well) to each well, incubate at 37°C for an appropriate time (try to keep the OD value within the range of 1.0-2.0), remove the culture plate cover of the 96-well plate, place it on a microplate reader (Molecular Devices Spectra MAX Plus), and read the value at 450 nm.

[0509] 4) Data processing

[0510] SoftMax Pro was used to edit the data and draw a four-parameter fitting curve.

[0511] 5) Experimental results and analysis

[0512] According to the four-parameter fitting curve ( Figure 4F , where the C value corresponds to IC 50), CB-18G has an inhibitory effect on the growth and proliferation of A549, Hela, SCLC-21H, U-2OS, T-47D and NCI-H460 tumor cell lines. Due to the different expression levels of the corresponding receptors of the coupled compounds in the cells, the inhibition levels vary. For the cell line Hela with relatively high expression of the receptor, the IC 50 The expression of CB-18G in the cell line NCI-H460 was lower than that in the cell line NCI-H460, which has a relatively low expression of the receptor.

[0513] 9. CB-50S tumor cell proliferation inhibition experiment

[0514] Sample information: CB-50S

[0515] Cell lines: KB human oral epidermoid carcinoma cells, NCI-H460 human lung cancer cells, RT4 human bladder cancer cells, T-47D human breast cancer cells, and LNCaP human prostate cancer cells

[0516] Main reagents: IMDM culture medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, CCK8

[0517] Experimental operation:

[0518] 1) Cell plating

[0519] Prepare cells in advance, digest with Trypsin, collect and count, and dilute KB cells to 3.5×10 4 cells / ml, LNCaP cells were diluted to 8.0×10 4 T-47D cells were diluted to 1.2×10 4 cells / ml, and NCI-H460 cells were diluted to 2.5×10 4 cells / ml, RT4 cells were diluted to 1.2×10 5 cells / ml, take a 96-well plate, add 100 μl of diluted cell solution to each well, set up negative and blank control wells on each plate, and place the 96-well plate with cells in a 37°C, 5% CO2 incubator for overnight culture.

[0520] 2) Dilution and sample addition

[0521] The sample was diluted with culture medium to the required concentration (see Table 16). 50 μl was added to each well of a 96-well plate after overnight culture, and three replicates were made. Negative control and blank control were set up and cultured in a 37°C, 5% CO2 incubator for 72 hours.

[0522] Table 16: Sample (CB-50S) concentration after dilution

[0523]

[0524]

[0525] 3) Chromogenic plate reader

[0526] Take CCK-8 colorimetric solution, add 15 μl (10% of the liquid volume in the well) to each well, incubate at 37°C for an appropriate time (try to keep the OD value within the range of 1.0-2.0), remove the culture plate cover of the 96-well plate, place it on a microplate reader (Molecular Devices Spectra MAX Plus), and read the value at 450 nm.

[0527] 4) Data processing

[0528] SoftMax Pro was used to edit the data and draw a four-parameter fitting curve.

[0529] 5) Experimental results and analysis

[0530] According to the four-parameter fitting curve ( Figure 4G , where the C value corresponds to IC 50 ), CB-50S has an inhibitory effect on the growth and proliferation of KB, LNCaP, T-47D, RT4 and NCI-H460 tumor cell lines. Due to the different expression levels of the corresponding receptors of the coupled compounds in the cells, the inhibition levels are different. For the cell lines KB, LNCaP, T-47D and RT4 with relatively high expression of receptors, the IC 50 It was significantly lower than that of the cell line NCI-H460, which has relatively low receptor expression. CB-50S showed a correlation between the anti-tumor effect and the expression level of cell-related receptors.

[0531] 10. Tumor cell proliferation inhibition experiment of conjugate compound CB-1020

[0532] Experimental purpose: To test the inhibitory effect of CB-1020 and related compounds on the proliferation of tumor cell lines LNCaP (human prostate cancer cells), SK-BR-3 (human breast cancer cells), NCI-H226 (human lung cancer cells), CFPAC-1 (pancreatic cancer cells) and PANC-1 (pancreatic cancer cells).

[0533] Related samples: MMAE and CB-1020

[0534] Experimental operation: Count the LNCaP, SK-BR-3, NCI-H226, CFPAC-1 and PANC-1 cells prepared in advance, and dilute the LNCaP, SK-BR-3 and CFPAC-1 cells to 4×104 cells / ml, NCI-H226 was diluted to 2.0×10 4 cells / ml, PANC-1 was diluted to 3.0×10 4 Cells / ml, 100μl / well plate 96-well plate, each plate set negative and blank control wells, after overnight adherence, add 50μl / well diluted sample to be tested, place in 37℃, 5% CO2 incubator, culture for 68-72 hours, add CCK8 colorimetric solution, add 15μl (10% of the liquid volume in the well) to each well, incubate at 37℃ for 45-70 minutes, place in microplate reader to read the value at 450nm. Use SoftMax Pro to edit the data and draw the 4-P fitting curve. The data are shown in the following table:

[0535] Table 17. Inhibitory effect of CB-1020 on cell line proliferation and expression levels of cell line receptor genes (refer to Depmap data)

[0536]

[0537] As can be seen from the table above, LNCaP and SK-BR-3 both express TRPV6 and FOLRH1, but LNCaP has a relatively higher expression of the two receptors. NCI-H226, CFPAC-1 and PANC-1 lowly or weakly express the two receptors. CB-1020 has an inhibitory effect on the growth and proliferation of LNCaP, SK-BR-3, NCI-H226, CFPAC-1 and PANC-1 tumor cell lines. The IC value of CB-1020 in the double-high expression cell line LNCaP of the receptor is 50 The ratio was higher than the IC of SK-BR-3, a cell line with moderate receptor expression. 50 Ratio; IC of SK-BR-3 50 The ratio was higher than that of CFPAC-1, whose receptor was relatively low, and NCI-H226 and PANC-1, whose receptors were weakly expressed. 50 The cell proliferation inhibition effect was positively correlated with the expression levels of the two receptors in the cells.

[0538] 11. Tumor cell proliferation inhibition experiment of conjugate compound CB-1320

[0539] Experimental purpose: To test the inhibitory effect of CB-1320 and related compounds on the proliferation of tumor cell lines LNCaP (human prostate cancer cells), SK-BR-3 (human breast cancer cells), MDA-MB-468 (human breast cancer cells) and CFPAC-1 (pancreatic cancer cells).

[0540] Related samples: MMAE and CB-1320

[0541] Experimental operation: Count the LNCaP, SK-BR-3, MDA-MB-468 and CFPAC-1 cells prepared in advance, and dilute the LNCaP, SK-BR-3, MDA-MB-468 and CFPAC-1 cells to 4×10 4 Cells / ml, 100μl / well plate 96-well plate, set up negative and blank control wells on each plate, after overnight adherence, add diluted sample to be tested at 50μl / well, place in 37℃, 5% CO2 incubator, culture for 68-72 hours, add CCK8 colorimetric solution, add 15μl (10% of the liquid volume in the well) to each well, incubate at 37℃ for 45-70 minutes, place in microplate reader and read the value at 450nm. Use SoftMax Pro to edit data and draw 4-P fitting curve. The specific data are shown in the following table:

[0542] Table 18. Inhibitory effect of CB-1320 on cell line proliferation and expression levels of cell line receptor genes (refer to Depmap data)

[0543]

[0544] As can be seen from the table above, the GNRHR expression levels of the four cell lines are roughly the same, and the FOLH1 expression level of LNCaP is significantly higher than that of other cell lines. CB-1320 has an inhibitory effect on the proliferation of the four tumor cell lines. CB-1320 has different inhibitory effects on cells with different receptor FOLH1 expression levels. In the cell line LNCap with relatively high expression of receptor FOLH1, IC 50 The ratio is significantly higher than the IC of other cell lines with relatively low receptor expression 50 ratio.

[0545] 12. Tumor cell proliferation inhibition experiment of conjugate compound CB-1820

[0546] Experimental purpose: To test the inhibitory effect of CB-1820 and related compounds on the proliferation of tumor cell lines LNCaP (human prostate cancer cells), MDA-MB-468 (human breast cancer cells), CFPAC-1 (pancreatic cancer cells) and PANC-1 (pancreatic cancer cells).

[0547] Related samples: MMAE and CB-1820

[0548] Experimental operation: Count the LNCaP, MDA-MB-468, CFPAC-1 and PANC-1 cells prepared in advance, and dilute the LNCaP, MDA-MB-468 and CFPAC-1 cells to 4×10 4 cells / ml; PANC-1 was diluted to 3.0×10 4 Cells / ml, 100μl / well plate 96-well plate, set up negative and blank control wells on each plate, after overnight adherence, add diluted sample to be tested at 50μl / well, place in 37℃, 5% CO2 incubator, culture for 68-72 hours, add CCK8 colorimetric solution, add 15μl (10% of the liquid volume in the well) to each well, incubate at 37℃ for 45-70 minutes, place in microplate reader and read the value at 450nm. Use SoftMax Pro to edit data and draw 4-P fitting curve. The specific values ​​are shown in the following table:

[0549] Table 19. Inhibitory effect of CB-1820 on cell line proliferation and expression levels of cell line receptor genes (refer to Depmap data)

[0550]

[0551] As shown in the table above, the SSTR2 expression levels of LNCap, MDA-MB-468, CFPAC-1, and PANC-1 are roughly the same. a The FOLH1 expression level of P cells was significantly higher than that of other cells. CB-1820 inhibited the proliferation of all four tumor cell lines. The degree of inhibition of CB-1820 on cells with different receptor FOLH1 expression levels was different. In the cell line LNCaP with relatively high receptor expression, IC 50 The ratio was significantly higher than the IC of other cell lines with relatively low receptor expression 50 The ratio showed a positive correlation between the cell proliferation inhibition effect and the expression level of cell-related receptor FOLH1.

[0552] 13. Tumor cell proliferation inhibition experiment of conjugate compounds CR19425, CR19426 and CR19428

[0553] Experimental purpose: To test the inhibitory effect of CR19428, CR19425, CR19426 and related compounds on the proliferation of tumor cell lines NCI--H226 (human lung cancer cells), CFPAC-1 (human pancreatic cancer cells) and MDA-MB-468 (human breast cancer cells).

[0554] Related samples: SN-38, Dxd0017, CR19428, CR19425, and CR19426

[0555] Experimental operation: Count the NCI-H226, CFPAC-1 and MDA-MB-468 cells prepared in advance, and the cell plating density of MDA-MB-468 and CFPAC-1 is 2×10 4 cells / ml, the plating density of NCI-H226 was 1×10 4 Cells / ml, 100μl / well plate 96-well plate, set up negative and blank control wells for each culture plate, after overnight adherence, add diluted sample to be tested at 50μl / well, place in 37℃, 5% CO2 incubator, culture for 68-72 hours, add CCK8 colorimetric solution, add 15μl (10% of the liquid volume in the well) to each well, incubate at 37℃ for 45-70min, place in microplate reader and read the value at 450nm. Use SoftMax Pro to edit data and draw 4-P fitting curve. The specific data are shown in the following table:

[0556] Table 20. Inhibitory effects of CR19428, CR19425 and CR19426 on cell line proliferation

[0557]

[0558] Example 5: Pharmacodynamic study of conjugate compounds in CDX model

[0559] 1. Pharmacodynamic study of CB-20BK in CDX model 1

[0560] 1) Sample preparation:

[0561] Weigh CB-20BK freeze-dried powder, dissolve it in PBS to prepare a sample stock solution, dilute the stock solution with injection saline to the working concentration sample solution, and set aside.

[0562] 2) CDX model construction

[0563] The main cell lines are: KB human oral epidermoid carcinoma cells, MIA paca-2 human pancreatic cancer cells, HCC1954 human breast cancer cells, CALU-3 human lung adenocarcinoma cells and DU145 human prostate cancer cells.

[0564] Model construction: cells were revived and cultured, collected, counted and injected into the subcutaneous part of the right limb of BALB / c-nude mice until the tumor grew to 80-160 mm 3 The mice tumor model was expanded by grouping the drugs or taking the rapidly expanded tumor blocks.

[0565] 3) Grouped medication observation

[0566] Grouping: When the average tumor size is about 80-160 mm 3 The mice were divided into groups, and a model control group and groups receiving different doses of medication were set up.

[0567] Drug administration: Tail vein injection.

[0568] Experimental observation and measurement: After tumor inoculation, routine monitoring includes tumor growth and the effect of treatment on the normal behavior of animals. Specific content includes the activity of experimental animals, food and water intake, weight gain and loss (weight is measured twice a week), eyes, hair and other abnormalities.

[0569] The weight of mice and the long diameter (a) and short diameter (b) of the tumor mass were measured twice a week. The tumor volume (TV) was calculated as follows: TV = 1 / 2 × a × b 2 .

[0570] 4) Experimental results and analysis

[0571] According to the changes in mouse tumor volume ( Figures 5A-5E ), it can be seen that CB-20BK has a good inhibitory effect on mouse KB, MIA aca-2, HCC1954, CALU-3 and DU145 cell line CDX tumor models.

[0572] 2. Pharmacodynamic study of CB-20BK in CDX model 2

[0573] Experimental purpose: To study the efficacy of the conjugate compound CB-20BK in subcutaneous xenograft models (CDX) of human LNCaP, DU145 and NCI-H460 cell lines

[0574] Key CDX models: LNCaP, DU145, and NCI-H460

[0575] Experimental protocol:

[0576] The prepared cells or tumor tissue pieces were inoculated subcutaneously in the anterior right limb of BALB / c-nude mice. When the tumor volume expanded to 80-160 mm 3 The mice were randomly divided into groups, and a model control group and a drug-treated group were set up. Drug administration began on the first day of grouping. The dosage was adjusted according to the mouse's most recent body weight, and the tail vein injection was performed with a dosing volume of 10 μl / g. After grouping and drug administration, monitoring included the effects of tumor growth and treatment on the normal behavior of the animals, including the activity of the experimental animals, food and water intake, weight gain or loss, eyes, fur and other abnormalities. After grouping and drug administration, the weight of the mice was measured twice a week, and the weight change rate was calculated; at the same time, the long and short diameters of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor growth rate, tumor volume inhibition rate and other indicators were calculated. The formula for tumor volume is TV = 0.5a × b2 , where a is the long diameter of the tumor and b is the short diameter of the tumor.

[0577] Table 21. Growth inhibition effect of CB-20BK on CDX model and expression levels of receptor genes in each model (refer to Sino-US Crown Biotechnology data)

[0578]

[0579] As can be seen from the above table, the test sample CB-20BK showed different degrees of tumor growth inhibition under the tail vein administration scheme. For the LNCaP, DU145 and NCI-H460 human cell line CDX models, compared with the negative control group, there was a certain anti-tumor growth effect. The FOLR1 and FOLH1 dual expression model LNCaP, at a dose of 3 mg / kg, was administered on the 1st, 8th and 15th days (D1, D8 and D15), and the TGI value was 95.68%, which had an excellent anti-tumor growth effect, which was significantly better than the DU145 and NCI-H460 models with relatively low expression of FOLR1 and FOLH1. The tumor inhibition effect showed a certain correlation with the expression level of cell-related receptors.

[0580] 3. Coupled compound CB-20BK Tumor inhibition experiment of xenograft PDX model 1

[0581] Experimental purpose: Pharmacodynamic study of the conjugate compound CB-20BK in PDX model

[0582] Main models: LU1206, LU1380 and LU0367

[0583] Experimental plan: The prepared tumor tissue was inoculated subcutaneously in the anterior right limb of BALB / c-nude mice. When the tumor volume expanded to 80-160mm 3 The mice were randomly divided into groups, and a model control group and a treatment group were set up. The drug was started on the first day of grouping. The dosage was adjusted according to the mouse's most recent body weight. The tail vein was injected with a dosage volume of 10 μl / g and a dosage of 3 mg / kg. After grouping and drug administration, monitoring included the effects of tumor growth and treatment on the normal behavior of the animals. The specific contents included the activity of the experimental animals, food and water intake, weight gain or loss, eyes, fur and other abnormalities. After grouping and drug administration, the weight of the mice was measured twice a week, and the weight change rate was calculated; at the same time, the long and short diameters of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor growth rate, tumor volume inhibition rate and other indicators were calculated. The formula for tumor volume is TV = 0.5a×b 2 , where a is the long diameter of the tumor and b is the short diameter of the tumor.

[0584] Table 22. Inhibitory effect of CB-20BK on the growth of lung cancer PDX models and the expression levels of receptor genes in each model (refer to Sino-US Crown Biotechnology data)

[0585]

[0586] From the data in Table 22, it can be seen that the test sample CB-20BK (3 mg / kg) showed certain anti-tumor effects on LU1206, LU1380 and LU0367 human lung cancer PDX models. The TGI value of FOLR1 and FOLH1 dual expression model LU1206 was 90.89%, and the TGI values ​​of LU1380 and LU0367 single expression models were 30.02% and 71.34%, respectively. In the tumor inhibition experiment, the tumor inhibition effect showed a certain correlation with the expression level of cell-related receptors.

[0587] 4. Coupled compound CB-20BK Tumor inhibition experiment of xenograft PDX model 2

[0588] Experimental purpose: Pharmacodynamic study of the conjugate compound CB-20BK in PDX model

[0589] Main models: BR1283 and BR0438

[0590] Experimental plan: The prepared tumor tissue was inoculated subcutaneously in the anterior right limb of BALB / c-nude mice. When the tumor volume expanded to 80-160mm 3 The mice were randomly divided into groups, and a model control group and a treatment group were set up. The drug was started on the first day of grouping. The dosage was adjusted according to the mouse's most recent body weight. The tail vein was injected with a dosage volume of 10 μl / g and a dosage of 3 mg / kg. After grouping and drug administration, monitoring included the effects of tumor growth and treatment on the normal behavior of the animals. The specific contents included the activity of the experimental animals, food and water intake, weight gain or loss, eyes, fur and other abnormalities. After grouping and drug administration, the weight of the mice was measured twice a week, and the weight change rate was calculated; at the same time, the long and short diameters of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor growth rate, tumor volume inhibition rate and other indicators were calculated. The formula for tumor volume is TV = 0.5a×b 2 , where a is the long diameter of the tumor and b is the short diameter of the tumor.

[0591] Table 23. Inhibitory effect of CB-20BK on the growth of breast cancer PDX models and the expression levels of receptor genes in each model (refer to Sino-US Crown Biotechnology data)

[0592]

[0593] As shown in the table above, the TGI values ​​of the test sample CB-20BK at a dose of 3 mg / kg in the FOLR1 and FOLH1 dual expression models BR1283 and BR0438 were 96.49% and 70.74%, respectively, showing excellent anti-tumor growth effects. Moreover, the TGI of CB-20BK was better in BR1283, where FOLR1 and FOLH1 were expressed more highly.

[0594] 5. Pharmacodynamic study of CB-20B in CDX model

[0595] 1) Sample preparation:

[0596] Weigh CB-20B freeze-dried powder, dissolve it in PBS to prepare a sample stock solution, dilute the stock solution with injection saline to the working concentration sample solution, and set aside.

[0597] 2) CDX model construction

[0598] The main cell lines are KB human oral epidermoid carcinoma cells, PC-9 human lung cancer cells and DU145 human prostate cancer cells.

[0599] Model construction: cells were revived and cultured, collected, counted and injected into the subcutaneous part of the right limb of BALB / c-nude mice until the tumor grew to 80-160 mm 3 The mice tumor model was expanded by grouping the drugs or taking the rapidly expanded tumor blocks.

[0600] 3) Grouped medication observation

[0601] Grouping: When the average tumor size is about 80-160 mm 3 The mice were divided into groups, and a model control group and groups receiving different doses of medication were set up.

[0602] Drug administration: Tail vein injection.

[0603] Experimental observation and measurement: After tumor inoculation, routine monitoring includes tumor growth and the effect of treatment on the normal behavior of animals. Specific content includes the activity of experimental animals, food and water intake, weight gain and loss (weight is measured twice a week), eyes, hair and other abnormalities.

[0604] The weight of mice and the long diameter (a) and short diameter (b) of the tumor mass were measured twice a week. The tumor volume (TV) was calculated as follows: TV = 1 / 2 × a × b 2 .

[0605] 4) Experimental results and analysis

[0606] According to the changes in mouse tumor volume ( Figures 6A-6C), it can be seen that CB-20B has a good inhibitory effect on mouse KB, PC-9 and DU145 cell line CDX tumor models.

[0607] 6. Pharmacodynamic study of CB-18G in CDX model

[0608] 1) Sample preparation:

[0609] Weigh CB-18G freeze-dried powder, dissolve it in PBS to prepare a sample stock solution, dilute the stock solution with injection saline to the working concentration sample solution, and set aside.

[0610] 2) CDX model construction

[0611] Main cell lines: KB human oral epidermoid carcinoma cells, PC-9 human lung cancer cells, SPC-A1 human lung adenocarcinoma cells, CALU-3 human lung adenocarcinoma cells and DU145 human prostate cancer cells

[0612] Model construction: Cells were revived and cultured, collected and counted, and the cell fluid was injected into the subcutaneous part of the right limb of BALB / c-nude mice until the tumor grew to 80-160 mm 3 The mice tumor model was expanded by grouping the drugs or taking the rapidly expanded tumor blocks.

[0613] 3) Grouped medication observation

[0614] Grouping: When the average tumor size is about 80-160 mm 3 The mice were divided into groups, and a model control group and groups receiving different doses of medication were set up.

[0615] Drug administration: Tail vein injection.

[0616] Experimental observation and measurement: After tumor inoculation, routine monitoring includes tumor growth and the effect of treatment on the normal behavior of animals. Specific content includes the activity of experimental animals, food and water intake, weight gain and loss (weight is measured twice a week), eyes, hair and other abnormalities.

[0617] The weight of mice and the long diameter (a) and short diameter (b) of the tumor mass were measured twice a week. The tumor volume (TV) was calculated as follows: TV = 1 / 2 × a × b 2 .

[0618] 4) Experimental results and analysis

[0619] According to the changes in mouse tumor volume ( Figures 7A-7E ), it can be seen that CB-18G has a good inhibitory effect on mouse KB, PC-9, SPC-A1, CALU-3 and DU145 cell line CDX tumor models.

[0620] 7. Tumor inhibition experiments of conjugate compounds CB-1020, CB-1320 and CB-1820 on subcutaneous xenograft models (CDX) of human HPAF-II, NCI-H226 and SCLC-21H cell lines

[0621] Experimental purpose: Pharmacodynamic study of ligand conjugates CB-1020, CB-1320 and CB-1820 in CDX model

[0622] Main CDX models: HPAF-II (human pancreatic cancer cells), NCI-H226 (human lung cancer cells) and SCLC-21H (small cell lung cancer cells)

[0623] Experimental protocol:

[0624] The prepared cells or tumor tissue pieces were inoculated subcutaneously in the anterior right limb of BALB / c-nude mice. When the tumor volume expanded to 80-160 mm 3 The mice were randomly divided into groups, and a model control group and a drug-treated group were set up. Drug administration began on the first day of grouping. The dosage was adjusted according to the mouse's most recent body weight, and the tail vein injection was performed with a dosing volume of 10 μl / g. After grouping and drug administration, monitoring included the effects of tumor growth and treatment on the normal behavior of the animals, including the activity of the experimental animals, food and water intake, weight gain or loss, eyes, fur and other abnormalities. After grouping and drug administration, the weight of the mice was measured twice a week, and the weight change rate was calculated; at the same time, the long and short diameters of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor growth rate, tumor volume inhibition rate and other indicators were calculated. The formula for tumor volume is TV = 0.5a × b 2 , where a is the long diameter of the tumor and b is the short diameter of the tumor.

[0625] Table 24. Inhibitory effects of CB-1020, CB-1320 and CB-1820 on CDX model and expression levels of receptor genes in each model (refer to Crown Bioscience data)

[0626]

[0627] CB-1020 has a significant anti-tumor growth effect on human HPAF-II, CB-1320 has a significant anti-tumor growth effect on NCI-H226 and SCLC-21H, and CB-1820 has a significant anti-tumor growth effect on the subcutaneous xenograft model of SCLC-21H cell line.

[0628] 8. Tumor inhibition experiment of the conjugate compound CR19428 on subcutaneous xenograft models (CDX) of human CALU-3, SCLC-21H and SPC-A1 cell lines

[0629] Experimental plan: The prepared cells were inoculated subcutaneously in the anterior right limb of BALB / c-nude mice. When the tumor volume expanded to 80-160mm 3 The mice were randomly divided into groups, and a model control group and a treatment group were set up. The drug was started on the first day of grouping. The dosage was adjusted according to the mouse's most recent body weight. The tail vein was injected with a dosage volume of 10 μl / g, and the drug was administered twice a week for 3 consecutive weeks. After grouping and drug administration, monitoring included the effects of tumor growth and treatment on the normal behavior of the animals. The specific contents included the activity of the experimental animals, food and water intake, weight gain or loss, eyes, fur and other abnormalities. After grouping and drug administration, the weight of the mice was measured twice a week, and the weight change rate was calculated; at the same time, the long and short diameters of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor growth rate, tumor volume inhibition rate and other indicators were calculated. The formula for tumor volume is TV = 0.5a×b 2 , where a is the long diameter of the tumor and b is the short diameter of the tumor.

[0630] Table 25. Inhibitory effect of CR19428 on lung cancer PDX models

[0631]

[0632] CR-19428 is a drug conjugate of the ligand of FOLR1 and FOLH1 and the payload Dxd. As shown in the table above, it has a significant anti-tumor growth effect on subcutaneous xenograft models of human CALU-3, SCLC-21H and SPC-A1 cell lines.

[0633] 9. Study on the effectiveness of the conjugate compound CBP-1018 in the lung cancer LU2505 model (PDX model)

[0634] This experiment is the third generation of lung cancer PDX model LU2505 (from an Asian female patient), which is a fast-growing tumor model. BALB / c nude mice were subcutaneously loaded with tumors, with a tumor volume of about 150mm 3 The animals were divided into groups: low, medium and high test sample groups, small molecule MMAE and targeted peptide 20BK-SM09 control groups, and blank preparation control groups, a total of 6 groups, 8 animals per group; the animals were given drugs on the 1st, 8th and 15th days after grouping, and continued to be observed for 14 days after the last drug. The active substance of the test sample CBP-1018 is CB-20BK, which is obtained by mixing CB-20BK with excipients and then freeze-drying.

[0635] Table 26. Pharmacodynamic results of CBP-1018 for injection in the LU2505 lung cancer model (first trial)

[0636]

[0637] Note: 1) Due to animal welfare requirements, the average tumor volume in the group reached 2000mm 3 Animals needed to be euthanized, so the killing time of each group was different. 2) The data of body weight, tumor volume and tumor inhibition rate were the data of day 22 (D22).

[0638] As shown in Table 26 and Fig. 8A As shown:

[0639] No abnormal clinical manifestations were observed in the animals of all groups after administration, and no death was observed; the body weight of the animals in each group increased slowly.

[0640] The animals in the blank control group, 20BK-SM09 group, and CBP-1018 low-dose group had an average tumor volume of more than 2000 mm 3 Euthanasia was performed on day 22, day 22 and day 26 (D26), respectively. Therefore, the data on body weight, tumor volume and tumor inhibition rate in Table 26 are all data on D22.

[0641] The effectiveness of CBP-1018 for injection has a clear dose-dependency: the low-dose group is ineffective, while the medium and high-dose groups are effective. The tumors in the high-dose group were completely cured on day 19 (D19), and no signs of growth were observed on day 29 (D29).

[0642] The peptide group 20BK-SM09 showed no obvious anti-tumor effect, suggesting that the targeted peptide alone is not sufficient to produce a clear anti-tumor effect.

[0643] The MMAE group had a clear tumor-suppressing effect. The dose of 0.375 mg / kg was equivalent to the MMAE contained in CBP-1018 at 1.5 mg / kg. By comparison, it was found that CBP-1018 at 1.5 mg / kg was significantly superior to the MMAE group, indicating the advantage of ligand targeting (82.60% vs 48.97%).

[0644] 10. Study on the effectiveness of the conjugate compound CBP-1018 in the lung cancer LU1206 model (PDX model)

[0645] This experiment is the fifth generation of lung cancer PDX model LU1206 (from an Asian female patient), which is a fast-growing tumor model. BALB / c nude mice were subcutaneously loaded with tumors, with a tumor volume of about 150mm 3 The mice were divided into groups: low, medium and high test sample groups, small molecule MMAE and targeted peptide 20BK-SM09 control groups, and blank preparation control group, for a total of 6 groups, 8 mice in each group; the drugs were administered on the 1st, 8th and 15th days after grouping, and the mice were observed for 14 days after the last dose.

[0646] Table 27. Pharmacodynamic results of CBP-1018 for injection in the LU1206 lung cancer model (first trial)

[0647]

[0648] As shown in Table 27 and Figure 8B As shown:

[0649] No abnormal clinical manifestations or deaths were observed in animals in all groups after administration, and all animals were euthanized on day 29. The body weights of animals in each group remained basically unchanged, slightly higher than on the first day of administration.

[0650] The effectiveness of CBP-1018 for injection has obvious dose correlation: the low-dose group is ineffective (tumor inhibition rate is 8.08%), while the medium and high-dose groups are effective, with tumor inhibition rates of 75.21% and 97.42%, respectively. The difference between the low and medium doses is relatively large.

[0651] The peptide group 20BK-SM09 showed no obvious anti-tumor effect, suggesting that a single targeted peptide is not sufficient to produce a clear anti-tumor effect in this model.

[0652] The MMAE group has a clear tumor inhibition effect. The dose of 0.375 mg / kg is the same molar amount of MMAE as that of CBP-1018 at 1.5 mg / kg. By comparison, CBP-1018 at 1.5 mg / kg was significantly better than the MMAE group, indicating the advantages of ligand targeting (tumor volume inhibition rate: 75.21% vs 36.03%, tumor weight inhibition rate: 78.38% vs 54.53%).

[0653] 11. Tissue distribution in tumor-bearing mice (PDX model LU2505)

[0654] 12 vaccinations Female mice bearing LU2505 lung cancer model tumors and 12 healthy male BALB / c nude mice were given a single tail vein injection of 1.5 mg / 140 μCi / kg of [ 3 H]CBP-1018 (isotope labeled on MMAE). Three males and three females were selected at 0.5 hours, 2 hours, 6 hours and 24 hours, respectively, and placed in an induction box to inhale an appropriate amount of carbon dioxide anesthesia and then blood was collected by cardiac puncture, and then euthanasia was immediately performed for sampling.

[0655] Table 28. Single intravenous administration [ 3 Total radioactivity in each tissue at different time points after H]CBP-1018

[0656]

[0657] As shown in the table above, there is no difference in tissue distribution between males and females. After administration, the drug is mainly distributed in the kidney, whole blood (mainly in plasma), liver and lungs. The highest concentration in each tissue is 0.5 hours, and then the drug is rapidly eliminated from each tissue, with the liver and tumor being the slowest. The slow elimination of the drug in the tumor can explain the advantage of CBP-1018 in effectiveness.

[0658] 12. Rat excretion test

[0659] Six normal SD rats, half male and half female, were given a single tail vein injection of 0.75 mg / 70 μCi / kg of [ 3 H]CBP-1018. Urine, feces, cage washing / cleaning fluid and carcass samples were collected from whole rats before and 0 to 168 hours after administration, and bile, urine, feces and cage washing / cleaning fluid samples were collected from BDC rats before and 0 to 72 hours after administration at specified time intervals. All the above samples were frozen in a low-temperature refrigerator (-10℃ to -30℃).

[0660] After adding an appropriate amount of scintillation fluid to each sample and mixing, the radioactivity was measured using a liquid scintillation counter. The radioactivity measured in samples such as bile, urine, feces, cage washing and cleaning fluid, and corpses was used to calculate the percentage of the dose. The radioactivity in plasma samples was used to calculate the total radioactivity per gram of sample, and WinNonLin software (version 7.0, Pharsight) was used to calculate the main pharmacokinetic parameters of total plasma radioactivity according to the non-compartmental model.

[0661] Table 29. Results of the whole rat material balance study after administration

[0662]

[0663] As can be seen from the table above, CBP-1018 is mainly excreted through urine, accounting for about 57% of the total dose, and less than 25% is excreted through feces. The result that it is mainly excreted through the kidneys from urine is consistent with the tissue distribution of nude mice in which a large amount of it is distributed in the kidneys.

[0664] 13. Plasma stability

[0665] CBP-1008 (whose active substance is LDC10B described in WO2017025047A1, CBP-1008 is obtained by mixing LDC10B with excipients and then freeze-drying. WO2017025047A1 is incorporated by reference in its entirety) and CBP-1018 at concentrations of 1 μg / mL, 10 μg / mL, and 100 μg / mL were incubated with plasma from different species at 37°C for 2 hours to observe the stability of the two compounds. The results show (Table 30) that the stability of CBP-1018 in plasma of various species, relative to the concentration at 0 hours, the remaining percentage after 2 hours of incubation is all above 90%. CBP-1008 is only stable in rat plasma (87.92-91.82%), and is unstable in plasma of other species, with only 0.751%-24.52% remaining.

[0666] The experimental results suggest that the plasma stability of CBP-1018 is better than that of CBP-1008.

[0667] Table 30. Summary of plasma stability data of CBP-1008 and CBP-1018 at different concentrations in various species (% relative to 0 hours)

[0668]

[0669] Note:

[0670] 1) The data in the table are the percentages of the plasma compound concentrations after 2 hours of plasma incubation to the concentrations at 0 hours;

[0671] 2) As the data belong to two tests, the number of significant digits after the decimal point is inconsistent. For the traceability and authenticity of the data, the data in the report are directly quoted without unification.

[0672] 14. Half-life in PK

[0673] Consistent with the plasma stability, the pharmacokinetic half-life of CBP-1018 in rats and crab-eating monkeys (approximately 1 hour) is significantly longer than that of CBP-1008 (approximately 20 minutes), suggesting that the stability of CBP-1018 is better than that of CBP-1008.

[0674] Table 31. Comparison of half-life (hours) of CBP-1008 and CBP-1018 in rat PK

[0675]

Claims

1. A conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and a prostate-specific membrane antigen ligand portion, respectively, wherein the synergistic molecule binds to a molecule selected from the group consisting of FOLR1, TRPV6, SSTR2 and GNRHR, wherein the two targeting molecules are a folate ligand and a prostate-specific membrane antigen ligand portion, a TRPV6 ligand and a prostate-specific membrane antigen ligand portion, a SSTR2 ligand and a prostate-specific membrane antigen ligand portion, or a GNRHR ligand and a prostate-specific membrane antigen ligand portion, wherein: The prostate-specific membrane antigen ligand comprises the following structure: The folate ligand is folic acid or a folic acid analog, wherein the folic acid analog is selected from the group consisting of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate and 5,10-methylenetetrahydrofolate, The TRPV6 ligand is P10, which is a peptide having an amino acid sequence of Cys-Lys-Glu-Phe-Leu-His-Pro-Ser-Lys-Val-Asp-Leu-Pro-Arg, The SSTR2 ligand has a structure shown in formula (I): The GNRHR ligand has the following structure:

2. A conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and a ligand portion having formula (I): The synergistic molecule binds to FOLR1, and the synergistic molecule is folic acid or a folic acid analog, wherein the folic acid analog is selected from the following group: 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate and 5,10-methylenetetrahydrofolate.

3. A conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule portion and P10, wherein the synergistic molecule binds to FOLR1, and the payload is camptothecin and any derivative thereof, wherein The synergistic molecule is folic acid or a folic acid analog, wherein the folic acid analog is selected from the group consisting of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate and 5,10-methylenetetrahydrofolate, The P10 is a peptide having the amino acid sequence Cys-Lys-Glu-Phe-Leu-His-Pro-Ser-Lys-Val-Asp-Leu-Pro-Arg.

4. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the conjugate compound or a pharmaceutically acceptable salt thereof comprises one, two, three, four or more effective loads.

5. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the effective load is selected from the group consisting of small molecule compounds, nucleotides, peptides and proteins. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 5, wherein the effective load is a small molecule compound.

7. The conjugate compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the small molecule compound is selected from the group consisting of camptothecin and any derivatives thereof, auristatin and any derivatives thereof, maytansine and any derivatives thereof, radionuclide complexes, cyclooxygenase-2 inhibitors, paclitaxel and any derivatives thereof, epothilone and any derivatives thereof, bleomycin and any derivatives thereof, dactinomycin and any derivatives thereof, plicamycin and any derivatives thereof, and mitomycin C.

8. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 7, wherein the small molecule compound is camptothecin and any derivatives thereof, auristatin and any derivatives thereof, a radionuclide complex or a cyclooxygenase-2 inhibitor.

9. The conjugate compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein the payload is connected to at least one of the targeting molecules via a linker. 10 . The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 9 , wherein the linker is a peptide linker, a disulfide linker, a pH-dependent linker or a combination of the above linkers. 11 . The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 10 , wherein the peptide linker can be cleaved by protease or reduction under a specific physiological environment.

12. The conjugate compound according to claim 10 or a pharmaceutically acceptable salt thereof, wherein the peptide linker is selected from the group consisting of cysteine, lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-lysine, cysteine-glutamate, aspartic acid-aspartic acid and aspartic acid-aspartic acid-lysine, and optionally, the carboxylic acids in the above amino acids are amidated.

13. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 10, wherein the disulfide linker is selected from the group consisting of DMDS, MDS, DSDM and NDMDS. 14 . The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 10 , wherein the pH-dependent linker is cis-aconitic anhydride.

15. The conjugate compound according to claim 9 or a pharmaceutically acceptable salt thereof, wherein the linker comprises the following structure: Alternatively, the linker is a combination of the above structure and a peptide linker.

16. The conjugate compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the two targeting molecules are connected via a spacer.

17. The conjugate compound according to claim 16 or a pharmaceutically acceptable salt thereof, wherein the spacer comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14, Arg-Arg, Ala-Ser-Asn, Ala-Ala-Ala, Ser-Ser-Arg, Pro-Arg and Pro-Leu-Gly.

18. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the conjugate compound is selected from the group consisting of the following compounds: Where M is the radionuclide.

19. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 2, wherein the conjugate compound is:

20. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 3, wherein the conjugate compound is 21. A pharmaceutical composition comprising the conjugate compound according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

22. The pharmaceutical composition according to claim 21, wherein The compositions are for intravenous, subcutaneous, oral, intramuscular or intraventricular administration.

23. Use of the conjugate compound according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 21 or 22 in the preparation of a medicament for delivering a payload to a subject in need thereof.

24. Use of the conjugate compound according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 21 or 22 in the preparation of a medicament for treating a disease in a subject.

25. The use according to claim 24, wherein the disease is selected from the group consisting of cancer, immune diseases, cardiovascular diseases, metabolic diseases and neurological diseases.

26. The use according to claim 25, wherein the cancer is selected from the group consisting of prostate cancer, breast cancer, lung cancer, kidney cancer, leukemia, ovarian cancer, stomach cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, testicular cancer, skin cancer, lymphoma and multiple myeloma.

27. The use according to claim 25, wherein the immune disease is an autoimmune disease.

28. The use according to claim 27, wherein the autoimmune disease is selected from the group consisting of connective tissue disease, systemic sclerosis, rheumatoid arthritis and systemic lupus erythematosus.

29. The use according to claim 25, wherein the cardiovascular disease is selected from the group consisting of angina pectoris, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmia and congenital heart disease.

30. The use according to claim 25, wherein the metabolic disease is selected from the group consisting of diabetes, gout, obesity, hypoglycemia, hyperglycemia and dyslipidemia.

31. The use according to claim 25, wherein the neurological disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, head injury, multiple sclerosis, vertigo, coma and epilepsy.

32. The use according to any one of claims 24 to 31, further comprising administering one or more therapeutic agents in combination with the conjugate compound or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition.

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