RU2820346C2 - Drug biligand conjugate and use thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biomedical chemistry. Disclosed are conjugated compounds or pharmaceutically acceptable salts thereof, in which the payload is conjugated with at least one of the targeting molecules. Targeting molecules are a moiety which is a synergistic molecule which binds to a molecule selected from a group consisting of FOLR1, TRPV6, SSTR2 and GNRHR, and a fragment representing a ligand to a prostate-specific membrane antigen. In another embodiment, the conjugate contains a fragment which is a ligand of formula (I), or P10, and the synergistic molecule binds to the FOLR1 molecule. Also disclosed is a pharmaceutical composition, a method for delivering a payload to a subject, a method of treating a disease, wherein the methods involve administering to a subject a therapeutically effective amount of a conjugated compound or a pharmaceutically acceptable salt or pharmaceutical composition thereof.

EFFECT: inventions enable to enhance the affinity and specificity of targeting drug compounds, thereby delivering the highly effective chemotherapeutic agents to the patient, extending the therapeutic window of such agents, and avoiding side effects.

37 cl, 8 dwg, 31 tbl, 5 ex

Description

Field of technology to which the invention relates

This application relates to the field of biomedical chemistry. More specifically, the present application relates to a bigand drug conjugate, a pharmaceutical composition containing a bigand drug conjugate, a method of delivering a payload to a subject in need thereof by using a bigand drug conjugate, and a method of treating a disease using a bigand drug conjugate.

BACKGROUND OF THE INVENTION

In general, diseased cells and normal cells differ significantly in both pathological and physiological characteristics, and one of the differences is that diseased cells contain specific or overexpressed components (such as antigens, chemical signals and receptors ) on its surface, whereas these components are absent or poorly expressed in normal cells. Based on this, antibody-drug conjugate (ADC) and polypeptide-drug conjugate (PDC) are being developed for the treatment of diseases. Currently, several ADC and PDC-based drugs are on the market or undergoing clinical trials; however, due to the principles underlying their design, ADC and PDC-based drugs have severe limitations in clinical use.

Due to the structural complexity and relatively large molecular weight of ADCs, their development faces many challenges, including a lack of suitable targets, manufacturing difficulties, and low drug stability. Currently, ADCs are mainly used in the field of tumor treatment. In some cases, the affinity of the targeting antibody for the surface antigen of the cancer cell can reach 10 ^-9 -10 ^-12 (Kd, mol/liter). Therefore, while having high specificity for target cells, ADCs also have high specificity for normal cells that have the same target receptor as the target cells. Moreover, ADCs can take a long time to metabolize in vivo (1 to 3 weeks), during which ADCs continually destroy normal cells, resulting in a significant increase in toxicity and side effects. Therefore, ADCs are more applicable to diseases characterized by very large differences in the abundance of cell surface antigens between tumor and normal cells. However, very few diseases known in the art can meet such a strict requirement.

PDCs have been used to treat a number of different diseases in clinical or preclinical studies, however, in this case, the chemotherapeutic drugs are simply bound to the polypeptides, or the polypeptides are added to nanoparticles or polymeric materials in which the chemotherapeutic drugs are included, which makes most of the polypeptides unable to penetrate into the body. cells due to their large molecular weight and the charges they have. Thus, most PDCs are currently only suitable for extracellular treatment, and the applications and effectiveness of PDCs are severely limited.

The drug conjugate compound may also be a ligand-drug conjugate (LDC), where the ligand may be a peptide or small molecule compound. However, in terms of bioavailability, stability, efficacy, toxicity, etc., various problems arise with LDCs. For example, due to high molecular weight, lipophilicity, or other properties, many ligands are unable to penetrate cells, which limits their therapeutic applications. In addition, ligands generally have low potency when conjugated to traditional chemotherapeutic agents (such as doxorubicin and paclitaxel), and when conjugated to highly potent drug molecules (such as MMAE and DM1), the ligands can cause high toxicity, thereby poisoning and even killing animals until a therapeutically effective amount is achieved to provide treatment of the tumor.

Thus, there is also a pressing need in the field to provide an improved LDC that can target highly expressed receptors widely present on the surface of disease-affected cells, expand the targeting range and treatment window, improve drug efficacy, and avoid drug side effects.

Brief description of the invention

In one aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof comprising a payload and two targeting molecules, wherein the two targeting molecules are respectively a synergistic molecule moiety and a prostate-specific membrane antigen ligand moiety.

In another aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof comprising a payload and two targeting molecules, wherein the two targeting molecules are respectively a synergistic molecule moiety and a ligand moiety represented by formula (I):

In another aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof comprising a payload and two targeting molecules, wherein the two targeting molecules are respectively a synergistic molecule moiety and P10, and the payload is camptothecin or any derivative thereof.

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

.

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

.

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

.

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

.

In some embodiments, the two targeting molecules contained in the conjugated compound or pharmaceutically acceptable salt thereof are different.

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

In some embodiments, the two targeting molecules contained in the conjugate compound or a pharmaceutically acceptable salt thereof are cell interacting molecules that interact with different molecules of the cell.

In some embodiments, the synergistic molecule contained in the conjugate compound or a pharmaceutically acceptable salt thereof is an endocytosis promoting molecule that is capable of mediating endocytosis.

In some embodiments, the synergistic molecule contained in the conjugate compound or a pharmaceutically acceptable salt thereof 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, claudin 18.2, APN, DLL3, CEACAM5, FZD10, TFRC, MET, IGFR1, SSTR2, CCKBR, LFA1, ICAM, GPR87, GM-CSF, GM-CSFR, TIM3, TLR family members, CD40 , CD40L, OX40, OX40L, GITRL, GITR, 4-BBL, 4-1BB, CD70, CD27, ICOSL, ICOS, HHLA2, CD28, CD86/80, CD28, MHCII antigen, 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.

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

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

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

In some embodiments, the synergistic molecule contained in the conjugate compound or a pharmaceutically acceptable salt thereof is folate or an analogue thereof. In some embodiments, the folate analog is selected from the group consisting of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate, and 5,10-methylenetetrahydrofolate.

In some embodiments, the conjugate compound or a pharmaceutically acceptable salt thereof contains one, two, three, four or more payloads. In some embodiments, the payload is selected from the group consisting of a small molecule compound, a nucleotide, a peptide, and a protein. 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 complex, cyclooxygenase-2 inhibitor, 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 or any derivative thereof, auristatin or any derivative thereof, maytansine or any derivative thereof, a radionuclide complex, or cyclooxygenase-2 inhibitor.

In some embodiments, the payload contained in a conjugate compound or a pharmaceutically acceptable salt thereof according to the present application is linked to at least one targeting molecule via a linker.

In some embodiments, the linker contained in the conjugate compound or a pharmaceutically acceptable salt thereof according to this application is a peptide linker, a disulfide linker, a pH-dependent linker, or a combination of the foregoing linkers.

In some embodiments, the peptide linker is cleavable when exposed to a particular physiological environment through protease cleavage or reduction. 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.

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

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

In some embodiments, the linker in a conjugate compound or a pharmaceutically acceptable salt thereof according to this application has the following structure:

,

,

,

,

,

,

,

,

,

,

,

,

or the linker is a combination of the above structures and a peptide linker.

In some embodiments, two targeting molecules contained in a conjugate compound or a pharmaceutically acceptable salt thereof according to the present application are linked to each other via a spacer. In some embodiments, a spacer described herein 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.

In some embodiments, the conjugate compound of this application is CB-20B, which has the following structural formula:

In some embodiments, the conjugate compound of this application is CB-20BK, which has the following structural formula:

In some embodiments, the conjugate compound of this application is CB-60S, which has the following structural formula:

In some embodiments, the conjugate compound of this application is CB-60SK, which has the following structural formula:

In some embodiments, the conjugate compound of this application is CB-20C, which has the following structural formula:

In some embodiments, the conjugate compound of this application is CB-1020, which has the following structural formula:

In some embodiments, the conjugate compound of this application is CB-1320, which has the following structural formula:

In some embodiments, the conjugate compound of this application is CB-1820, which has the following structural formula:

In some embodiments, the conjugate compound of this application is CR19428, which has the following structural formula:

In some embodiments, the conjugate compound of this application is 20R-SM09, which has the following structural formula:

In some embodiments, the conjugate compound of this application is CB-20R, which has the following structural formula:

,

where M represents a radionuclide.

In some embodiments, the conjugate compound of this application is CB-18G, which has the following structural formula:

In some embodiments, the conjugate compound of this application is CR19426, which has the following structural formula:

In some embodiments, the conjugate compound of this application is CB-10S, which has the following structural formula:

In some embodiments, the conjugate compound of this application is CR19425, which has the following structural formula:

In some embodiments, the conjugate compound of this application is CB-50S, which has the following structural formula:

In another aspect, the present application discloses a pharmaceutical composition comprising a conjugate compound or a pharmaceutically acceptable salt thereof according to the present application and a pharmaceutically acceptable carrier.

In some embodiments, the composition is administered intravenously, subcutaneously, orally, intramuscularly, or intraventricularly.

In another aspect, this application discloses a method of delivering a payload to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate compound or a pharmaceutically acceptable salt thereof according to this application or a pharmaceutical composition according to this application.

In another aspect, this application discloses a method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of a conjugate compound or a pharmaceutically acceptable salt thereof according to this application or a pharmaceutical composition according to this application.

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

In yet another aspect, the present application discloses the use of a conjugate compound or a pharmaceutically acceptable salt thereof of the present application or a pharmaceutical composition of the present application in the preparation of a medicament for treating a disease in a subject.

In yet another aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof according to the present application or a pharmaceutical composition according to the present application for treating a disease in a subject.

In some embodiments, the disease is selected from the group consisting of cancer, an immune system disease, a cardiovascular disease, a metabolic disease, and a neurological disease.

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

In some embodiments, the immune system 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.

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, arrhythmia, and congenital heart disease.

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

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 graphic materials

In fig. Figure 1 shows the chemical structural formulas of the conjugated 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.

In fig. Figure 2A shows the binding and endocytosis curve of Cy5-pep-20BK by different cells (from top to bottom: LNCaP cells, SKOV3 cells, DU145 cells and NCI-H460 cells) over time. In fig. Figure 2B shows the binding and endocytosis curve of Cy5-pep-20AK by different cells (from top to bottom: LNCaP cells, SKOV3 cells, DU145 cells and NCI-H460 cells) over time.

In fig. Figure 3 shows fluorescence images of the binding and endocytosis of Cy5-FA by various cells over time, where the fluorescence shown as a solid circle represents the fluorescence of cell nuclei, and the fluorescence distributed as spots is the fluorescence of Cy5-FA.

In fig. 4A shows the inhibitory activity of the conjugated compound CB-20BK to increase the number of these tumor cells. In fig. 4B shows the inhibitory activity of the conjugated compound CB-20B to increase the number of these tumor cells. In fig. 4C shows the inhibitory activity of the conjugated compound CB-10S to increase the number of these tumor cells. In fig. 4D shows the inhibitory activity of the conjugated compound CB-60S to increase the number of these tumor cells. In fig. 4E shows the inhibitory activity of the conjugated compound CB-60SK to increase the number of these tumor cells. In fig. 4F shows the inhibitory activity of the conjugated compound CB-18G to increase the number of these tumor cells. In fig. 4G shows the inhibitory activity of the conjugated compound CB-50S to increase the number of these tumor cells.

In fig. 5A-5E show the tumor suppressive effects of the conjugated compound CB-20BK in mice.

In fig. 6A-6C show the tumor suppressive effects of the conjugated compound CB-20B in mice.

In fig. 7A-7E show the tumor suppressive effects of the conjugated compound CB-18G in mice.

In fig. 8A-8B show the effects of CBP-1018 injection on tumor volume values for the LU2505 cell lung cancer model and the LU1206 cell lung cancer model.

Detailed Description of Embodiments

Although various aspects and embodiments will be disclosed herein, it will be appreciated that one skilled in the art can make various equivalent changes and modifications to the aspects and embodiments without departing from the spirit and scope of the present application. The various aspects and embodiments disclosed herein are for purposes of illustration only and are not intended to be limiting, the true scope being set forth in the appended claims. All publications, patents and patent applications cited in this application are incorporated by reference in their entirety. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this application belongs.

As used herein and in the accompanying claims, the singular number is intended to refer to the plural unless the context clearly requires otherwise. The singular forms, "one or more" and "at least one" may be used interchangeably herein. It should also be noted that the terms "comprise", "include" and "have" can be used interchangeably.

As used herein and in the accompanying claims, the term “analog” includes structural analog and functional analog. A structural analogue refers to a class of compounds with similar chemical structures that may contain one or more different atoms or one or more different functional groups. A functional analog refers to a class of compounds that have the same or similar chemical, biological or pharmacological effects. For example, folate analogs include 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate, and 5,10-methylenetetrahydrofolate.

As used herein and in the accompanying claims, the term “derivative” refers to a relatively complex compound derived from a parent compound molecule in which one or more atoms or groups of atoms have been 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, hymatecan, carenitecin , Silatecan, Lurtotecan, Exatecan, Diphlomothecan, Belotecan, Lurtotecan and S39625.

In one aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof comprising a payload and two targeting molecules, wherein the two targeting molecules are respectively a synergistic molecule moiety and a prostate-specific membrane antigen ligand moiety.

In another aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof comprising a payload and two targeting molecules, wherein the two targeting molecules are respectively a synergistic molecule moiety and a ligand moiety represented by formula (I):

In another aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof comprising a payload and two targeting molecules, wherein the two targeting molecules are respectively a synergistic molecule moiety and P10, and the payload is camptothecin or any derivative thereof.

As used herein, the term "payload" refers to a molecule or material to be delivered to a target cell or tissue. Without limitation, a payload can be any molecule or material intended for use in diagnosing, treating, or preventing a disease in a subject. In some embodiments, the payload has a molecular weight of less than or equal to about 5 kDa. In some embodiments, the payload has a molecular weight of less than or equal to about 1.5 kDa. In some embodiments, the payload is a drug or diagnostic reagent that is recognized as safe and effective for use by relevant drug approval organizations (such as the FDA, EMEA, or NMPA).

In some embodiments, the payload of the present application is a small molecule compound, a nucleotide (such as DNA, plasmid DNA, RNA, siRNA, antisense oligonucleotide, and aptamer), peptide, or protein (such as an enzyme). In some embodiments, the payload is a small molecule compound.

In some embodiments, payloads herein include, but are not limited to, an anticancer drug, a radioactive substance, a vitamin, an anti-AIDS drug, an antibiotic, an immunosuppressant, an antiviral drug, an enzyme inhibitor, a neurotoxin, an opioid, a cell-extracellular matrix interaction regulator, a vasodilator, antihypertensive drug, hypnotic drug, antihistamine, anticonvulsant drug, muscle relaxant, anti-Parkinson's disease drug, anticonvulsant and muscle contraction drug, antiparasitic drug and/or antiprotozoal drug, analgesic drug, antipyretic drug, steroidal or non-steroidal anti-inflammatory drug drug, antiangiogenic factor, secretory inhibitory factor, anticoagulant and/or antithrombotic agent, local anesthetic, prostaglandin, antidepressant, antipsychotic, antiemetic or imaging agent.

In some embodiments, the payload of the present application has a free amino or carboxyl group prior to conjugation to the conjugate compound of the present application, and the payload is conjugated to the conjugate compound through an acylation reaction between the above amino or carboxyl group and a corresponding moiety (e.g., a linker ) conjugated compound. In some embodiments, modification of the above-mentioned free amino or carboxyl group (e.g., by conjugation to a conjugate compound of the present application) can significantly reduce the activity of the payload (e.g., by at least 50%, 60%, 70%, 80%, 90 %, 95%, 98% or 99%).

As used herein, the term “low molecular weight compound” refers to a compound having a molecular weight of less than or equal to approximately 2 kDa. In some embodiments, the small molecule compound has a molecular weight of less than or equal to about 1.5 kDa. In some preferred embodiments, the low molecular weight compound has a molecular weight of less than or equal to about 1 kDa, 800 Da, 700 Da, 600 Da, or 500 Da. In some embodiments, the small molecule compound of this 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, a cyclooxygenase inhibitor, 2 (e.g., celecoxib), radionuclide complex, 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 is camptothecin or any derivative thereof, auristatin or 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 medicament for alleviating or treating an autoimmune disease.

As used herein, the term “camptothecin” refers to a cytotoxic alkaloid primarily derived from Camptotheca acuminata (Nyssaceae) and exhibiting potent antitumor activity. Camptothecin and its derivative according to the present application include camptothecin and its derivative, which already exist or will be obtained in the future. Camptothecin and its derivative herein 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, Hymatecan, Karenitecin, Silatecan, Lurtotecan, Exatecan, Diphlomothecan, Belotecan, Lurtotecan and S39625.

As used herein, the term "austatin and any derivative thereof/austatin or any derivative thereof" refers to the natural antitumor product aplysiatoxin 10 and a number of its derivatives, wherein such compounds interfere with the self-assembly of components at the microscopic level to arrest cells in the mitotic phase, which leads to high cellular lethality. Auristatin and any derivative thereof as used herein includes auristatin and any derivative thereof that already exist or will be produced in the future. Auristatin and its derivative herein include, but are not limited to, auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), monomethyl auristatin D (MMAD), AFP and AFHPA.

As used herein, the term “cyclooxygenase-2 inhibitor” refers to a class of specific cyclooxygenase-2 inhibitors. Cyclooxygenase-2 is involved in the development and infiltration of malignant tumors through a number of different mechanisms. Cyclooxygenase-2 inhibitors can suppress the migration and adhesion of tumor cells, as well as intravascular infiltration, thereby suppressing the occurrence and development of malignant tumors. The cyclooxygenase-2 inhibitors of the present application include cyclooxygenase-2 inhibitors that already exist or will be produced in the future. Cyclooxygenase-2 inhibitors include, but are not limited to, celecoxib, rofecoxib, parecoxib, valdecoxib and etoricoxib.

As used herein, the term “radionuclide complex” refers to a special class of complexes containing radionuclides, wherein the chelating agent in the complex can be chelated by a radionuclide and can provide a coupling moiety that binds more stably to the target substance. As used herein, the term “radionuclide” refers to an element that can spontaneously emit radiation (such as α-rays, β-rays or γ-rays). Radionuclides according to this application include all radionuclides for treatment and diagnostics that already exist or will be produced in the future. Radionuclides according to this application include, without limitation, 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 and ²¹¹ At . In some embodiments, the chelating agent is a macrocyclic chelating agent. The chelating agents of this application include, but are not limited to, H2dedpa, 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-ester DOTA-tris(tBu), DO3AM-acetic acid, DO3AM-N-(2-aminoethyl)ethanamide, DO3AtBu-N-(2-aminoethyl)ethanamide, DOTA-di(tBu)ester, DOTA NHS ester -tris(tBu)-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, cyclene, DO2AtBu, DO3AtBu, DO3AEt, DO3AM, DOTAEt, DOTPrEt, cis-glyoxal-cyclene, mono-N-benzyl-cyclene, trans-N -dibenyl-cyclen, TriBOC-cyclen, mono-N-benzyl-TACN, DiBOC-TACN, cross-bridged cycles (CB-cycles), (13)anN4, 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, CB-cyclam derivatives, CB-TE2A, methylamino-(13)anN4, bis-(13)anN4, oxo-(13)anN4, mono-N-benzyl-(13)anN4, TriBOC-(13)anN4 , TRITRAM, TRI3AEt, TRI3AtBu, TRITAM, TRITA, mono-N-benzyl-cyclam, formaldehyde-cyclam, cis-glyoxal-cyclam, dioxocyclam, oxocyclam, trans-N-dibenzyl-cyclam, TriBOC-cyclam, DOTP, DOTMA, TETA , DOTAM, DiAmSar, CB-cycles, CB-TE2A, NOTA, NOTAM, NH ₂ -NODA-GA, iodine-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, NH ₂ -MPAA-NODA, NH ₂ -NODA-GA, iodine-NODA-GA and TACN.

In some embodiments, the conjugate compound or a pharmaceutically acceptable salt thereof according to this application contains a single payload. In some embodiments, a conjugate compound or a pharmaceutically acceptable salt thereof according to this application contains two or more payloads. For example, the conjugate compound or a pharmaceutically acceptable salt thereof according to the present application contains 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 conjugated compound containing multiple payloads, each of the payloads may be identical to the others or different from each other. In some embodiments, the at least two payloads are different from each other.

As used herein, the term “targeting molecule” refers to any molecule or moiety capable of targeting a conjugate compound of this application to a target site, target tissue, target organ, target cell, or intracellular region of the target. In some embodiments, the targeting molecule provides the conjugate compound of the present application with a higher level of distribution to the target site, target tissue, target organ, target cell, or intracellular region of the target compared to the non-target site, non-target tissue , a non-target organ, a non-target cell, or a non-target intracellular region, e.g., at least 10% higher, 20% higher, 50% higher, 80% higher, 100% higher, 150% higher higher, 200% higher, 300% higher, 400% higher, 500% higher, etc. In some embodiments, the targeting molecule provides for a conjugate compound containing the targeting molecule, compared to the conjugate compound, not containing a targeting molecule, a higher level of distribution in the target site, target tissue, target organ, target cell or intracellular region of the target, for example, at least 10% higher, 20% higher, 50% higher , 80% higher, 100% higher, 150% higher, 200% higher, 300% higher, 400% higher, 500% higher, etc. In some embodiments, the targeting molecule may induce or promote specific binding of a conjugated compound containing such targeting molecules to a target molecule, induce or stimulate endocytosis of the conjugated compound by the target cell, and induce or stimulate accumulation of the conjugate compound in the vicinity of the target cell and/or entry into the target cell.

In some embodiments, the conjugate compound of the present application contains at least two targeting molecules. In some embodiments, two or more targeting molecules contained in a conjugated compound according to the present application are identical or different. In some embodiments, at least two targeting molecules of the two or more targeting molecules contained in a conjugated compound according to the present application are different. In some embodiments, all of the two or more targeting molecules contained in a conjugated compound according to the present application are different from each other. In some embodiments, at least two targeting molecules of the two or more targeting molecules contained in a conjugated compound of the present application may specifically bind to different proteins or cell surface markers. In some embodiments, two or more targeting molecules contained in a conjugated compound of the present application may specifically bind to different proteins or cell surface markers.

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

As used herein, the term “synergistic molecule” refers to any molecule or moiety that is capable of acting in a synergistic manner with other targeting molecules contained in a conjugated compound of the present application to provide a higher level of inducing or promoting specific binding of the conjugated compound to the molecule. target, inducing or promoting endocytosis of the conjugated compound by the target cell, inducing or promoting accumulation of the conjugated compound in the vicinity of the target cell, and/or entry into the target cell, and/or otherwise causing specific binding of the conjugated compound to the target cell and its retention. In some embodiments, the synergistic molecule provides a conjugate compound of this application with a higher level of distribution to a target site, target tissue, target organ, target cell, or intracellular region of the target compared to a non-target site, non-target tissue , a non-target organ, a non-target cell, or a non-target intracellular region, e.g., at least 10% higher, 20% higher, 50% higher, 80% higher, 100% higher, 150% higher higher, 200% higher, 300% higher, 400% higher, 500% higher, etc. In some embodiments, the synergistic molecule provides for a conjugate compound containing the synergistic molecule, compared to the conjugate compound, not containing a synergistic molecule, a higher level of distribution in the target site, target tissue, target organ, target cell or intracellular region of the target, for example, at least 10% higher, 20% higher, 50% higher , 80% higher, 100% higher, 150% higher, 200% higher, 300% higher, 400% higher, 500% higher, etc. In some embodiments, the synergistic molecule provides for the conjugated of a compound containing a synergistic molecule, compared to a conjugate compound not containing the synergistic molecule, having a higher level of activity on the target cell, e.g., at least 10% higher, 20% higher, 50% higher, 80 % higher, 100% higher, 150% higher, 200% higher, 300% higher, 400% higher, 500% higher, etc.

In some embodiments, the synergistic molecule of the present application is a cell interacting molecule.

As used herein, the term “cell interacting molecule” refers to a molecule that is capable of interacting with the cell surface material of a target cell to induce or promote specific binding to the cell of a conjugated compound containing such cell interacting molecules to induce or promote endocytosis conjugated compound by the target cell and/or inducing or promoting accumulation of the conjugated compound near the target cell and/or penetration into the target cell.

The molecule interacting with the cell can be a small molecular chemical molecule or a large biomolecule. In some embodiments, the cell-interacting molecule is a small molecule compound or polypeptide. In some embodiments, the cell interacting molecule is a small molecule compound or polypeptide containing 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, or 9 amino acids.

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

Ligands of this application may include a number of different chemical or biological molecules that may have a specific binding affinity for a selected target, where the selected target may be, for example, a cell surface receptor, a cell surface antigen, a cell, a tissue, an organ, etc. In some embodiments, the ligand may specifically bind to a protein or marker expressed on the surface of a target cell. In some embodiments, the ligand of the present application binds to a protein or cell surface marker with an affinity of 10 ^-6 -10 ^-11 M (K _d value). In some embodiments, the ligand of the present application binds to a protein or cell surface marker with an affinity of at least 10 ^-7 , at least 10 ^-8 and at least 10 ^-9 M (K _d value). In some embodiments, the ligand of the present application binds to a protein or cell surface marker with an affinity of less than 10 ^-6 , less than 10 ^-7 and less than 10 ^-8 M (K _d value). In some embodiments, a ligand of the present application binds to a protein or cell surface marker with a certain affinity, wherein the determined affinity refers to the affinity of the ligand for a target protein or cell surface marker that is at least two, three, four, five , six, eight, ten, twenty, fifty, one hundred or more times higher than the affinity for the non-target protein or cell surface marker. In some embodiments, the expression of a protein or cell surface marker of the present application in target cells (eg, cancer cells) is significantly higher than in normal cells. As used herein, the term “significantly” refers to statistically significant differences or significant differences that can be recognized by one skilled in the art.

In some embodiments, the level of expression of a protein or cell surface marker according to this application in target cells (eg, cancer cells) is 2-1,000,000 times higher than in normal cells; for example, an expression level in target cells (e.g., cancer cells) of 2-10, 2-100, 2-1000, 2-10000, 2-100000, or 2-1000000 (which could be any value within the above numerical range , and at the same time including the end values of this range) times higher than in normal cells. In some embodiments, the expression level of the cell surface receptor in target cells (e.g., cancer cells) is at least 10 times higher, or 100 times higher, or 1000 times higher, or 10,000 times higher, or 100,000 times higher than in normal cells. In some embodiments, compared to the level of the protein or cell surface marker on target cells (e.g., cancer cells), the level of the cell surface receptor on normal cells is reduced by at least 50%, 60%, 70%, 80%, 90%, 95% or 99%. In some embodiments, a protein or cell surface marker described herein is not detectable in normal cells.

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

In some embodiments, the cell surface receptor of the present application is selected from the group consisting of transferrin receptor (TFR), low density lipoprotein receptor (LDLR), folate receptor (FR), growth hormone inhibitory hormone receptor, uric acid kinase receptor, factor receptor tumor necrosis receptor (TNFR), integrin receptor (LFA-1), SST-14 receptor (SSTR2), GNRH receptor (GNRHR), TRPV6 receptor and integrin α.

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

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

In some embodiments, the cell interacting molecule of the present application may 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, claudin 18.2, APN, DLL3, CEACAM5, FZD10, TFRC, MET, IGFR1, SSTR2, CCKBR, LFA1, ICAM, GPR87, GM-CSF, GM-CSFR, TIM3, TLR family members, CD40, CD40L, OX40, OX40L, GITRL, GITR, 4-BBL, 4-1BB, CD70, CD27, ICOSL, ICOS, HHLA2, CD28, CD86/80, CD28, MHCII antigen, 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.

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

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

In some other embodiments, the conjugate compound or a pharmaceutically acceptable salt thereof according to this application comprises P10 and a moiety that is a synergistic molecule, wherein the synergistic molecule binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA) and GNRHR.

In some embodiments, one of the synergistic molecules in a conjugate compound or a pharmaceutically acceptable salt thereof according to the present application is a moiety that is an endocytosis promoting molecule that is capable of mediating endocytosis. As used herein, the term “endocytosis” means that a conjugate compound or a pharmaceutically acceptable salt thereof interacts with a target cell and is then capable of mediating its own endocytosis, internalization, or uptake by the target cell. As used herein, the term “endocytosis promoting molecule” refers to a molecule that interacts with a target cell and is then capable of mediating endocytosis, internalization or uptake of a conjugated compound or a pharmaceutically acceptable salt thereof according to this application by the target cell.

In some embodiments, the endocytosis promoting molecule is selected from the group consisting of folate and an analog thereof, an endocytosis-promoting peptide, and a cell penetrating peptide.

In some embodiments, the endocytosis promoting molecule of the present application is folate or an analog thereof.

Folate is useful for forming chemical bonds with other groups due to its low molecular weight, non-immunogenicity and good stability. Folate can bind with high affinity to the folate receptor expressed on the cell surface, mediating cellular uptake of folate. Although the folate receptor is expressed at very low levels in most normal cells, it is expressed at high levels in many cancer cells to meet the high folate demand of rapidly dividing cells in low folate conditions (see Kelemen LE, Int J Cancer , 2006; 119 :243-50; Kane MA, et al., J Clin Invest. 81: 1398-406; Matsue H, et al., Proc Natl Acad Sci USA 89: 6006-9; ., Annu Rev Nutr. 2011; 31: 177-201). Folate is capable of specifically binding to the folate receptor on the cell surface and also mediating endocytosis of the conjugated compound or its pharmaceutically acceptable salt into target cells.

In some embodiments, the folate analog is selected from the group consisting of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate, and 5,10-methylenetetrahydrofolate.

In some embodiments, the endocytosis promoting molecule is a peptide capable of mediating endocytosis.

In some embodiments, the peptide capable of mediating endocytosis comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and Arg-Gly-Asp (referred to as RGD), as well as homologous peptides characterized by 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%, at least 99% amino acid sequence homology with any of SEQ ID NO: 16-18, where the homologous peptides are functional equivalents of the peptides indicated under SEQ ID NO: 16-18, respectively.

In some embodiments, a peptide capable of mediating endocytosis as described herein comprises a conservative amino acid substitution at only one amino acid site compared to the sequences of SEQ ID NOs: 16-20 and RGD. In some embodiments, a peptide capable of mediating endocytosis as described herein comprises a conservative amino acid substitution at 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid sites compared to the sequences of SEQ ID NO: 16 -20.

Provided that its biological activity is not affected, the peptide capable of mediating endocytosis as described herein may also contain non-naturally occurring amino acids, including, for example, β-fluoroalanine, 1-methylhistidine, γ-methyleneglutamic acid, α-methylleucine, 4,5-dehydrolysine, hydroxyproline, 3-fluorophenylalanine, 3-aminotyrosine, 4-methyltryptophan, etc.

The percentage of homology can be determined by various methods well known in the art. For example, sequence comparisons can be achieved using the following publicly available tools: BLASTp software (available on the National Center for Biotechnology Information (NCBI) website: http://blast.ncbi.nlm.nih.gov/Blast.cgi; also see .: Altschul SF et al., J. Mol. Biol. , 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res. , 25:3389-3402 (1997)), ClustalW2 (available online) European Bioinformatics Institute: http://www.ebi.ac.uk/Tools/msa/clustalw2/; also see: Higgins DG et al., Methods in Enzymology , 266:383-402 (1996); ., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and Tcoffee (available on the website of the Swedish Institute of Bioinformatics; also see: 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 sequence alignment is performed using software, the default parameters available in the software can be used, or otherwise, the parameters can be adjusted according to the purpose of the alignment. All this is within the knowledge of a person skilled in the art.

As used herein, the term “functional equivalent” refers to a derived peptide that retains biological activity substantially similar to that of the parent peptide sequence from which the derived peptide is derived. The functional equivalent may be a natural derivative or obtained synthetically. Examples of functional equivalents include amino acid sequences involving substitutions, deletions or additions of one or more amino acids, provided that the biological activity of the peptide is maintained. The amino acid used for replacement desirably has chemical and physical properties similar to those of the amino acid being replaced. Desirable analogous chemical-physical properties include charge similarity, bulkiness, hydrophobicity, hydrophilicity, and the like.

In some embodiments, the functional equivalents include a conservative substitution of an amino acid residue. Conservative amino acid residue substitution refers to a substitution occurring between amino acids with similar properties, for example, a substitution between polar amino acids (for example, a substitution between glutamine and asparagine), a substitution between hydrophobic amino acids (for example, a substitution between leucine, isoleucine, methionine and valine), a substitution between amino acids with the same charges (for example, a substitution between arginine, lysine and histidine or a substitution between glutamic acid and aspartic acid), etc.

In some embodiments, the endocytosis promoting molecule is a cell penetrating peptide. Cell penetrating peptides (CPPs), also known as protein transduction domains (PTDs), are short peptides (usually less than 40 amino acids) capable of entering cells in a receptor-independent manner. Cell-penetrating peptides, when conjugated with payloads, are capable of mediating transmembrane transport of payloads and have protein transduction activity. In some embodiments, the cell penetrating peptide described herein is selected from the group consisting of a tumor homing peptide, a mitochondrial penetrating peptide, an activated cell penetrating peptide, and an antibacterial peptide. In some embodiments, the cell penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19 (RRRRRRRRR, referred to as R9) and SEQ ID NO: 20 (GRKKRRQRRRPPQ, which is a Tat peptide, i.e., penetrating cell peptide based on the HIV transcription transactivator protein).

In some embodiments, one targeting molecule in a conjugate compound or a pharmaceutically acceptable salt thereof according to the present application is a moiety that is a ligand to a prostate-specific membrane antigen.

As used herein, the term “prostate-specific membrane antigen” refers to a type Ⅱ transmembrane glycoprotein that is present in the membrane of prostate epithelial cells and consists of 750 amino acids, comprising 19 amino acids in the intracellular region, 24 amino acids in the transmembrane region, and 707 amino acids in the extracellular region. Prostate-specific membrane antigen is expressed in normal prostate epithelial cells, but is expressed at much higher levels in prostate cancer cells. Compared with traditional prostate-specific antigen used for clinical detection, prostate-specific membrane antigen is a more sensitive and specific tumor marker of prostate cancer, and in particular, it is expressed at high levels in both hormone-refractory prostate cancer and metastatic prostate cancer. gland, and also has high sensitivity and specificity for identifying differences between prostate cancer and other types of malignant tumors. Moreover, in a number of different non-prostate solid tumors (such as lung cancer, bladder cancer, gastrointestinal cancer, pancreatic cancer, kidney cancer and colorectal cancer), prostate-specific membrane antigen is also highly expressed and specifically expressed on tumor vascular endothelial cells.

As used herein, the term “prostate-specific membrane antigen ligand” refers to an antibody, aptamer, and small molecule that is capable of specifically recognizing and binding to a prostate-specific membrane antigen. Ligands to prostate-specific membrane antigen according to the present application include ligands to prostate-specific membrane antigen that already exist or will be produced in the future, as well as fragments of the above-mentioned ligands, provided that these fragments still retain the ability to bind to prostate-specific membrane antigen. Antibody-based ligands are the most common ligands for prostate-specific membrane antigen, 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). An aptamer is a single-stranded DNA or RNA that is obtained by technical screening using an exponential ligand enrichment system and can bind to prostate-specific membrane antigens with high affinity and high specificity. Such prostate-specific membrane antigen ligands include, but are not limited to, the xPSM-A10 aptamer and its derivative, and the xPSM-A9 aptamer and its derivative (e.g., 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 , Cancer Res, 2002, 62(14):4029-4033). Compared with antibody-based ligands and aptamer-based ligands, small molecule prostate-specific membrane antigen ligands have the advantages of small molecular weight, high permeability, low immunogenicity and ease of synthesis, and include, but are not limited to, small molecule glutamine urea ligands and phosphoramidate small molecule ligands.

In some embodiments, the small molecule ligands to prostate-specific membrane antigen of the present application may 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)hydroxyphosphinyl]methyl]glutaric acid; 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-[[propylhydroxyphosphinyl]oxy]glutaric acid; 2-[[butylhydroxyphosphinyl]oxy]glutaric acid; 2-[[phenylhydroxyphosphinyl]oxy]glutaric acid; 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-hydroxyl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-methyl)carbamoyl]methyl]glutaric acid; 2-[[(n-butyl-n-hydroxyl)carbamoyl]methyl]glutaric acid; 2-[[(n-benzyl-n-hydroxyl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-phenyl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-2-phenylethyl)carbamoyl]methyl]glutaric acid; 2-[[(n-ethyl-n-hydroxyl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-propyl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-3-phenylpropyl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-4-pyridyl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(methyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(benzyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(phenyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(2-phenylethyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(ethyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(propyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(3-phenylpropyl)amide]methyl]glutaric acid and 2-[[n-hydroxyl(4-pyridyl)amide]methyl]glutaric acid; 2-[(thionyl)methyl]glutaric acid; 2-[(methylthionyl)methyl]glutaric acid; 2-[(ethylthionyl)methyl]glutaric acid; 2-[(propylthionyl)methyl]glutaric acid; 2-[(butylthionyl)methyl]glutaric acid; 2-[(phenylthionyl]methyl]glutaric acid; 2-[[(2-phenylethyl)thionyl]methyl]glutaric acid; 2-[[(3-phenylpropyl)thionyl]methyl]glutaric acid; 2-[[(4- pyridyl)thionyl]methyl]glutaric acid; 2-[(benzylthionyl)methyl]glutaric acid; 2-[(methylsulfonyl)methyl]glutaric acid; glutaric acid; 2-[(propylsulfonyl)methyl]glutaric acid; 2-[(butylsulfonyl)methyl]glutaric acid; 2-[[(2-phenylethyl)sulfonyl]methyl]glutaric acid; acid; 2-[[(3-phenylpropyl)sulfonyl]methyl]glutaric acid; 2-[[(4-pyridyl)sulfonyl]methyl]glutaric acid; )methyl]glutaric acid; 2-[(methylsulfoxyminyl)methyl]glutaric acid; 2-[(propylsulfoxyminyl)methyl]glutaric acid; 2-[(phenylsulfoxyminyl]methyl]glutaric acid;2-[[(2-phenylethyl)sulfoxyminyl]methyl]glutaric acid; 2-[[(3-phenylpropyl)sulfoxyminyl]methyl]glutaric acid; 2-[[(4-pyridyl)sulfoxyminyl]methyl]glutaric acid and 2-[(benzylsulfoxyminyl)methyl]glutaric acid; n-[methylhydroxyphosphinyl]glutamic acid; n-[ethylhydroxyphosphinyl]glutamic acid; n-[propylhydroxyphosphinyl]glutamic acid; n-[butylhydroxyphosphinyl]glutamic acid; n-[phenylhydroxyphosphinyl]glutamic acid; n-[(phenylmethyl)hydroxyphosphinyl]glutamic acid; n-[((2-phenylethyl)methyl)hydroxyphosphinyl]glutamic acid and N-methyl-N-[phenylhydroxyphosphinyl]glutamic acid. The prostate-specific membrane antigen ligands of this application also include all small molecule prostate-specific membrane antigen ligands disclosed in PCT applications WO 2010/108125 and WO 2006/093991, the two aforementioned patent applications being incorporated herein in their entirety.

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

In some embodiments, the small molecule prostate-specific membrane antigen ligand of the present application has the following structure:

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

.

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

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

.

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

In some embodiments, one targeting molecule in a conjugate compound or a pharmaceutically acceptable salt thereof according to this application is a ligand moiety represented by formula (I):

or a fragment constituting a ligand having at least 70%, at least 80%, at least 85% or at least 90% amino acid sequence homology with respect to it, or comprising no more than 3, 2 or 1 amino acid substitution (for example, conservative substitutions) regarding it.

In some embodiments, one targeting molecule in a conjugate compound or a pharmaceutically acceptable salt thereof according to this application is P10 or a moiety that is a ligand characterized by 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 with respect to it, or involving no more than 3, 2, or 1 amino acid substitution (eg, conservative substitutions) with respect to it.

As used herein, the term “P10” refers to a peptide having the amino acid sequence Cys-Lys-Glu-Phe-Leu-His-Pro-Ser-Lys-Val-Asp-Leu-Pro-Arg.

In some embodiments, the conjugate compound of the present application comprises targeting molecules selected from the group consisting of (1) a folate receptor ligand and a prostate-specific membrane antigen ligand; (2) a ligand to TRPV6 and a ligand to prostate-specific membrane antigen; (3) a ligand to GNRHR and a ligand to prostate-specific membrane antigen; (4) a ligand for SSTR2 and a ligand for prostate-specific membrane antigen; (5) a folate receptor ligand and an SSTR2 ligand or (6) a TRPV6 ligand and a folate receptor ligand.

In some embodiments, the two targeting molecules in a conjugate compound or a pharmaceutically acceptable salt thereof provided herein are a synergistic molecule moiety and a prostate-specific membrane antigen ligand moiety, respectively. In some embodiments, the synergistic molecule is capable of mediating endocytosis. In some embodiments, the two targeting molecules in a conjugate compound or a pharmaceutically acceptable salt thereof provided herein are folate or an analog thereof and a prostate-specific membrane antigen ligand moiety, respectively. Without wishing to be bound by theory, specific folate or analogues thereof and prostate-specific membrane antigen ligand fragment are selected that have better stability than combinations of ligands presented in the prior art.

In some embodiments, the two targeting molecules in a conjugate compound or a pharmaceutically acceptable salt thereof provided herein are a synergistic molecule moiety and a ligand moiety represented by formula (I), respectively. In some embodiments, the synergistic molecule is capable of mediating endocytosis. In some embodiments, the two targeting molecules in a conjugate compound or a pharmaceutically acceptable salt thereof provided herein are folate or an analog thereof and the ligand moiety represented by formula (I), respectively.

In some embodiments, the two targeting molecules in a conjugate compound or a pharmaceutically acceptable salt thereof provided herein are a synergistic molecule moiety and P10, respectively. In some embodiments, the synergistic molecule is capable of mediating endocytosis. In some embodiments, the two targeting molecules in a conjugate compound or a pharmaceutically acceptable salt thereof provided herein are folate or an analog thereof and P10, respectively.

In some embodiments, a conjugated compound provided herein comprises only one payload conjugated to two targeting molecules. In some embodiments, a conjugated compound provided herein comprises multiple payloads conjugated to two targeting molecules.

As used herein, the term “conjugated” refers to the linking through a covalent bond of two chemical groups, either directly forming a covalent bond between two chemical groups, or indirectly linking two chemical groups through a linker.

In some embodiments, the conjugate compound or a pharmaceutically acceptable salt thereof comprises payload(s) (eg, 1 payload) and two targeting molecules, wherein the payload is directly covalently linked to at least one of the targeting molecules. In some embodiments, the payload is directly covalently linked to two targeting molecules.

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

As used herein, the term “linker” refers to a molecule or fragment that covalently links the payload to a targeting molecule. Linkers contain a functional group for linking the payload to at least one targeting molecule. In some embodiments, the functional group may contain two reactive moieties, one intended to bind to the payload and the other intended to bind to the targeting molecule. In some embodiments, the functional groups are different from each other. In some embodiments, the functional groups include a group containing a thiol-reactive moiety and an amine-reactive moiety. In some embodiments, the functional groups are identical to each other. In some embodiments, the functional groups are maleimide groups. In some embodiments, the linker comprises an amino acid. In some embodiments, the carboxylic acid in the amino acid contained in the linker is amidated. In some embodiments, the linker comprises a short chain polyethylene glycol (eg, containing 2-10, 2-8, 3-8, 4-8, 4-7, 4-6, or 5 repeat units).

In some embodiments, the linker of the present application is a multivalent linker capable of contacting at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload and at least at least one targeting molecule. The payloads associated with the multivalent linker may be identical or different, and the targeting molecules associated with the multivalent linker may be identical or different.

In one aspect, the linker must be sufficiently stable to avoid inadvertent release of payloads during circulation in the bloodstream to increase the effective amount of payload delivered to target cells or tissues, as well as to avoid toxicity. In another aspect, the linker must be capable of releasing payloads near or within target cells to effectively kill the target cells or block the functions of the target cells. In some embodiments, the linker contains at least one cleavable functional group. Preferably, the functional group to be cleaved is sufficiently stable outside the target cell, but upon entering the target cell is cleaved to release the payload(s). In some embodiments, the cleaved functional group is cleaved at least 10, 20, 30, 50, 100 or more times more efficiently in target cells than in blood or serum.

The cleavable linker can be cleaved by hydrolysis, an enzymatic or reduction reaction, or by a change in pH. In some embodiments, the linker is cleavable when exposed to a specific physiological environment (eg, an appropriate pH environment). In some embodiments, the linker is cleavable in an acidic environment with a pH of approximately 6.5 or lower or by reagents such as enzymes. In some embodiments, the linker is sensitive to factors that mediate cleavage, such as pH, redox potential, or the presence of destructive molecules.

In some embodiments, the linker is non-cleavable. Non-cleavable linkers, as used herein, refer to linkers that remain substantially intact during intracellular metabolism.

In some embodiments, the linker is a peptide linker consisting of straight or branched chain amino acids linked by peptide bonds. In some embodiments, the peptide linker is a cleavable protease that is highly or specifically expressed near or within target cells, such as cathepsin B in a lysosome or endosome. The peptide linker used herein may have various lengths. Typically, the peptide linker according to the present application has a length of from 1 to 50 amino acids. In some embodiments, the peptide linker has a length of 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 peptide liqueur has a length of 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 as described herein can be any integer value within the above numerical range, including the ends of that range. In some embodiments, a peptide linker that is 1, 2, 3, 4, or 5 amino acids in length is preferred. 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 a carboxylic acid a in the above amino acids is amidated.

In some embodiments, the linker is a disulfide linker containing a disulfide bond. The disulfide bond can be cleaved in an intracellular reducing environment while remaining stable in the bloodstream. The disulfide linker of the present application may be DSDM, DMDS, MDS or NDMDS. The structures of these disulfide linkers are shown in Table 1 below.

Table 1. Structures of DSDM, DMDS, MDS and NDMDS

Name Structure DSDM DMDS M.D.S. NDMDS

In some embodiments, the linker is a pH-dependent linker. The pH-dependent linker described in this application is cleavable in an environment with a certain pH. In some embodiments, the pH-dependent linker may be stable under alkaline conditions, but cleavable under acidic conditions, for example, at a pH of 6.5 or lower. In some embodiments, the pH-dependent linker is cis-aconitic anhydride.

In some embodiments, the linker in the conjugate compound or a pharmaceutically acceptable salt thereof is

,

or is a combination of the above structure and a peptide linker (eg, binding to a targeting molecule via a peptide linker containing 1-3 amino acids).

In some embodiments, the linker in the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

,

or is a combination of the above structure and a peptide linker (eg, binding to a targeting molecule via a peptide linker containing 1-3 amino acids).

In some embodiments, the linker in the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

,

or is a combination of the above structure and a peptide linker (eg, binding to a targeting molecule via a peptide linker containing 1-3 amino acids).

In some embodiments, the linker in the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

,

or is a combination of the above structure and a peptide linker (eg, binding to a targeting molecule via a peptide linker containing 1-3 amino acids).

In some embodiments, the linker in the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

,

or is a combination of the above structure and a peptide linker (eg, binding to a targeting molecule via a peptide linker containing 1-3 amino acids).

In some embodiments, the linker in the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

,

or is a combination of the above structure and a peptide linker (eg, binding to a targeting molecule via a peptide linker containing 1-3 amino acids).

In some embodiments, the linker in the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

,

or is a combination of the above structure and a peptide linker (eg, binding to a targeting molecule via a peptide linker containing 1-3 amino acids).

In some embodiments, the linker in the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

,

or is a combination of the above structure and a peptide linker (eg, binding to a targeting molecule via a peptide linker containing 1-3 amino acids).

In some embodiments, the linker in the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

,

or is a combination of the above structure and a peptide linker (eg, binding to a targeting molecule via a peptide linker containing 1-3 amino acids).

In some embodiments, the linker in the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

,

or is a combination of the above structure and a peptide linker (eg, binding to a targeting molecule via a peptide linker containing 1-3 amino acids).

In some embodiments, the linker in the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

or is a combination of the above structure and a peptide linker (eg, binding to a targeting molecule via a peptide linker containing 1-3 amino acids).

In some embodiments, the linker in the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:

,

or is a combination of the above structure and a peptide linker (eg, binding to a targeting molecule via a peptide linker containing 1-3 amino acids).

In some embodiments, the linker of this application may include any one or combination of linkers as described above.

In some embodiments, the payload is conjugated directly or indirectly to a first targeting molecule, and the first targeting molecule is conjugated directly or indirectly to a second targeting molecule. In some embodiments, the payload is conjugated directly to each of the first and second targeting molecules. In some embodiments, the payload is conjugated indirectly to each of the first and second targeting molecules. In some embodiments, the payload is conjugated indirectly to a first targeting molecule, such as through a linker, and the first targeting molecule is conjugated directly or indirectly to a second targeting molecule. In some embodiments, the payload is conjugated to a first targeting molecule via a first linker, and wherein the payload is conjugated to a second targeting molecule via a second linker. In some embodiments, the linker is a multivalent linker that binds to at least one (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload and two targeting molecules.

In some embodiments, two targeting molecules are linked to each other via a spacer. In some embodiments, the spacer is cleaved by a protease that is specifically expressed in or subject to expression in target cells. Such proteases include, for example, those listed in Table 2 below. In some embodiments, the spacer comprises an amino acid sequence selected from any of the amino acid sequences listed in Table 2 below.

Table 2. List of enzymatically cleavable sequences

Protease Amino acid sequence of the recognition site SEQ ID NO. Cathepsin B R.R. - Legumain ASN - Matripase KSRAEDE SEQ ID NO: 1 MMP-2 PLGLAG SEQ ID NO: 2 Prostate-specific antigen SSLY SEQ ID NO: 3 Stromelizin-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 VGVF SEQ ID NO: 14

As used herein, the terms “cleavable” or “cleaved” refer to a metabolic or reaction process with a conjugated compound as provided herein, whereby the linker between the payload and the targeting molecule or the spacer between the targeting molecules is disrupted to release the free payload or targeting molecule. The linker and spacer are either cleaved by a protease or are cleaved when exposed to a certain physiological environment, for example, an environment with a certain pH value.

In some embodiments, the conjugate compound has the structure of Formula I, II, III, or IV, shown below, wherein n, m, p, and q are independently 0 or 1, thereby indicating that the linker and spacer are independently present or absent. "Molecule" in the following formula is short for "targeting molecule."

In some embodiments, a conjugate compound or a pharmaceutically acceptable salt thereof provided herein 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, a conjugate compound or a pharmaceutically acceptable salt thereof provided herein comprises one payload as provided herein, one ligand that specifically binds to a protein or cell surface marker as provided herein, one synergistic molecule , as provided in this application, and a linker or spacer, as provided in this application.

In some embodiments, the conjugate compound has the structure of Formula V, VI, VII, or VIII shown below, wherein n, m, p, q, and s are independently 0 or 1, thereby indicating that a linker, multivalent linker, and spacer are independently present or none.

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

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

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

In some embodiments, the conjugate compound of this 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 shown in Fig. 1). In some embodiments, the conjugate compound of the present application is prepared by linking a linker-drug moiety to a ligand moiety through a covalent bond. The linker-drug moiety of the present application contains a payload and a linker, and the ligand moiety of the present application contains two targeting molecules and an optional spacer or linker, wherein the two moieties form a conjugated compound of the present application by react and form a covalent bond, and the covalent bond may be formed between a linker in the linker-drug moiety and a ligand molecule in the ligand moiety, or it may be formed between a linker in the linker-drug moiety , and a spacer or linker in the fragment representing the ligand.

The conjugated compound of the present application, CB-20B, is formed by linking the linker-drug moiety LT1002 to the ligand moiety 20B-SM09 via a covalent bond. The conjugate compound of the present application, CB-20BK, is formed by linking the linker-drug moiety of LT1002 to the ligand moiety of 20BK-SM09 via a covalent bond. The conjugate compound of the present application, CB-60S, is formed by linking the linker-drug moiety of LT2000C to the ligand moiety of 60S-SM09 via a covalent bond. The conjugate compound of the present application, CB-60SK, is formed by linking the drug-linker moiety LT2000C to the ligand moiety 60SK-SM09 via a covalent bond. The conjugate compound of the present application, CB-20C, is formed by linking the drug-linker moiety LD1001 to the ligand moiety 20BK-SM09 via a covalent bond. The conjugate compound of the present application, CB-1020, is formed by linking the drug-linker moiety LT1002 to the ligand moiety 1020BK-SM09 via a covalent bond. The conjugate compound of the present application, CB-1320, is formed by linking the drug-linker moiety LT1002 to the ligand moiety 1320BK-SM09 via a covalent bond. The conjugate compound of the present application, CB-1820, is formed by linking the drug-linker moiety LT1002 to the ligand moiety 1820BK-SM09 via a covalent bond. The conjugate compound of the present application, CR19428, is formed by linking the linker-drug moiety CR19423 to the ligand moiety 20BK-SM09 via a covalent bond. The conjugate compound of the present application, CB-20R, is formed by complexing 20R-SM09 with the radionuclide ion M. The conjugate compound of the present application, CB-18G, is formed by coupling the linker-drug moiety LT1002 to the ligand, 18G-SM09 through a covalent bond. The conjugated compound of the present application, CR19426, is formed by linking the linker-drug moiety CR19423 to the ligand moiety 18G-SM09 via a covalent bond. The conjugated compound of the present application, CB-10S, is formed by linking the linker-drug moiety LT1000 to the ligand moiety CBSM09 via a covalent bond. The conjugated compound of the present application, CR19425, is formed by linking the linker-drug moiety CR19423 to the ligand moiety CBSM09 via a covalent bond. The conjugate compound of the present application, CB-50S, is formed by linking the linker-drug moiety LT1000N3 to the ligand moiety 50S-SM09 via a covalent bond. Each structure is shown in Table 3 below.

Table 3. Structures of the linker-drug fragment and the ligand fragment

Reduction Structure LD1001 LT1000 LT1000N3 LT1002 LT2000C CBSM09 18G-SM09 20B-SM09 20BK-SM09 20R-SM09 50S-SM09 60S-SM09 60SK-SM09 1020BK-SM09 1320BK-SM09 1820BK-SM09 CR19423

In some embodiments, the conjugate compound or a pharmaceutically acceptable salt thereof provided herein is released into the bloodstream and into the extracellular space (extracellular space). Since the linker is very stable in the extracellular environment and the drug molecules cannot be released, the toxicity of the drug molecules is blocked. The conjugate is a drug with no or low toxicity and will not be toxic to normal cells.

In some embodiments, a conjugate compound or a pharmaceutically acceptable salt thereof provided herein binds to multiple receptors or antigens and other active molecules that are simultaneously expressed at high levels on diseased cells, and their synergistic effect significantly increases the affinity of the conjugate compound for the cells -targets, reducing the likelihood of binding to normal cells. Therefore, a drug based on a highly effective toxin, such as the MMAE/Dxd/SN38/radionuclide complex, can be used to improve the effectiveness of drugs, expand the therapeutic window, and prevent drug side effects.

In some embodiments, the conjugate compound or a pharmaceutically acceptable salt thereof provided herein is internalized into target cells and the linker can then be cleaved by changes in the internal environment of the cells (by specific enzyme cleavage, pH changes, disulfide bond reduction, etc. .) with the release of drug molecules (equivalent to the removal of modifying groups of drug molecules), which has a therapeutic effect on tumor cells.

In some embodiments, a conjugate compound or a pharmaceutically acceptable salt thereof according to the present application can be used to specifically deliver a payload to target cells within the target tissue environment. In general, two targeting molecules in a conjugate compound or a pharmaceutically acceptable salt thereof have three advantages. First, two targeting molecules can act in multiple modes (often synergistically), leading to improved therapeutic effects while reducing side effects. Second, the binding of two targeting molecules increases the affinity and avidity of the conjugated compound or its pharmaceutically acceptable salt for target receptors or target cells, thereby increasing their specificity and avoiding off-target toxicity. Finally, when properly designed, the combination of two targeting molecules can realize the multifunctional properties required for a drug conjugate.

The conjugated compound or a pharmaceutically acceptable salt thereof according to the present application achieves unexpected technical effects, including without limitation the following: (1) the combination of a ligand capable of binding to cell surface receptors and an endocytosis promoting molecule capable of mediating endocytosis allows the conjugated compound to specifically penetrate into target cells; (2) the conjugate compound or a pharmaceutically acceptable salt thereof enhances the affinity and specificity of targeting of drug compounds, thereby delivering highly effective chemotherapeutic agents such as MMAE to the patient, expanding the therapeutic window of such agents and avoiding side effects; (3) the linker can prevent the release of the payload outside the target cells (eg, into the circulatory system, intercellular substance, etc.), which ensures the stability of the conjugated compound while in the bloodstream and reduces the toxicity of the drug; wherein, upon entry into target cells, the linker is cleaved to release the payload and produce the intended drug effect, and the development of multidrug resistance (MDR) can be avoided; and (4) a wide range of drugs can be delivered in the form of a conjugate compound according to the present application, and, therefore, the fields of application of the corresponding drugs are expanded. Therefore, the conjugate compound or a pharmaceutically acceptable salt thereof according to the present application not only expands the targeting spectrum and therapeutic window of LDC-based drugs, but also reduces the toxicity and side effects of certain drugs.

As used herein, the terms “polypeptide,” “protein,” and “peptide” can refer to a single amino acid or a polymer of amino acids. The polypeptide, protein or peptide described herein may contain naturally occurring amino acids and non-naturally occurring amino acids or analogs and mimetics thereof. The polypeptide, protein, or peptide can be produced by any method well known in the art, such as, but not limited to, isolation and purification from natural materials, recombinant expression, chemical synthesis, etc.

In another aspect, this application discloses a pharmaceutical composition comprising a conjugate compound or a pharmaceutically acceptable salt thereof provided herein and a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable" means, within the bounds of reasonable medical judgment, suitable for contact with cells of humans and other animals without undue toxicity, irritation, allergic reaction, etc. and commensurate with a reasonable benefit/risk ratio.

As used herein, the term “pharmaceutically acceptable salt” refers to the relatively non-toxic inorganic and organic acid addition salts and base addition salts of the conjugate compound of this application. Exemplary acid addition salts include hydrobromides, hydrochlorides, sulfates, bisulfates, phosphates, nitrates, acetates, oxalates, valerates, oleates, palmitates, stearates, laurates, borates, benzoates, lactates, phosphates, tosylates, citrates, maleates, fumarates, succinates, tartrates , naphthylates, mesylates, glucoheptonates, lacthiobionates, sulfamates, malonates, salicylates, propionates, methylene bis- b -hydroxynaphthoates, gentisates, isethionates, di-p-toluoyl tartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, quinatesla cyclohexylsulfonates, uryl sulfonate salts and 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 derived from metal bases, which include, 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 that have sufficient basicity to form a stable salt and preferably include the following amines, which are often used in medicinal chemistry due to their low toxicity and acceptability for medical use: ammonia, ethylenediamine, N-methylglucamine, lysine, arginine , ornithine, choline, N, N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabiethylamine, N-ethylpiperidine, benzylamine, monium , tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids such as lysine and arginine, dicyclohexylamine and the like.

As used herein, the term “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable solvent, suspension, or any other pharmacologically inert carrier medium for delivering the conjugate compound provided herein to a subject without interfering with the structure and properties of the conjugate compound. Such carriers allow the conjugate compound to be formulated, for example, into tablets, pills, capsules, liquids, gels, syrups, pastes, suspensions and troches for oral administration by a subject. Such carriers allow the conjugate compound to be formulated as injections, infusions or topical preparations.

Pharmaceutically acceptable carriers for use in the pharmaceutical composition provided herein may include, without limitation, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous carrier vehicles (such as sodium chloride injection, Ringer's solution for injection, isotonic saline dextrose injection, sterile water for injection or lactated dextrose Ringer's solution for injection), non-aqueous carrier media (eg, fixed oils derived from plants, cottonseed oil, corn oil, sesame oil, or peanut oil), antimicrobial agents, isotonic agents (such as sodium chloride or dextrose), buffers (such as phosphate or citrate buffers), antioxidants (such as sodium bisulfate), anesthetics (such as procaine hydrochloride), suspensions/dispersions (such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone), chelating agents (such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycoltetraacetic acid)), emulsifiers (such as polysorbate 80 (Tween-80)), diluents, auxiliaries, excipients or non-toxic auxiliary materials, other components well known in the art areas, or various combinations thereof. Suitable components may include, for example, fillers, binders, buffers, preservatives, lubricants, flavoring agents, thickeners, coloring agents or emulsifiers.

In some embodiments, the pharmaceutical composition is an injectable preparation. Injectable preparations include sterile aqueous solutions or dispersions, suspensions or emulsions. In all cases, injectable preparations must be sterile and in liquid form to ensure ease of administration. They must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. Carriers can be solvents or dispersion media containing, for example, water, ethanol, polyol (eg glycerin, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof and/or vegetable oils. Injectable preparations must maintain adequate fluidity. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by the use of surfactants, etc. Prevention of the action of microorganisms can be achieved using various antibacterial and antifungal agents, for example parabens, chlorobutanol, phenol, sorbic acid, thimerosal and so on.

In some embodiments, the pharmaceutical composition is an oral preparation. Oral preparations include, but are not limited to, capsules, starches, pills, tablets, lozenges (using a flavored base, usually sucrose and gum acacia or tragacanth), powders, granules, or presented as solutions or suspensions in aqueous or non-aqueous liquids , or present in the form of liquid oil-in-water or water-in-oil emulsions, or presented in the form of elixirs or syrups, or in the form of lozenges (using an inert base such as gelatin and glycerin or sucrose and gum acacia) and/or in the form of mouthwashes.

In solid dosage forms 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) fillers or diluents such as starches, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders such as carboxymethylcellulose, alginate varieties, gelatin varieties, polyvinylpyrrolidone, sucrose and/or gum acacia; (3) humectants such as glycerin; (4) disintegration agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) dissolution retarders such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as acetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof; and (10) coloring matter.

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, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, isopropanol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan fatty acid esters and mixtures thereof. In addition to inert diluents, the oral composition may also include auxiliary agents such as wetting agents, emulsifiers and suspensions, sweeteners, flavoring agents, coloring agents, flavoring agents and preservatives.

In some embodiments, the pharmaceutical composition is an oral spray formulation or a nasal spray formulation. Spray formulations include, but are not limited to, aqueous aerosols, non-aqueous suspensions, liposomal formulations, or solid granular formulations. Aqueous aerosols are prepared by mixing aqueous solutions or suspensions of agents with traditional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary depending on the requirements for specific compounds, but in general they include nonionic surfactants (Tween varieties or polyethylene glycol), oleic acid, lecithin, amino acids such as glycine, buffer solution, salts, sugar or sugar alcohol. Aerosols are usually prepared from isotonic solutions and can be delivered using nebulizers.

In some embodiments, the pharmaceutical composition can be used by mixing with one or more other drugs. In some embodiments, the pharmaceutical composition contains at least one other drug. In some embodiments, the other drugs are antineoplastic drugs, cardiovascular drugs, anti-inflammatory drugs, antiviral drugs, digestive drugs, nervous system drugs, respiratory drugs, immune system, dermatological drugs, metabolic drugs and the like.

In some embodiments, the pharmaceutical compositions can be administered to a subject in need thereof by suitable routes, including, but not limited to, oral, injection (such as intravenous, intramuscular, subcutaneous, intradermal, intracardiac, intrathecal, intrapleural and intraperitoneal injection), mucosal (such as nasal and intraoral), sublingual, rectal, transdermal, intraocular and pulmonary administration. In some embodiments, the pharmaceutical composition can be administered intravenously, subcutaneously, orally, intramuscularly, or intraventricularly.

Due to the properties of some payloads, such as high toxicity and high hydrophilicity, it is desirable to deliver payloads to a subject in need in a more specific and more efficient manner. For example, in the treatment of cancer, it is desirable to specifically deliver chemotherapeutic agents to cancer cells without toxicity to normal cells. Therefore, in another aspect, this application discloses a method of delivering a payload to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate compound or a pharmaceutically acceptable salt thereof provided herein or a pharmaceutical composition provided herein. The payloads described herein can be any pharmaceutical agent that produces biological or therapeutic responses in a tissue, system, animal, individual or human that a researcher, veterinarian, physician or other clinician seeks to achieve by preventing, suppressing, reducing the intensity or treating the disease.

As used herein, the term “object” refers to human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammalian animals. The subject may also be a livestock animal such as cattle, pigs, sheep, poultry and horses, or domestic animals such as dogs and cats. The subject may be male (eg, man) or female (eg, woman), may be elderly, and may be an adult, adolescent, child, or infant. A person may be of Caucasian, African, Asian, Semitic descent, or a person with other racial characteristics or a combination of such racial characteristics.

As used herein, the term “therapeutically effective amount” refers to an amount of a conjugate compound or a pharmaceutically acceptable salt or pharmaceutical composition thereof that alleviates to some extent one or more symptoms of a disease or disorder in a subject, partially or completely restores to normal one or more physiological or biochemical parameters associated with or causing a disease or disorder, and/or reduces the likelihood of onset of the disease or disorder. Such amounts will generally vary depending on a number of factors, which can be determined and explained to the extent of the disclosure provided herein by those skilled in the art. These factors include, without limitation, the individual subject and his age, weight, height, general physical condition and medical history, the particular compound used, the drug carrier and route of administration chosen, as well as the nature and severity of the condition being treated.

In some embodiments, the amount of the conjugate compound or a pharmaceutically acceptable salt or pharmaceutical composition thereof is sufficient to suppress a disease or disorder in a subject or to prophylactically suppress or prevent the onset of a disease or disorder in a subject. Although the therapeutically effective amount may vary between subjects, it is generally in the range of 0.01 to 100 mg/kg, for example, 0.01 to 90 mg/kg, 0.01 to 80 mg/kg, 0. 01 to 70 mg/kg, from 0.01 to 60 mg/kg, from 0.01 to 50 mg/kg, from 0.01 to 40 mg/kg, from 0.01 to 30 mg/kg, from 0, 01 to 20 mg/kg, from 0.01 to 10 mg/kg, from 0.01 to 5 mg/kg, from 0.01 to 4 mg/kg, from 0.01 to 3 mg/kg, from 0. 01 to 2 mg/kg, 0.01 to 1 mg/kg and 0.01 to 0.1 mg/kg. A therapeutically effective amount as described herein may be any value within the above numerical range, including the endpoints of that range.

In another aspect, this application discloses a method of delivering a payload to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate compound or a pharmaceutically acceptable salt thereof provided herein or a pharmaceutical composition provided herein.

In another aspect, this application discloses a method of treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of a conjugate compound or a pharmaceutically acceptable salt thereof provided herein or a pharmaceutical composition provided herein.

In some embodiments, the disease is cancer, including, without limitation, prostate cancer, breast cancer, lung cancer, kidney cancer, leukemia, ovarian cancer, gastrointestinal cancer, uterine cancer, endometrial carcinoma, liver cancer, thyroid cancer, pancreatic cancer, colon cancer, colorectal cancer, esophageal cancer, skin cancer, lymphoma and multiple myeloma.

In some embodiments, cancer cells of cancers are characterized by the expression of cell surface receptors or antigens mentioned herein. In some embodiments, the cancer cells of the cancers have a high level of expression (e.g., according to Depmap (see: https://depmap.org/portal/), the expression of the corresponding gene 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) cell surface receptors or antigens mentioned in this application. In some embodiments, the cancer cells of the cancers have high levels of 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 system disease, such as an autoimmune disease, including, but not limited to, connective tissue disease, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

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, arrhythmia, and congenital heart disease.

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

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.

In some embodiments, the method provided herein further comprises administering one or more therapeutic agents in combination with a conjugate compound or a pharmaceutically acceptable salt or pharmaceutical composition thereof. In some embodiments, the therapeutic agents target an anticancer therapeutic target, induce or enhance an immune response against cancer, or are chemotherapeutic agents.

The present application will be described in more detail with the help of specific examples. The following examples are offered for illustrative purposes only and are not intended to limit the present invention in any way. One skilled in the art will readily recognize many non-critical parameters that can be changed or modified to obtain substantially the same results.

EXAMPLES

The following examples are intended to further illustrate the present application. The advantages and features of the present application will become clear from the description. However, these illustrative materials are presented by way of example only and should not be construed as limiting the scope of this application.

Example 1: Preparation of conjugated compounds

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

1. 10 g of Rink Amide-AM resin (hereinafter referred to as “Rink resin”, from Sunresin New Materials Co. Ltd., Cat. No. 183599-10-2) with a degree of substitution of 0.45 mmol/g was weighed and loaded onto the column for carrying out a solid-phase reaction; DCM was added and nitrogen gas was bubbled through the solvent to allow the resin to swell for 30 minutes; the solvent was pumped out; The Fmoc protecting groups on the resin were removed with DBLK, and then the resin was washed 5 times with DMF. 4.79 g (9 mmol) Fmoc-Lys(Dde)-OH and 1.47 g (10.8 mmol) HOBt were weighed and dissolved in DMF. To the above solution at 0°C in an ice bath, 1.67 ml (10.8 mmol) of DIC was added and stirred to ensure activation for 5 minutes. The solution was added to the above reaction column and reacted for 3 hours, and then the solvent was pumped out and the resin in the reaction column was washed 3 times. Fmoc protecting groups were then removed using DBLK.

2. The above operations were repeated and Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH and intermediate 108 were sequentially conjugated according to structures.

(Intermediate 108)

3. Dde-protecting groups were deprotected twice with 2% hydrazine hydrate/DMF, each time for 10 minutes, and then the resin was washed 5 times with DMF. Fmoc-Glu-OtBu and pteroic acid were conjugated sequentially. Finally, the resin was condensed twice with methanol and the solvent was pumped off to obtain 17.4 g of protected peptide resin.

4. 17.4 g of the peptide resin obtained in the previous step was added to a 250 ml one-neck flask; 139 ml of lysis buffer (TFA: H ₂ O: TIS=95: 3: 2 (volume ratio)) was previously prepared and 2.1 g of DTT was weighed and added to the lysis buffer. Lysis buffer was added to the above flask and the mixture was reacted at room temperature for 2.5 hours and filtered; the resin continued to be washed with 30 ml TFA; the above filtrate was combined and the mixture was added to 834 ml of absolute ether to precipitate a yellow solid and centrifuged to obtain a solid, which was washed with absolute ether and dried in vacuo to obtain 6.4 g of crude product as a yellow solid and yield 93.4% and 82.3% purity according to HPLC results. The product was separated by preparative HPLC (conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid in water, B: acetonitrile, elution gradient: (15-25)%B, elution time: 60 minutes; fractions were collected); and the fraction containing the corresponding product was lyophilized to obtain 4.73 g of 20BK-SM09 with a purity of 98.8%.

5. 4.09 g (3.11 mmol) Mc-Val-Cit-PAB-MMAE (LT1002) was weighed and added to a 1000 ml one-neck flask, and 500 ml of phosphate buffer and 100 ml of acetonitrile were added; the solution was stirred and maintained at pH=7.2 until a clear solution was obtained; and 4.73 g (3.11 mmol) of intermediate 20BK-SM09 was added and the mixture was allowed to react at room temperature for 2 hours, during which time the reaction was monitored by HPLC. After completion of the reaction, the mixture was filtered and the filtrate was separated using preparative HPLC (conditions: C18 column, mobile phase A: ammonium bicarbonate solution (pH=7.2), B: acetonitrile, elution gradient: (25-35)%B, elution time : 60 minutes; fractions were collected); the fraction containing the corresponding product was lyophilized to obtain 6.96 g of product CB-20BK with a purity of 98.8% and a yield of 78.8%.

In the same way, conjugate compounds CB-18G, CB-20B, CB-10S, CB-20C, FA-MMAE (with the structure shown below), CB-20AK (with the structure shown below), CB-1020, CB-1320 can be obtained and CB-1820, performing similar steps in the above methods.

(FA-MMAE)

(CB-20AK)

Synthesis of conjugated compounds CB-50S, CB-60S and CB-60SK

1. 10 g of Wang resin (from Sunresin New Materials Co. Ltd., Cat. No. 1365700-43-1) with a degree of substitution of 1.1 mmol/g was weighed and loaded into a solid-phase reaction column; DMF was added and nitrogen gas was bubbled through the solvent to allow the resin to swell for 30 minutes; 14.3 g (22 mmol) Fmoc-Arg(pbf)-OH, 3.56 g (26.4 mmol) HOBt and 0.27 g (2.2 mmol) DMAP were weighed and dissolved in DMF; at 0°C in an ice bath, add 4.1 ml (26.4 mmol) DIC and stir to activate for 5 minutes; the solution was added to the reaction column and reacted for 3 hours, and then the solvent was pumped out and the resin was washed 3 times.

2. 10.4 ml of acetic anhydride and 8.9 ml of pyridine were dissolved in 50 ml of DMF and mixed, and after washing in the above steps, the resin was added; the mixture was blocked at room temperature for 5 hours and washed three times with DMF; and the resin was condensed with methanol, and then the solvent was pumped off to obtain Fmoc-Arg(pbf)-Wang resin, the degree of substitution of which was determined to be 0.53 mmol/g.

3. 3.8 g (2 mmol) of Fmoc-Arg(pbf)-Wang resin (degree of substitution = 0.53 mmol/g) was weighed and loaded into the reaction column; the resin was washed 3 times with DMF and then allowed to swell for 30 minutes, which was achieved by adding DMF. The Fmoc protecting groups were then removed with DBLK and the resin was washed 6 times with DMF. 2.0 g (6 mmol) Fmoc-Pro-OH and 0.97 g (7.2 mmol) HOBt were weighed and dissolved in DMF; at 0°C in an ice bath, add 1.1 ml (7.2 mmol) DIC and stir to activate for 5 minutes; the mixture was added to the reaction column and reacted for 2 hours; and then the Fmoc protecting groups were removed using DBLK.

4. The above operations were repeated for 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 were conjugated sequentially according to the structures. The resin was condensed twice with methanol and the solvent was pumped off to obtain 8.4 g of peptide resin.

5. 8.4 g of the peptide resin obtained in the previous step was added to a 250 ml one-neck flask; 67 ml of lysis buffer (TFA: H ₂ O: TIS=95: 3: 2 (volume ratio)) was previously prepared and 0.92 g of DTT was weighed and added to the lysis buffer. Lysis buffer was added to the flask and the mixture was reacted at room temperature for 2.5 hours; the resin was filtered and washed with 20 ml TFA; the filtrate was combined and the mixture was added to 402 ml of absolute ether to precipitate a yellow solid and centrifuged to give a solid which was washed with absolute ether and dried in vacuo to give 4.06 g of crude product as a yellow solid in a yield of 97 .3% and 84.6% purity according to HPLC results. The product was separated by preparative HPLC (conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid in water, B: acetonitrile, elution gradient: (20-29)%B, elution time: 60 minutes; fractions were collected); and the fraction containing the corresponding product was lyophilized to obtain 2.86 g of 50S-SM09 with a purity of 97.6%.

6. 1.29 g (1.37 mmol) LT1000N3 was weighed and added to a 500 ml one-neck flask, and 270 ml mixed solvent (CAN: H ₂ O=1:4) and 393 mg (2.74 mmol) CuBr were added and mixed. 2.86 g (1.37 mmol) of intermediate 50S-SM09 was added and the mixture was allowed to react at room temperature for 2-3 hours, during which time the reaction was monitored by HPLC. After completion of the reaction, the mixture was filtered and the filtrate was separated using preparative HPLC (conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid in water, B: acetonitrile, elution gradient: (22-40)%B, elution time: 60 minutes; fractions were collected); and the fraction containing the corresponding product was lyophilized to obtain 3.17 g of CB-50S with a purity of 98.6% and a yield of 76.4%.

In the same way, the conjugated compounds CB-60S and CB-60SK can be prepared by performing similar steps in the above methods.

Synthesis CB-20R

1. 5 g of Rink resin with a degree of substitution of 0.45 mmol/g was weighed and loaded into a solid-phase reaction column; DCM was added and nitrogen gas was bubbled through the solvent to allow the resin to swell for 30 minutes; the solvent was pumped out; The Fmoc protecting groups on the resin were removed with DBLK, and then the resin was washed 5 times with DMF. 2.4 g (4.5 mmol) Fmoc-Lys(Dde)-OH and 0.74 g (5.4 mmol) HOBt were weighed and dissolved in DMF; at 0°C in an ice bath, 0.84 ml (5.4 mmol) DIC was added and stirred to ensure activation for 5 minutes; the mixture was added to the reaction column and reacted for 3 hours; and the solvent was pumped out and the resin was washed 3 times. Fmoc protecting groups were then removed using DBLK.

2. The above operations were repeated and Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH and intermediate 108 were sequentially conjugated according to structures.

3. Dde-protecting groups were deprotected twice with 2% hydrazine hydrate/DMF, each time for 10 minutes, and then the resin was washed 5 times with DMF. DOTA-tris(tBu)-ester was conjugated. The Dde protecting groups were then deprotected twice with 2% hydrazine hydrate/DMF, each time for 10 minutes, and then the resin was washed 5 times with DMF. Fmoc-Glu-OtBu and pteroic acid were conjugated sequentially, and finally the resin was double-condensed with methanol and the solvent was pumped off to obtain 9.2 g of protected peptide resin.

4. 9.2 g of peptide resin obtained in the previous step was added to a 250 ml one-neck flask; 74 ml of lysis buffer (TFA: H ₂ O: TIS=95: 3: 2 (volume ratio)) was previously prepared and 1.05 g of DTT was weighed and added to the lysis buffer. Lysis buffer was added to the flask and the mixture was reacted at room temperature for 2.5 hours; the resin was filtered and washed with 20 ml TFA; the filtrate was combined and the mixture was added to 560 ml of absolute ether to precipitate a yellow solid and centrifuged to give a solid which was washed with absolute ether and dried in vacuo to give 3.8 g of crude product as a yellow solid in a yield of 87 .4% and 81.2% purity according to HPLC results. The product was separated by preparative HPLC (conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid in water, B: acetonitrile, elution gradient: (15-25)%B, elution time: 60 minutes; fractions were collected); and the fraction containing the synthesized product was lyophilized to obtain 2.9 g of 20R-SM09 with a purity of 97.8%.

5. Complexed 20R-SM09 with the radionuclide ion M to produce CB-20R. Specifically, radiolabeled ¹⁷⁷ Lu (approximately 50 MBq) was mixed with 100 μl of 0.5 M sodium acetate buffer (pH=5). 40 μl of an aqueous solution of 1 mM CB-20R dissolved in 10% DMSO, 2 μl of saturated ascorbic acid solution and 100 μl of a solution containing ¹⁷⁷ Lu were mixed, and the mixture was heated to 95°C for 10 minutes. The label was detected by radio-HPLC (5 minutes; 0%-100% ACN in water; C18 column).

Synthesis of compound CR19425

1. Under _N2 protection, 441.4 mg of CR19420 (with the structure shown for the above reaction step) and 8.0 ml of DMF were added to the reaction flask, stirred, dissolved and cooled in an ice bath; then 459.2 mg of HATU and 380 μl of DIPEA were added and stirred for half an hour; and then 500.0 mg of CR19419 (with the structure shown for the above reaction step) and 190 μl of DIPEA were added, and the mixture was reacted at room temperature until the reaction was completed. After completion of the reaction, the reaction solution was poured into an aqueous solution of acetic acid; the solid was precipitated and filtered, and the filter cake was washed with aqueous acetic acid and water and dried in vacuo to obtain 835.7 mg of CR19421 (with the structure shown for the above reaction step) as a brown powder and with a purity of 90.0% according to HPLC results and a yield of 90.6%.

2. Under protection No. ₂ , 835.7 mg of CR19421 and 16 ml of 10% piperidine in DMF were added to the reaction flask and the reaction was carried out at room temperature for half an hour. After completion of the reaction, the reaction solution was poured into TFA/MTBE; the solid was precipitated and filtered, and the filter cake was washed with MTBE and dried in vacuo to obtain 599.7 mg of CR19422 (with the structure shown for the above reaction step) as a gray-green powder with a purity of 84.7% according to the results HPLC and yield 83.0%.

3. Under _N2 protection, 599.7 mg CR19422, 586.7 mg CR19424 (with the structure shown for the above reaction step) and 15 ml DMF were added to the reaction flask, stirred, dissolved and cooled in an ice bath; then 495.7 mg of HATU and 410 μl of DIPEA were added and the reaction was carried out at room temperature. After completion of the reaction, the reaction solution was subjected to preparative purification and acetonitrile was removed under reduced pressure from the pure product solution; the residue was extracted with a mixed solvent of dichloromethane and methanol, concentrated and dried to obtain 674.9 mg of CR19423 (with the structure shown for the above reaction step) as a yellow powder and with a purity of 88.4% by HPLC and a yield of 63.1 %.

4. Under protection No. ₂ , 14.8 mg of CR19423, 3.0 ml of PBS buffer (pH=6.6) and 3.0 ml of acetonitrile were added to the reaction flask, mixed and dissolved; then 32.9 mg of CBSM09 was added and the mixture was adjusted to pH 6.6-6.8 with Na ₂ HPO ₄ and reacted for half an hour. After completion of the reaction, the reaction solution was preparatively purified and the pure product was lyophilized to obtain 21.8 mg of CR19425 as a yellow powder with a purity of 95.6% by HPLC and a yield of 48.8%.

Synthesis of compound CR19426

1. Under protection No. ₂ , 25.2 mg of CR19423, 3.0 ml of PBS buffer (pH=6.6) and 3.0 ml of acetonitrile were added to the reaction flask, mixed and dissolved; then 55.5 mg of 18G-SM09 was added and the mixture was adjusted to pH 6.6-6.8 with Na ₂ HPO ₄ and reacted for half an hour. After completion of the reaction, the reaction solution was preparatively purified and the pure product was lyophilized to obtain 44.6 mg of CR19426 as a yellow powder with a purity of 95.4% by HPLC and a yield of 55.2%.

Synthesis of compound CR19428

1. Under protection No. ₂ , 486 mg of CR19423, 3.0 ml of PBS buffer (pH=6.6) and 3.0 ml of acetonitrile were added to the reaction flask, mixed and dissolved; then 712 mg of 20BK-SM09 was added and the mixture was adjusted to pH 6.6-6.8 with Na ₂ HPO ₄ and reacted for half an hour. After completion of the reaction, the reaction solution was preparatively purified and the pure product was lyophilized to obtain 508 mg of CR19428 as a yellow powder with a purity of 96.7% by HPLC and a yield of 42.3%.

Example 2. Determination of the affinity of conjugated compounds towards target proteins

1. Determination of the binding affinity of CB-20BK to the FOLR1 protein

Instruments, materials and reagents for the experiment

BIAcore T200 (GE)

Chip CM5 (GE, cat. no. 29104988)

Buffer: 10X HBS-EP+ Buffer (GE Cat. No. BR100669), diluted 10X with deionized water before use.

Amino immobilization kit (GE, cat. no. BR100050)

Regeneration reagents: 10 mM glycine 2.0 (GE, cat. no. BR100355)

10 mM glycine 3.0 (GE, cat. no. BR100357)

Stages of the experiment

The experiment was performed according to the instructions for BIAcore T200 (GE) to determine the affinity of the analytes CB-20BK, CB-20AK, folate (FA) and FA-MMAE for FOLR1. During the experiment, the CM5 chip was conjugated with a ligand to FOLR1 (R&D System, cat. no. 5646-FR). The experiment results are shown in Table 4.

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

ka kd KD FA (folate) 3.34 × 10 ⁶ M ^-1 s ^-1 2.26 × 10 ^-4 s ^-1 6.77 × 10 ^-11 M FA-MMAE 1.79 × 10 ⁶ M ^-1 s ^-1 1.15 × 10 ^-4 s ^-1 6.43 × 10 ^-11 M CB-20AK N/D N/D N/D CB-20BK 1.03 × 10 ⁶ M ^-1 s ^-1 1.31 × 10 ^-4 s ^-1 1.27 × 10 ^-10 M

"N/D" means that no specific binding was detected.

Table 4 shows that CB-20BK specifically binds to FOLR1 with high affinity. The binding affinity of CB-20BK to FOLR1 is slightly weaker than the binding affinity of FA or FA-MMAE to FOLR1. CB-20AK does not contain the folate moiety of CB-20BK and was not experimentally found to specifically bind to FOLR1.

2. Determination of the binding affinity of CB-20BK to the FOLH1 protein

Instruments, materials and reagents for the experiment

^GatorTM (Probe Life)

SA probe (Probe Life, cat. no. 1906018)

Buffer: Buffer Q (Probe Life), 10 mM, pH=7.4

Stages of the experiment

(1) Synthesis steps of biotin-CB-20BK analyte

1) 5.1 g of Rink resin with a degree of substitution of 0.45 mmol/g was weighed and loaded into a solid-phase reaction column; add DCM and bubble nitrogen gas to allow resin to swell for 30 minutes; the solvent was pumped out; Fmoc protecting groups were removed with DBLK and then the resin was washed 5 times with DMF. 2.45 g of Fmoc-Lys(Dde)-OH and 0.75 g of HOBt were weighed and dissolved in DMF; at 0°C in an ice bath, 0.83 ml of DIC was added to ensure activation for 5 minutes; the mixture was added to the reaction column and reacted for 3 hours; and the solvent was pumped out and the resin was washed 3 times. Fmoc protecting groups were then removed using DBLK.

2) The above operations were repeated and Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH and intermediate 108 were sequentially conjugated according to structures.

3) Dde-protecting groups were deprotected twice with 2% hydrazine hydrate/DMF, each time for 10 minutes, and then the resin was washed 5 times with DMF. Fmoc-Lys(biotin)-OH, Fmoc-Glu-OtBu and pteroic acid were conjugated sequentially, and after conjugation of pteroic acid, the resin was washed twice with DMF; and finally, the resin was condensed with methanol and the solvent was pumped off to obtain 9.7 g of protected peptide resin.

4) 9.7 g of peptide resin obtained in the previous step was added to a 250 ml one-neck flask; 77.6 ml of lysis buffer (TFA: H2O: TIS=95: 3: 2 (volume ratio)) was previously prepared and 1.1 g of DTT was weighed and added to the lysis buffer. Lysis buffer was added to the flask and the mixture was reacted at room temperature for 2.5 hours; the resin was filtered and washed with 20 ml TFA; the filtrate was combined and the mixture was added to 466 ml of methyl tert-butyl ether to precipitate a yellow solid and centrifuged to obtain a solid, which was washed with methyl tert-butyl ether and dried in vacuo to obtain 4.6 g of a yellow solid with a purity of 83.2% according to HPLC results. The product was separated by preparative HPLC and lyophilized to yield 2.1 g of biotin-20BK-SM09 with a purity of at least 95%.

5) 500 mg of Mc-Val-Cit-PAB-MMAE (LT1002) was weighed and added to a 250 ml one-neck flask, and 65 ml of phosphate buffer and 20 ml of acetonitrile were added; the solution was stirred and maintained at pH=7.2 until a clear solution was obtained; and 785 mg of intermediate biotin-20BK-SM09 was added and the mixture was allowed to react at room temperature for 2 hours, during which time the reaction was monitored by HPLC. After completion of the reaction, the mixture was filtered, separated by preparative HPLC and lyophilized to obtain 723 mg of biotin-CB-20BK with a purity of 96.8% and a yield of 59.4%.

(2) The experiment was performed according to the instructions for Gator ^TM (Probe Life) to determine the binding affinity of biotin-CB-20BK to FOLH1. In the experiment, the SA probe binds to the ligand biotin-CB-20BK, and the analyte is FOLH1 (Sino Biologicals, cat. no. 15877-H07H). The experiment results are shown in Table 5.

Table 5. Binding affinity of CB-20BK to FOLH1

ka kd KD FOLH1 1.19 × 10 ⁴ M ^-1 s ^-1 1.70 × 10 ^-4 s ^-1 6.98 × 10 ^-9 M

Table 5 shows that CB-20BK binds to FOLH1 with high affinity.

3. FOLH1 associates with the CB-20BK-FOLR1 complex

Materials and reagents for the experiment

BIAcore T200 (GE)

Chip CM5 (GE, cat. no. 29104988)

Buffer: 10X HBS-EP+ Buffer (GE Cat. No. BR100669), diluted 10X with deionized water before use.

Amino immobilization kit (GE, cat. no. BR100050)

Regeneration reagents: 10 mM glycine 2.0 (GE, cat. no. BR100355)

10 mM glycine 3.0 (GE, cat. no. BR100357)

Stages of the experiment

The CM5 chip was conjugated to a ligand, FOLR1 (R&D System, cat. no. 5646-FR).

Analyte: CB-20BK, FA-MMAE and FOLH1 (Sino Biologicals, Cat. No. 15877-H07H).

According to the BIAcore T200 (GE) instruction manual, FOLR1 was conjugated to the CM5 chip; where CB-20BK or FA-MMAE was loaded first and then FOLH1 was loaded; and detection of FOLH1 binding to the CB-20BK-FOLR1 complex was performed. The experiment results are shown in Table 6.

Table 6. Binding of the CB-20BK-FOLR1 complex to FOLH1 solution at various concentrations

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

Table 6 shows that for the high concentration FOLH1 solution, the amount of FOLH1 bound to the CB-20BK-FOLR1 complex is significantly higher than the amount of FOLH1 bound to the FA-MMAE-FOLR1 complex, showing a continuous increasing trend. FOLH1 binding to the FA-MMAE-FOLR1 complex tends to saturate at high concentrations. This experiment confirmed that CB-20BK can bind to the FOLR1 and FOLH1 receptors with high affinity.

Example 3. Study of binding and endocytosis of ligand conjugates by target cells

1. Experiment on the binding and endocytosis of the conjugated compound CB-20BK by cells

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

(1) 2 g of Rink resin with a degree of substitution of 0.45 mmol/g was weighed and loaded onto a solid-phase reaction column; DCM was added and nitrogen gas was bubbled through the solvent to allow the resin to swell for 30 minutes; the solvent was pumped out; The Fmoc protecting groups were removed with DBLK and then the resin was washed 5 times with DMF. 0.96 g (1.8 mmol) Fmoc-Lys(Dde)-OH and 0.3 g (2.2 mmol) HOBt were weighed and dissolved in DMF; at 0°C in an ice bath, add 0.33 ml (2.2 mmol) DIC and stir to activate for 5 minutes; the mixture was added to the reaction column and reacted for 3 hours; and the solvent was pumped out and the resin was washed 3 times. Fmoc protecting groups were then removed using DBLK.

(2) The above operations were repeated and Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH and intermediate 108 were sequentially conjugated according to the structures:

(3) Dde-protecting groups were deprotected twice with 2% hydrazine hydrate/DMF, each time for 10 minutes, and then the resin was washed 5 times with DMF. Fmoc-Lys(Dde)-OH, Fmoc-Glu-OtBu and pteroic acid were conjugated sequentially.

(4) Dde-protecting groups were deprotected twice with 2% hydrazine hydrate/DMF, each time for 10 minutes, and then the resin was washed 5 times with DMF; the resin was conjugated with Cy5-COOH and then washed twice with DMF; and finally, the resin was condensed twice with methanol and the solvent was pumped off to obtain 3.6 g of protected peptide resin.

(5) 3.6 g of the peptide resin obtained in the previous step was added to a 50 ml one-neck flask; 29 ml of lysis buffer (TFA: H ₂ O: TIS=95: 3: 2 (volume ratio)) was previously prepared and 0.4 g of DTT was weighed and added to the lysis buffer. Lysis buffer was added to the flask and the mixture was reacted at room temperature for 2.5 hours; the resin was filtered and washed with 8 ml TFA; the filtrate was combined and the mixture was added to 173 ml of absolute ether to precipitate a yellow solid and centrifuged to give a solid which was washed with absolute ether and dried in vacuo to give 1.9 g of crude product as a blue solid in a yield of 89 .6% and 76.3% purity according to HPLC results. The product was separated by preparative HPLC (conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid in water, B: acetonitrile, elution gradient: (20-28)%B, elution time: 50 minutes; fractions were collected); and the fraction containing the corresponding product was lyophilized to obtain 736 mg of Cy5-pep-20BK with a purity of 93.4%.

In the same way, the compounds Cy5-FA and Cy5-pep-20AK can be prepared by performing similar steps in the above methods.

(Cy5-pep-20BK)

(Cy5-pep-20AK)

(Cy5-FA)

Experiment to detect binding and endocytosis by cells for Cy5-pep-20AK/Cy5-pep-20BK samples by flow cytometry

Sample information: Cy5-pep-20AK and Cy5-pep-20BK.

Cell lines: LNCaP human prostate cancer cells, Du145 human prostate cancer cells, SKOV3 human ovarian cancer cells, and NCI-H460 human lung cancer cells.

Basic reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and PBS.

Experimental procedures

(1) Cell culture: cells were trypsinized, collected and counted, and then the cells were added to several culture flasks containing complete medium; The culture flasks were placed in a 37°C, 5% CO ₂ incubator to culture the cells, and finally approximately 5 × 10 ⁶ cells were collected into a centrifuge tube.

(2) Sample incubation: Cy5-pep-20AK/Cy5-pep-20BK samples were diluted to 8 nmol/L with PBS; the cell suspension was centrifuged at 1000 rpm for 5 minutes; the supernatant was removed and the residue was washed once with PBS; the cells were evenly suspended and their numbers were equally divided into different centrifuge tubes according to the experimental design; PBS was removed by centrifugation; 200 μL of sample working solution was added to each tube and incubated at 37°C for 15, 30, 60 and 90 minutes, respectively, and 1 tube to which only PBS was added was used as a blank control; Moreover, all operations were performed without exposure to light.

(3) Washing and loading: the working solution was removed by centrifugation at 1000 rpm for 5 minutes, and the cells were washed 3 times with PBS, and an appropriate amount of PBS was added to suspend the cells to 1 × 10 ⁶ cells/ml; the Beckman CytoFLEX flow cytometer was turned on in advance to complete the initial purification process; cell samples were loaded sequentially, and the fluorescence of 10,000 live cells was read in the APC channel; Moreover, all operations were performed without exposure to light.

(4) Data analysis: The absolute MFI (mean fluorescence intensity) value of APC fluorescence of each cell sample was obtained, as well as the relative MFI value compared with the blank control, and a line graph of the data was plotted according to the relative MFI value.

Results and analysis

Table 7. Expression levels of FOLR1 and FOLH1 in different cell lines

(with reference to Depmap data, https://depmap.org/portal/)

RNA-Seq LNCaP SKOV3 Du145 NCI-H460 FOLR1 +/- 6+ +/- +/- FOLH1 10+ + +/- +/-

According to Depmap, data corresponding to 0 or negative result is expressed as "-", data representing 0.001-0.499 is expressed as "+/-", data representing 0.500-1.499 is expressed as "+", data representing 1.500 -2.499, expressed as "2+", and so on.

The fluorescence intensity of the cells was determined after 15 minutes, 30 minutes, 60 minutes and 90 minutes of incubation, respectively, and the results were plotted and shown in FIG. 2A and 2B. Binding and endocytosis of the Cy5-pep-20BK sample by cells can be seen in the figures. Compared with the DU145 cell line with relatively low FOLR1 expression and the NCI-H460 cell line with relatively low FOLH1 expression, the LNCaP cell line with relatively high FOLR1 expression and the SKOV3 cell line with relatively high FOLH1 expression are characterized by significantly higher CB fluorescence intensity -20BK, which was bound and endocytosed. Since Cy5-pep-20AK contains only a ligand for FOLH1 and does not contain a ligand for FOLR1, which is FA, Cy5-pep-20AK shows high levels of binding and endocytosis in LNCaP cells with high levels of FOLH1 expression and shows moderate levels of binding and endocytosis in the case of SKOV3 cells with a moderate level of FOLH1 expression; in these two cell lines, the level of binding and endocytosis for Cy5-pep-20AK was significantly lower than for Cy5-pep-CB-20BK. The extent of ligand conjugate binding and internalization by cells is positively correlated with cellular receptor expression levels.

2. Experiment on binding and endocytosis of single-ligand Cy5-FA conjugate by cells

Sample Information: Cy5-FA

Cell lines: Hela cervical cancer cells, SKOV3 human ovarian cancer cells and A549 human lung cancer cells.

Basic reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, PBS and anti-fluorescence quenching medium containing DAPI.

Experimental procedures

(1) Cells growing on coverslips: coverslips were placed in a 24-well plate; cells were trypsinized, collected and counted, and then cells were diluted with complete cell culture medium to approximately 2 × 10 ⁵ cells/ml; 500 μL of diluted cell solution was added to each well of a 24-well plate; and the plate was placed in an incubator at 37°C, 5% CO ₂ for 48 hours for culture.

(2) Sample incubation: Cy5-FA sample was diluted in IMDM medium to 73 nmol/L; the culture medium in the 24-well plate was removed; 200 μL of sample working solution was added to the coverslips containing cells in each well and incubated at 37°C for 15, 30, and 60 minutes, respectively, and 1 well containing only IMDM medium was used as a blank control; Moreover, all operations were performed without exposure to light.

(3) Wash and pour: the working solution in the 24-well plate was removed and the plate was washed 3 times with pre-warmed PBS at 37°C; the cell coverslips were removed and 5 μl of histological anti-fluorescence quenching medium containing DAPI was added dropwise to the slides and then covered with the cell coverslips to complete the embedding; Moreover, all operations were performed without exposure to light.

(4) Interpretation of images for samples. A Leica DM2500 fluorescence microscope was turned on to preheat the fluorescence exciter for 15 minutes; At one position, cell nuclei and Cy5 fluorescein were photographed in the corresponding fluorescence channel, and image merging was completed using software.

Cellular binding and endocytosis of the Cy5-FA sample at 15 minutes and 30 minutes can be seen in FIG. 3, and the fluorescence intensity in the Hela and SKOV-3 cell lines with a high level of FOLR1 expression is significantly higher than in the A549 cell line with a relatively low level of FOLR1 expression. The extent of ligand conjugate binding and internalization by cells is positively correlated with cellular receptor expression levels.

Example 4. Experiment to suppress cell proliferation in the presence of conjugated compounds

1. Experiment to suppress tumor cell proliferation in the presence of CB-20BK

Sample information: CB-20BK.

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 liver cancer cells and LNCaP human prostate cancer cells .

Basic reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8.

Experimental procedures

1) Cell seeding

Cells were prepared in advance, trypsinized, collected, and counted; using complete cell culture medium, KB cells were diluted to 2.5 × 10 ⁴ cells/ml, T-47D cells were diluted to 1.3 × 10 ⁴ cells/ml, 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, and LNCaP cells were diluted to 8.0 × 10 ⁴ cells/ml; 100 μL of diluted cell solution was added to each well of 96-well plates; and negative control and blank control wells were prepared for each plate. 96-well plates with added cells were placed in a 37°C, 5% CO ₂ incubator for overnight culture.

2) Dilution and addition of samples

Samples were diluted with culture medium to the required concentrations (see Table 8). 50 μl of diluted samples was added to each well of 96-well plates that were cultured overnight, providing 3 replicates per well; prepared negative controls and blank controls; and the plates were placed in an incubator at 37°C, 5% CO ₂ for 72 hours for cultivation.

Table 8. Sample concentration (CB-20BK) after dilution

Tube number 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

3) Development of staining and reading of the tablet

15 μl (10% of the liquid volume in the well) of a color development solution from CCK-8 was added to each well; the plates were incubated at 37°C for an appropriate time (trying to maintain the OD value in the range of 1.0-2.0); The lids of the 96-well plates were removed and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).

4) Data processing

Data were edited using SoftMax Pro and a curve was generated using the four-parameter fitting method.

5) Experimental results and their analysis

According to the curve plotted by the four-parameter fitting method (Fig. 4A, where the C value corresponds to IC ₅₀ ), CB-20BK has an inhibitory effect on the growth and proliferation of tumor cell lines KB, T-47D, NCI-H460, CALU-3 , HuH-7 and LNCaP. Since the expression levels of the respective conjugate receptors are different in cells, the levels of inhibition vary. The IC ₅₀ for the T-47D, KB, LNCaP, HuH-7 and CALU-3 cell lines with relatively high levels of receptor expression is significantly lower than for the NCI-H460 cell line with relatively low levels of receptor expression. CB-20BK demonstrates a correlation between tumor suppressive effects and cellular receptor expression levels.

2. Experiment to suppress the proliferation of tumor cell lines LNCaP (human prostate cancer cells) and 22RV1 (human prostate cancer cells) with the conjugated compound CB-20BK and related compounds

Because different cells have different sensitivities to the cell proliferation-inhibiting toxicity of the ligand conjugate payload, quantitative assay results can sometimes be biased. A series of cell lines with known levels of receptor expression of the various ligands were selected and tested for response to the cell proliferation-inhibitory effects of the ligand conjugate and the individual payload. The IC ₅₀ ratio for a single payload and for a ligand conjugate in the same cell line was used to eliminate such biases in the results.

Related samples: MMAE, CB-20BK, CB-20AK and FA-MMAE.

Experimental Procedures LNCaP and 22RV1 cells were prepared in advance, counted, and seeded in 96-well cell culture plates at cell densities of 4 x ¹⁰⁴ cells/mL and 2.0 x ¹⁰⁴ cells/mL, respectively (100 μL/well). ; negative control and blank control wells were prepared for each plate. After culturing the adherent cells overnight, diluted samples to be tested were added at 50 μL/well. The plates were placed in an incubator at 37°C, 5% CO ₂ and cultured for 68-72 hours. 15 μl (10% of the liquid volume in the well) of a color development solution from CCK8 was added to each well; and the plates were incubated at 37°C for 45-70 minutes, and each plate was read at 450 nm on a microplate reader. Data were edited using SoftMax Pro and a curve was generated using the 4-parameter fitting method. The calculated data are presented in Table 9.

Table 9. Suppressive effects of MMAE, FA-MMAE, CB-20BK and CB-20AK on cell line proliferation and receptor gene expression levels in cell lines (with reference to Depmap data)

Cell line FOLR1 gene expression level FOLH1 gene expression level IC ₅₀ (MMAE) IC ₅₀ (FA-MMAE) IC ₅₀ (CB-20AK) IC ₅₀ (CB-20BK) IC ₅₀ ratio (MMAE/FA-MMAE) IC ₅₀ ratio (MMAE/CB-20AK) IC ₅₀ ratio (MMAE/CB-20BK) LNCaP +/- 10+ 0.00255 0.264 0.102 0.0958 0.010 0.025 0.0267 22RV1 +/- 6+ 0.00648 1.01 1.05 0.502 0.006 0.006 0.013

From the above table, it can be seen that there is not much difference in the expression level of FOLR1 between LNCaP and 22RV1, and the expression level of FOLH1 in the case of LNCaP is significantly higher than the expression level of FOLH1 in the case of 22RV1. CB-20BK (with FOLH1 ligand and FOLR1 ligand) and CB-20AK (with only FOLH1 ligand) have suppressive effects on the proliferation of both LNCaP and 22RV1 tumor cell lines. The suppressive effects of CB-20BK and CB-20AK on cells with different levels of FOLH1 receptor expression differ. The IC ₅₀ ratio for the LNCaP cell line with a relatively high level of expression of the FOLH1 receptor is 2-4 times higher than the IC ₅₀ ratio for the 22RV1 cell line with a relatively low level of expression of this receptor. FA-MMAE also has a suppressive effect on the proliferation of LNCaP and 22RV1 tumor cell lines. Since the expression level of FOLR1 in LNCaP and 22RV1 is very low, the expression in LNCaP cells (FOLR1 gene expression level is 0.3219 with reference to Depmap data) is slightly higher than in 22RV1 (FOLR1 gene expression level is 0.0704 with reference to Depmap data), and the IC ₅₀ ratio for FA-MMAE between LNCaP and 22RV1 differs only by a factor of 1.5. The above data indicate that the inhibitory effects on cell proliferation are positively correlated with the expression levels of cell-expressed receptors FOLH1 and FOLR1.

3. Experiment to suppress the proliferation of tumor cell lines PANC-1 (human pancreatic cancer cells) and CFPAC-1 (human pancreatic cancer cells) with the conjugated compound CB-20BK and related compounds

Related designs: MMAE and CB-20BK.

Experimental Procedures PANC-1 and CFPAC-1 cells were prepared in advance, counted, and seeded in 96-well cell culture plates at a cell density of 4 × 10 ⁴ cells/mL (100 μL/well); negative control and blank control wells were prepared for each plate. After culturing the adherent cells overnight, diluted samples to be tested were added at 50 μL/well. The plates were placed in an incubator at 37°C, 5% CO ₂ and cultured for 68-72 hours. 15 μl (10% of the liquid volume in the well) of a color development solution from CCK8 was added to each well; and the plates were incubated at 37°C for 45-70 minutes, and each plate was read at 450 nm on a microplate reader. Data were edited using SoftMax Pro and a curve was generated using the 4-parameter fitting method. The calculated data is presented in the following table.

Table 10. Suppressive effects of CB-20BK on cell line proliferation and receptor gene expression levels in cell lines

(with reference to Depmap data)

Cell line FOLR1 gene expression level FOLH1 gene expression level IC ₅₀
(MMAE) IC ₅₀
(CB-20BK) IC ₅₀ (MMAE)/
IC ₅₀ (CB-20BK) CFPAC-1 4+ +/- 0.015 0.557 0.027 PANC-1 +/- +/- 0.002 0.126 0.020

From Table 10, it can be seen that the expression level of FOLH1 in the case of CFPAC-1 is very low, and the expression level of FOLR1 in the case of CFPAC-1 is significantly higher than in the case of the PANC1 cell line. CB-20BK has a suppressive effect on the proliferation of PANC-1 and CFPAC-1 tumor cell lines. The suppressive effects on cells with different levels of FOLR1 receptor expression differ. The IC ₅₀ ratio for the CFPAC-1 cell line with relatively high levels of FOLR1 receptor expression is significantly higher than the IC ₅₀ ratio for PANC-1 with relatively low levels of FOLR1 receptor expression, and the inhibitory effects on cell proliferation are positively correlated with cellular FOLR1 receptor expression levels .

Experiments 2 and 3 provide evidence that the two ligands in CB-20BK and their receptors (FOLR1 and FOLH1) expressed on the cell surface play an important role in the compound's inhibition of tumor cell proliferation.

4. Experiment to suppress tumor cell proliferation in the presence of CB-20B

Sample information: CB-20B.

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

Basic reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8.

Experimental procedures

1) Cell seeding

Cells were prepared in advance, trypsinized, collected, and counted; A549 cells, HuH-7 cells, KB cells and DU145 cells diluted with complete cell culture medium were seeded at a density of 2 × 10 ⁴ cells/ml, T-47D cells were seeded at a density of 1 × 10 ⁵ cells/ml and cells LNCaP was seeded at a density of 6 × ¹⁰⁴ cells/ml in 96-well plates; 100 μl of diluted cell solution was added to each well; and negative control and blank control wells were prepared for each plate. 96-well plates with added cells were placed in a 37°C, 5% CO ₂ incubator for overnight culture.

2) Dilution and addition of samples

Samples were diluted with culture medium to the required concentrations (see Table 11). 50 μl of diluted samples was added to each well of 96-well plates that were cultured overnight, providing 3 replicates per well; prepared negative controls and blank controls; and the plates were placed in an incubator at 37°C, 5% CO ₂ for 72 hours for cultivation.

Table 11. Sample concentration (CB-20B) after dilution

Tube number 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

3) Development of staining and reading of the plate

15 μl (10% of the liquid volume in the well) of a color development solution from CCK-8 was added to each well; the plates were incubated at 37°C for an appropriate time (trying to maintain the OD value in the range of 1.0-2.0); The lids of the 96-well plates were removed and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).

4) Data processing

Data were edited using SoftMax Pro and a curve was generated using the four-parameter fitting method.

5) Experimental results and their analysis

As plotted against the curves generated by the four-parameter fitting method (Figure 4B, where the C value corresponds to the _IC50 ), CB-20B has an inhibitory effect on the growth and proliferation of tumor cell lines A549, HuH-7, KB, LNcap, DU145 and T -47D. Since the expression levels of the respective conjugate receptors are different in cells, the levels of inhibition vary. The IC ₅₀ for the T-47D, LNcap, KB, HuH-7 and DU145 cell lines with relatively high levels of receptor expression is significantly lower than for the A549 cell line with relatively low levels of receptor expression. CB-20B demonstrates a correlation between tumor suppressive effects and cellular receptor expression levels.

5. Experiment to suppress tumor cell proliferation in the presence of CB-10S

Sample information: CB-10S.

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.

Basic reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8.

Experimental procedures

1) Cell seeding

Cells were prepared in advance, trypsinized, collected, and counted; using complete cell culture medium, KB cells were diluted to 3.5 × 10 ⁴ cells/ml, LNCaP cells were diluted to 8.0 × 10 ⁴ cells/ml, T-47D cells were diluted to 1.2 × 10 ⁴ cells/ml , NCI-H460 cells were diluted to 2.5 × 10 ⁴ cells/ml and RT4 cells were diluted to 1.2 × 10 ⁴ cells/ml; 100 μl of diluted cell solution was added to each well of 96-well plates; and negative control and blank control wells were prepared for each plate. 96-well plates with added cells were placed in a 37°C, 5% CO ₂ incubator for overnight culture.

2) Dilution and addition of samples

Samples were diluted with culture medium to the required concentrations (see Table 12). 50 μl of diluted samples was added to each well of 96-well plates that were cultured overnight, providing 3 replicates per well; prepared negative controls and blank controls; and the plates were placed in an incubator at 37°C, 5% CO ₂ for 72 hours for cultivation.

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

Tube number Concentration after dilution (µmol/l) 1 302 2 43 3 6 4 0.9 5 0.1 6 0.02 7 0.003 8 0.0004 9 0.00005 10 0.000007

3) Development of staining and reading of the plate

15 μl (10% of the liquid volume in the well) of a color development solution from CCK-8 was added to each well; the plates were incubated at 37°C for an appropriate time (trying to maintain the OD value in the range of 1.0-2.0); The lids of the 96-well plates were removed and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).

4) Data processing

Data were edited using SoftMax Pro and a curve was generated using the four-parameter fitting method.

5) Experimental results and their analysis

As plotted against the curves generated by the four-parameter fitting method (Figure 4C, where the C value corresponds to the _IC50 ), CB-10S has an inhibitory effect on the growth and proliferation of tumor cell lines KB, LNCaP, T-47D, RT4 and NCI-H460 . Since the expression levels of the respective conjugate receptors are different in cells, the levels of inhibition vary. The IC ₅₀ for the KB, LNcap, T-47D and RT4 cell lines with relatively high levels of receptor expression is significantly lower than for the NCI-H460 cell line with relatively low levels of receptor expression. CB-10S demonstrates a correlation between tumor suppressive effects and cellular receptor expression levels.

6. Experiment to suppress tumor cell proliferation in the presence of CB-60S

Sample information: CB-60S.

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 liver cancer cells and LNCaP human prostate cancer cells .

Basic reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8.

Experimental procedures

1) Cell seeding

Cells were prepared in advance, trypsinized, collected, and counted; using complete cell culture medium, KB cells were diluted to 2.0 × 10 ⁴ cells/ml, T-47D cells were diluted to 1.5 × 10 ⁴ cells/ml, NCI-H460 cells were diluted to 2.0 × 10 ⁴ cells /ml, CALU-3 cells were diluted to 3.0 × 10 ⁴ cells/ml, HuH-7 cells were diluted to 4.0 × 10 ⁴ cells/ml, and LNCaP cells were diluted to 8.0 × 10 ⁴ cells/ml; 100 μL of diluted cell solution was added to each well of 96-well plates; and negative control and blank control wells were prepared for each plate. 96-well plates with added cells were placed in a 37°C, 5% CO ₂ incubator for overnight culture.

2) Dilution and addition of samples

Samples were diluted with culture medium to the required concentrations (see Table 13). 50 μl of diluted samples was added to each well of 96-well plates that were cultured overnight, providing 3 replicates per well; prepared negative controls and blank controls; and the plates were placed in an incubator at 37°C, 5% CO ₂ for 72 hours for cultivation.

Table 13. Sample concentration (CB-60S) after dilution

Tube number 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

3) Development of staining and reading of the plate

15 μl (10% of the liquid volume in the well) of a color development solution from CCK-8 was added to each well; the plates were incubated at 37°C for an appropriate time (trying to maintain the OD value in the range of 1.0-2.0); The lids of the 96-well plates were removed and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).

4) Data processing

Data were edited using SoftMax Pro and a curve was generated using the four-parameter fitting method.

5) Experimental results and their analysis

According to the curve plotted by the four-parameter fitting method (Fig. 4D, where the C value corresponds to IC ₅₀ ), CB-60S has an inhibitory effect on the growth and proliferation of tumor cell lines KB, T-47D, NCI-H460, CALU-3 , HuH-7 and LNCaP. Since the expression levels of the respective conjugate receptors are different in cells, the levels of inhibition vary. The IC ₅₀ for cell lines LNCaP and HuH-7 with relatively high levels of receptor expression is significantly lower than for cell lines NCI-H460, KB, T-47D and CALU-3 with relatively low levels of receptor expression. CB-60S demonstrates a correlation between tumor suppressive effects and cellular receptor expression levels.

7. Experiment to suppress tumor cell proliferation in the presence of CB-60SK

Sample information: CB-60SK.

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 liver cancer cells and LNCaP human prostate cancer cells .

Basic reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8.

Experimental procedures

1) Cell seeding

Cells were prepared in advance, trypsinized, collected, and counted; using complete cell culture medium, KB cells were diluted to 2.5 × 10 ⁴ cells/ml, T-47D cells were diluted to 1.3 × 10 ⁴ cells/ml, 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, and LNCaP cells were diluted to 8.0 × 10 ⁴ cells/ml; 100 μl of diluted cell solution was added to each well; and negative control and blank control wells were prepared for each plate. 96-well plates with added cells were placed in a 37°C, 5% CO ₂ incubator for overnight culture.

2) Dilution and addition of samples

Samples were diluted with culture medium to the required concentrations (see Table 14). 50 μl of diluted samples was added to each well of 96-well plates that were cultured overnight, providing 3 replicates per well; prepared negative controls and blank controls; and the plates were placed in an incubator at 37°C, 5% CO ₂ for 72 hours for cultivation.

Table 14. Sample concentration (CB-60SK) after dilution

Tube number 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

3) Development of staining and reading of the plate

15 μl (10% of the liquid volume in the well) of a color development solution from CCK-8 was added to each well; the plates were incubated at 37°C for an appropriate time (trying to maintain the OD value in the range of 1.0-2.0); The lids of the 96-well plates were removed and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).

4) Data processing

Data were edited using SoftMax Pro and a curve was generated using the four-parameter fitting method.

5) Experimental results and their analysis

According to the curve plotted by the four-parameter fitting method (Fig. 4E, where the C value corresponds to IC ₅₀ ), CB-60SK has an inhibitory effect on the growth and proliferation of tumor cell lines KB, T-47D, NCI-H460, CALU-3 , HuH-7 and LNCaP. Since the expression levels of the respective conjugate receptors are different in cells, the levels of inhibition vary. The IC ₅₀ for cell lines LNCaP and HuH-7 with relatively high levels of receptor expression is significantly lower than for cell lines T-47D, NCI-H460, CALU-3 and KB with relatively low levels of receptor expression. CB-60SK demonstrates a correlation between tumor suppressive effects and cellular receptor expression levels.

8. Experiment to suppress tumor cell proliferation in the presence of CB-18G

Sample information: CB-18G.

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.

Basic reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8.

Experimental procedures

1) Cell seeding

Cells were prepared in advance, trypsinized, collected, and counted; A549 cells, Hela cells, SCLC-21H cells and U-2 OS cells diluted with complete cell culture medium were seeded at a density of 2 × 10 ⁴ cells/ml, and T-47D cells and NCI-H460 cells were seeded at a density 3 × 10 ⁴ cells/ml in 96-well plates; 100 μl of diluted cell solution was added to each well of 96-well plates; and negative control and blank control wells were prepared for each plate. 96-well plates with added cells were placed in a 37°C, 5% CO ₂ incubator for overnight culture.

2) Dilution and addition of samples

Samples were diluted with culture medium to the required concentrations (see Table 15). 50 μl of diluted samples was added to each well of 96-well plates that were cultured overnight, providing 3 replicates per well; prepared negative controls and blank controls; and the plates were placed in an incubator at 37°C, 5% CO ₂ for 72 hours for culture.

Table 15. Sample concentration (CB-18G) after dilution

Tube number 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

3) Development of staining and reading of the plate

15 μl (10% of the liquid volume in the well) of a color development solution from CCK-8 was added to each well; the plates were incubated at 37°C for an appropriate time (trying to maintain the OD value in the range of 1.0-2.0); The lids of the 96-well plates were removed and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).

4) Data processing

Data were edited using SoftMax Pro and a curve was generated using the four-parameter fitting method.

5) Experimental results and their analysis

According to the curve plotted by the four-parameter fitting method (Fig. 4F, where the C value corresponds to the _IC50 ), CB-18G has an inhibitory effect on the growth and proliferation of tumor cell lines A549, Hela, SCLC-21H, U-2 OS, T-47D and NCI-H460. Since the expression levels of the respective conjugate receptors are different in cells, the levels of inhibition vary. The IC ₅₀ for the Hela cell line with a relatively high level of receptor expression is significantly lower than for the NCI-H460 cell line with a relatively low level of receptor expression. CB-18G demonstrates a correlation between tumor suppressive effects and cellular receptor expression levels.

9. Experiment to suppress tumor cell proliferation in the presence of CB-50S

Sample information: CB-50S.

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.

Basic reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8.

Experimental procedures

1) Cell seeding

Cells were prepared in advance, trypsinized, collected, and counted; using complete cell culture medium, KB cells were diluted to 3.5 × 10 ⁴ cells/ml, LNCaP cells were diluted to 8.0 × 10 ⁴ cells/ml, T-47D cells were diluted to 1.2 × 10 ⁴ cells/ml , NCI-H460 cells were diluted to 2.5 × 10 ⁴ cells/ml and RT4 cells were diluted to 1.2 × 10 ⁵ cells/ml; 100 μl of diluted cell solution was added to each well of 96-well plates; and negative control and blank control wells were prepared for each plate. 96-well plates with added cells were placed in a 37°C, 5% CO ₂ incubator for overnight culture.

2) Dilution and addition of samples

Samples were diluted with culture medium to the required concentrations (see Table 16). 50 μl of diluted samples was added to each well of 96-well plates that were cultured overnight, providing 3 replicates per well; prepared negative controls and blank controls; and the plates were placed in an incubator at 37°C, 5% CO ₂ for 72 hours for culture.

Table 16. Sample concentration (CB-50S) after dilution

Tube number Concentration after dilution (µmol/l) 1 314 2 45 3 6 4 0.9 5 0.1 6 0.02 7 0.003 8 0.0004 9 0.00005 10 0.000008

3) Development of staining and reading of the plate

15 μl (10% of the liquid volume in the well) of a color development solution from CCK-8 was added to each well; the plates were incubated at 37°C for an appropriate time (trying to maintain the OD value in the range of 1.0-2.0); The lids of the 96-well plates were removed and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).

4) Data processing

Data were edited using SoftMax Pro and a curve was generated using the four-parameter fitting method.

5) Experimental results and their analysis

According to the curve plotted by the four-parameter fitting method (Fig. 4G, where the C value corresponds to IC ₅₀ ), CB-50S has an inhibitory effect on the growth and proliferation of tumor cell lines KB, LNCaP, T-47D, RT4 and NCI-H460 . Since the expression levels of the respective conjugate receptors are different in cells, the levels of inhibition vary. The IC ₅₀ for the KB, LNcap, T-47D and RT4 cell lines with relatively high levels of receptor expression is significantly lower than for the NCI-H460 cell line with relatively low levels of receptor expression. CB-50S demonstrates a correlation between tumor suppressive effects and cellular receptor expression levels.

10. Experiment on suppressing the proliferation of tumor cells in the presence of the conjugated compound CB-1020

Purpose of the experiment: testing for the inhibitory effects 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).

Related samples: MMAE and CB-1020.

Experimental Procedures LNCaP, SK-BR-3, NCI-H226, CFPAC-1, and PANC-1 cells were prepared in advance and counted; using culture medium (IMDM + 10% FBS + 1x L-glutamine + 1x P/S), LNCaP, SK-BR-3 and CFPAC-1 cells were diluted to 4 × 10 ⁴ cells/ml, NCI- cells H226 was diluted to 2.0 × 10 ⁴ cells/ml and PANC-1 cells were diluted to 3.0 × 10 ⁴ cells/ml; the diluted cell solution was seeded into 96-well plates at 100 μl/well; and negative control and blank control wells were prepared for each plate. After culturing the adherent cells overnight, diluted samples to be tested were added at 50 μL/well. The plates were placed in an incubator at 37°C, 5% CO ₂ and cultured for 68-72 hours. 15 μl (10% of the liquid volume in the well) of a color development solution from CCK8 was added to each well; and the plates were incubated at 37°C for 45-70 minutes, and each plate was read at 450 nm on a microplate reader. Data were edited using SoftMax Pro and a curve was generated using the 4-parameter fitting method. The data is presented in the following table.

Table 17. Inhibitory effects of CB-1020 on cell line proliferation and receptor gene expression levels in cell lines

(with reference to Depmap data)

Cell line TRPV6 gene expression level FOLH1 gene expression level IC ₅₀
(MMAE) IC ₅₀
(CB-1020) IC ₅₀ (MMAE)/IC ₅₀ (CB-1020) LNCap 5+ 10+ 0.003 0.168 0.0179 SK-BR-3 4+ 2+ 0.002 0.287 0.0070 CFPAC-1 + +/- 0.015 3,000 0.005 PANC-1 +/- +/- 0.002 0.458 0.0044 NCI-H226 +/- - 0.006 1,570 0.00384

From the table above, it can be seen that both LNCaP and SK-BR-3 express TRPV6 and FOLRH1, with LNCap having relatively higher expression levels of these two receptors. NCI-H226, CFPAC-1 and PANC-1 express these two receptors at low or reduced levels. CB-1020 has an inhibitory effect on the growth and proliferation of tumor cell lines LNCaP, SK-BR-3, NCI-H226, CFPAC-1 and PANC-1. The IC ₅₀ ratio of CB-1020 for the LNCaP cell line with relatively high expression levels of the two receptors is higher than the IC ₅₀ ratio for the SK-BR-3 cell line with an average level of receptor expression; however, the IC ₅₀ ratio for SK-BR-3 is higher than the IC ₅₀ ratios for CFPAC-1 with a relatively low level of receptor expression and for cell lines NCI-H226 and PANC-1 with a reduced level of expression of two receptors. The suppressive effect on cell proliferation is positively correlated with the expression level of these two receptors in the cells.

eleven. Experiment on suppressing the proliferation of tumor cells in the presence of the conjugated compound CB-1320

Purpose of the experiment: testing for the inhibitory effects 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 (breast cancer cells human glands) and CFPAC-1 (pancreatic cancer cells).

Related samples: MMAE and CB-1320.

Experimental Procedures LNCaP, SK-BR-3, MDA-MB-468, and CFPAC-1 cells were prepared in advance and counted; using culture medium (IMDM+10% FBS+1x L-glutamine+1x P/S), LNCaP, SK-BR-3, MDA-MB-468 and CFPAC-1 cells were diluted to 4 × 10 ⁴ cells /ml; the diluted cell solution was seeded into 96-well plates at 100 μl/well; and negative control and blank control wells were prepared for each plate. After culturing the adherent cells overnight, diluted samples to be tested were added at 50 μL/well. The plates were placed in an incubator at 37°C, 5% CO ₂ and cultured for 68-72 hours. 15 μl (10% of the liquid volume in the well) of a color development solution from CCK8 was added to each well; and the plates were incubated at 37°C for 45-70 minutes, and each plate was read at 450 nm on a microplate reader. Data were edited using SoftMax Pro and a curve was generated using the 4-parameter fitting method. Specific data is shown in the following table.

Table 18. Suppressive effects of CB-1320 on cell line proliferation and receptor gene expression levels in cell lines

(with reference to Depmap data)

Cell line GNRHR gene expression level FOLH1 gene expression level IC ₅₀
(MMAE) IC ₅₀
(CB-1320) IC ₅₀ (MMAE)/IC ₅₀ (CB-1320) LNCaP +/- 10+ 0.003 0.060 0.043 SK-BR-3 +/- 2+ 0.002 0.093 0.016 CFPAC-1 +/- +/- 0.015 1,090 0.014 MDA-MB-468 +/- +/- 0.002 0.212 0.011

From the table above, it can be seen that the expression levels of GNRHR for the 4 cell lines are approximately equivalent, and the expression level of FOLH1 in the case of LNCaP is significantly higher than for the other cell lines. CB-1320 has an inhibitory effect on the proliferation of 4 tumor cell lines. The suppressive effects of CB-1320 on cells with different levels of FOLH1 receptor expression vary. The IC ₅₀ ratio for the LNCaP cell line with relatively high levels of expression of the FOLH1 receptor is significantly higher than the IC ₅₀ ratio for other cell lines with relatively low levels of expression of this receptor.

12. Experiment to suppress the proliferation of tumor cells in the presence of the conjugated compound CB-1820

Purpose of the experiment: testing for the inhibitory effects 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).

Related samples: MMAE and CB-1820.

Experimental Procedures LNCaP, MDA-MB-468, CFPAC-1, and PANC-1 cells were prepared in advance and counted; using culture medium (IMDM+10% FBS+1x L-glutamine+1x P/S), LNCaP, MDA-MB-468 and CFPAC-1 cells were diluted to 4 × 10 ⁴ cells/ml and PANC- cells 1 was diluted to 3.0 × 10 ⁴ cells/ml; the diluted cell solution was seeded into 96-well plates at 100 μl/well; and negative control and blank control wells were prepared for each plate. After culturing the adherent cells overnight, diluted samples to be tested were added at 50 μL/well. The plates were placed in an incubator at 37°C, 5% CO ₂ and cultured for 68-72 hours. 15 μl (10% of the liquid volume in the well) of a color development solution from CCK8 was added to each well; and the plates were incubated at 37°C for 45-70 minutes, and each plate was read at 450 nm on a microplate reader. Data were edited using SoftMax Pro and a curve was generated using the 4-parameter fitting method. Specific data is shown in the following table.

Table 19. Suppressive effects of CB-1820 on cell line proliferation and receptor gene expression levels in cell lines

(with reference to Depmap data)

Cell line SSTR2 gene expression level FOLH1 gene expression level IC ₅₀
(MMAE) IC ₅₀
(CB-1820) IC ₅₀ (MMAE)/IC ₅₀ (CB-1820) LNCap +/- 10+ 0.003 0.021 0.1429 MDA-MB-468 +/- +/- 0.002 0.062 0.0323 CFPAC-1 +/- +/- 0.015 0.356 0.0421 PANC-1 +/- +/- 0.002 0.055 0.0364

From the above table, it can be seen that the expression levels of SSTR2 for LNCap, MDA-MB-468, CFPAC-1 and PANC-1 are approximately equivalent, and the expression level of FOLH1 in the case of LNCaP is significantly higher than the FOLH1 expression level for other cell lines. CB-1820 has an inhibitory effect on the proliferation of 4 tumor cell lines. The suppressive effects of CB-1820 on cells with different levels of FOLH1 receptor expression vary. The IC ₅₀ ratio for the LNCaP cell line with relatively high levels of expression of this receptor is significantly higher than the IC ₅₀ ratio for other cell lines with relatively low levels of expression of this receptor, and the inhibitory effects on cell proliferation are positively correlated with expression levels of the cellular receptor FOLH1.

13. Experiment on suppressing the proliferation of tumor cells in the presence of conjugated compounds CR19425, CR19426 and CR19428

Purpose of the experiment: Testing for the inhibitory effects 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 (cancer cells human mammary gland).

Related samples: SN-38, Dxd0017, CR19428, CR19425 and CR19426.

Experimental Procedures NCI-H226, CFPAC-1, and MDA-MB-468 cells were prepared in advance and counted; MDA-MB-468 and CFPAC-1 cells were seeded at a density of 2 × 10 ⁴ cells/ml (100 μl/well), and NCI-H226 cells were seeded at a density of 1 × 10 ⁴ cells/ml (100 μl/well) in 96 - well plates; and negative control and blank control wells were prepared for each plate. After culturing the adherent cells overnight, diluted samples to be tested were added at 50 μL/well. The plates were placed in an incubator at 37°C, 5% CO ₂ and cultured for 68-72 hours. 15 μl (10% of the liquid volume in the well) of a color development solution from CCK8 was added to each well; and the plates were incubated at 37°C for 45-70 minutes, and each plate was read at 450 nm on a microplate reader. Data were edited using SoftMax Pro and a curve was generated using the 4-parameter fitting method. Specific data is shown in the following table.

Table 20. Suppressive effects of CR19428, CR19425 and CR19426 on cell line proliferation

Cell line IC ₅₀
(SN-38) IC ₅₀
(Dxd0017) IC ₅₀
(CR19428) IC ₅₀
(CR19425) IC ₅₀
(CR19426) CFPAC-1 0.0055 0.0064 0.9060 0.6210 1.6600 MDA-MB-468 0.0136 0.0061 1.0700 0.9510 2.3000 NCI-H226 0.0173 0.0080 1.6200 1.0100 4.3900

Example 5: Pharmacodynamic Study of Conjugate Compounds Using CDX Models

1. Pharmacodynamic Study 1 for CB-20BK Conducted in CDX Models

1) Sample preparation

Lyophilized CB-20BK powder was weighed, dissolved in PBS and prepared as a sample stock solution, and the stock solution was diluted with physiological saline injection to a working concentration sample solution for subsequent use.

2) Creating a CDX model

Major cell lines: human oral epidermoid carcinoma KB cells, human pancreatic cancer MIA PaCa-2 cells, human breast cancer HCC1954 cells, human lung adenocarcinoma CALU-3 cells, and human prostate cancer DU145 cells.

Creating a model. Cells were recovered, cultured, harvested, counted, and injected subcutaneously into the right flank of athymic BALB/c mice. When the tumor grew and increased to 80-160 mm ³ , group assignment and administration were carried out, or the rapidly increasing tumor masses were transferred and injected into mice in order to carry out their growth.

3) Allocation to groups, administration and observation

Distribution into groups. When the tumor had grown to an average size of approximately 80-160 mm ³ , the groups were allocated to include a model control group and different dose groups.

Introduction. Administration to mice was performed by tail vein injection.

Observation and measurement performed as part of an experiment. Following tumor inoculation, the effects of tumor growth and treatment on normal animal behavior parameters were monitored in a conventional manner, including experimental animal activity, food and fluid intake status, weight gain or loss (with weight measured twice weekly), and regarding other abnormal conditions associated with the eyes, hair, etc.

The weight of the mice, as well as the long axis size (a) and short axis size (b) of the tumor mass were measured twice a week. The formula for calculating tumor volume (TV) is: TV=1/2 × a × b ² .

4) Experimental results and their analysis

From the changes in tumor volume values in mice (Fig. 5A-5E), it can be seen that CB-20BK has a good suppressive effect against CDX tumor models based on the cell lines KB, MIA PaCa-2, HCC1954, CALU-3 and DU145 in mice.

2. Pharmacodynamic Study 2 for CB-20BK Conducted in CDX Models

The purpose of the experiment: to conduct a pharmacodynamic study of the conjugated compound CB-20BK on a subcutaneous xenograft (CDX) model of humanized cell lines LNCaP, DU145 and NCI-H460.

Main CDX models: LNCaP, DU145 and NCI-H460.

Experimental design

Cells or tumor tissue mass were obtained and subcutaneously inoculated into the anterior right lateral region of athymic BALB/c mice. When the tumor volume increased to 80-160 mm ³ , randomization was performed into groups and included a control group and a treatment group in relation to the model. Mice were administered from the first day of group assignment, with the dose adjusted according to the last weight measured. Administration to mice was performed by tail vein injection at an injected volume of 10 μL/g. Following group assignment and administration, the effects of tumor growth and treatment were monitored with respect to normal animal behavior parameters, particularly including experimental animal activity, food and fluid intake status, weight gain or loss status, and other abnormal conditions associated with with eyes, hair, etc. After group assignment and administration, the weight of the mice was measured twice a week to calculate the rate of weight change; at the same time, the long and short axis size of the tumor was measured with a caliper, and the tumor volume, the relative proliferation rate of tumor cells, the degree of tumor volume suppression and other indicators were calculated. The formula for calculating tumor volume is TV=0.5 a × b ² where a is the long axis size of the tumor and b is the short axis size of the tumor.

Table 21. Suppressive effects of CB-20BK on growth in CDX models and receptor gene expression levels in each model

(with reference to data obtained from Crown Bioscience Inc.)

Model FOLR1 FOLH1 Tumor growth inhibition rate (TGI) (%) Dose/time of administration LNCaP 5+ 10+ 95.68 3 mg/kg in D1, 8, 15 DU145 - - 52 5 mg/kg in D1, 4, 7 NCI-H460 - - 32 10 mg/kg in D1, 8, 15

From the table above, it can be seen that the test sample CB-20BK exhibits varying degrees of suppressive effects on tumor growth in the tail vein administration regimen. In relation to CDX models based on humanized cell lines LNCaP, DU145 and NCI-H460, the test sample has a certain tumor growth suppression effect when compared with the negative control group. In the case of the LNCaP-based model with dual expression of FOLR1 and FOLH1, the test sample administered on day 1, day 8 and day 15 (D1, D8 and D15) at a dose of 3 mg/kg has a TGI value of 95.68%, while demonstrates excellent tumor growth suppression effect, which is significantly superior to that of DU145- and NCI-H460-based models with relatively low expression levels of FOLR1 and FOLH1. The suppressive effect on tumor growth is characterized by a certain degree of correlation with the level of expression of cellular receptors.

3. Tumor Inhibition Experiment 1 for CB-20BK Conjugate Conducted in HuPrime Xenograft PDX Models ^®

Purpose of the experiment. Pharmacodynamic study of the conjugate compound CB-20BK conducted in PDX models.

Main models: LU1206, LU1380 and LU0367.

Experimental design. A mass of tumor tissue was obtained and subcutaneously inoculated into the anterior right lateral region of athymic BALB/c mice. When the tumor volume increased to 80-160 mm ³ , randomization was performed into groups and included a control group and a treatment group in relation to the model. Mice were administered from the first day of group assignment, with the dose adjusted according to the last weight measured. Administration to mice was performed by tail vein injection at an injected volume of 10 μl/g and an injected dose of 3 mg/kg. Following group assignment and administration, the effects of tumor growth and treatment were monitored with respect to normal animal behavior parameters, particularly including experimental animal activity, food and fluid intake status, weight gain or loss status, and other abnormal conditions associated with with eyes, hair, etc. After group assignment and administration, the weight of the mice was measured twice a week to calculate the rate of weight change; at the same time, the long and short axis size of the tumor was measured with a caliper, and the tumor volume, the relative proliferation rate of tumor cells, the degree of suppression of tumor volume and other indicators were calculated. The formula for calculating tumor volume is TV=0.5 a × b ² where a is the long axis size of the tumor and b is the short axis size of the tumor.

Table 22. Suppressive effects of CB-20BK on growth in PDX models of lung cancer and receptor gene expression levels in each model

(with reference to data obtained from Crown Bioscience Inc.)

Model FOLR1 gene expression level FOLH1 gene expression level TGI (%) Dose LU1206 4+ 2+ 90.89 3 mg/kg LU1380 - 4+ 30.02 3 mg/kg LU0367 - 5+ 71.34 3 mg/kg

From the data in Table 22, it can be seen that the test sample CB-20BK (3 mg/kg) shows a certain antitumor effect in the case of PDX models based on humanized lung cancer cells LU1206, LU1380 and LU0367. The TGI value for the LU1206-based model with dual expression of FOLR1 and FOLH1 is 90.89%, and the TGI values for the LU1380 and LU0367 single-expression models are 30.02% and 71.34%, respectively. In a tumor growth inhibition experiment, the suppressive effect on tumor growth is characterized by a certain degree of correlation with the level of expression of cellular receptors.

4. Tumor Inhibition Experiment 2 for CB-20BK Conjugate Conducted in HuPrime Xenograft PDX Models ^®

Purpose of the experiment. Pharmacodynamic study of the conjugate compound CB-20BK conducted in PDX models.

Main models: BR1283 and BR0438.

Experimental design. A mass of tumor tissue was obtained and subcutaneously inoculated into the anterior right lateral region of athymic BALB/c mice. When the tumor volume increased to 80-160 mm ³ , randomization was performed into groups and included a control group and a treatment group in relation to the model. Mice were administered from the first day of group assignment, with the dose adjusted according to the last weight measured. Administration to mice was performed by tail vein injection at an injected volume of 10 μl/g and an injected dose of 3 mg/kg. Following group assignment and administration, the effects of tumor growth and treatment were monitored with respect to normal animal behavior parameters, particularly including experimental animal activity, food and fluid intake status, weight gain or loss status, and other abnormal conditions associated with with eyes, hair, etc. After group assignment and administration, the weight of the mice was measured twice a week to calculate the rate of weight change; at the same time, the long and short axis size of the tumor was measured with a caliper, and the tumor volume, the relative proliferation rate of tumor cells, the degree of suppression of tumor volume and other indicators were calculated. The formula for calculating tumor volume is TV=0.5 a × b ² where a is the long axis size of the tumor and b is the short axis size of the tumor.

Table 23. Suppressive effects of CB-20BK on growth in PDX models of breast cancer and receptor gene expression levels in each model

(with reference to data obtained from Crown Bioscience Inc.)

Model FOLR1 gene expression level FOLH1 gene expression level TGI (%) Dose BR1283 7+ 6+ 96.49 3 mg/kg BR0438 5+ 4+ 70.74 3 mg/kg

From the above table, it can be seen that the TGI values of CB-20BK test sample at 3 mg/kg for FOLR1 and FOLH1 dual expression models BR1283 and BR0438 are 96.49% and 70.74%, respectively, demonstrating excellent suppression effect tumor growth. Moreover, the TGI for CB-20BK is higher in the case of BR1283 with higher expression levels of FOLR1 and FOLH1.

5. Pharmacodynamic Study of CB-20B Conducted in CDX Models

1) Sample preparation

Lyophilized CB-20B powder was weighed, dissolved in PBS and prepared as a sample stock solution, and the stock solution was diluted with physiological saline injection to a working concentration sample solution for subsequent use.

2) Creating a CDX model

Major cell lines: human oral epidermoid carcinoma KB cells, human lung cancer PC-9 cells, and human prostate cancer DU145 cells.

Creating a model. Cells were recovered, cultured, harvested, counted, and injected subcutaneously into the right flank of athymic BALB/c mice. When the tumor grew and increased to 80-160 mm ³ , group assignment and administration were carried out, or the rapidly increasing tumor masses were transferred and injected into mice in order to carry out their growth.

3) Allocation to groups, administration and observation

Distribution into groups. When the tumor had grown to an average size of approximately 80-160 mm ³ , the groups were allocated to include a model control group and different dose groups.

Introduction. Administration to mice was performed by tail vein injection.

Observation and measurement performed as part of an experiment. Following tumor inoculation, the effects of tumor growth and treatment on normal animal behavior parameters were monitored in a conventional manner, including experimental animal activity, food and fluid intake status, weight gain or loss (with weight measured twice weekly), and regarding other abnormal conditions associated with the eyes, hair, etc.

The weight of the mice, as well as the long axis size (a) and short axis size (b) of the tumor mass were measured twice a week. The formula for calculating tumor volume (TV) is: TV=1/2 × a × b ² .

4) Experimental results and their analysis

From the changes in the tumor volume value in mice (Fig. 6A-6C), it can be seen that CB-20B has a good suppressive effect against the CDX tumor models based on the KB, PC-9 and DU145 cell lines in mice.

6. Pharmacodynamic Study of CB-18G Conducted in CDX Models

1) Sample preparation

Lyophilized CB-18G powder was weighed, dissolved in PBS and prepared as a sample stock solution, and the stock solution was diluted with physiological saline injection to a working concentration sample solution for subsequent use.

2) Creating a CDX model

Major cell lines: human oral epidermoid carcinoma KB cells, human lung cancer PC-9 cells, human lung adenocarcinoma SPC-A1 cells, human lung adenocarcinoma CALU-3 cells, and human prostate cancer DU145 cells.

Creating a model. Cells were recovered, cultured, collected and counted, and the cell solution was injected subcutaneously into the right flank of BALB/c nude mice. When the tumor grew and increased to 80-160 mm ³ , group assignment and administration were carried out, or the rapidly increasing tumor masses were transferred and injected into mice in order to carry out their growth.

3) Group assignment, administration and observation

Distribution into groups. When the tumor had grown to an average size of approximately 80-160 mm ³ , the groups were allocated to include a model control group and different dose groups.

Introduction. Administration to mice was performed by tail vein injection.

Observation and measurement performed as part of an experiment. Following tumor inoculation, the effects of tumor growth and treatment on normal animal behavior parameters were monitored in a conventional manner, including experimental animal activity, food and fluid intake status, weight gain or loss (with weight measured twice weekly), and regarding other abnormal conditions associated with the eyes, hair, etc.

The weight of the mice, as well as the long axis size (a) and short axis size (b) of the tumor mass were measured twice a week. The formula for calculating tumor volume (TV) is: TV=1/2 × a × b ² .

4) Experimental results and their analysis

From the changes in tumor volume values in mice (Fig. 7A-7E), it can be seen that CB-18G has a good suppressive effect against CDX tumor models based on the cell lines KB, PC-9, SPC-A1, CALU-3 and DU145 in mice.

7. Tumor inhibition experiment for conjugate compounds CB-1020, CB-1320 and CB-1820 conducted in a subcutaneous xenograft (CDX) model based on humanized cell lines HPAF-II, NCI-H226 and SCLC-21H

Purpose of the experiment. Pharmacodynamic study of ligand conjugates CB-1020, CB-1320 and CB-1820 in CDX models

The main CDX models are HPAF-II (human pancreatic cancer cells), NCI-H226 (human lung cancer cells), and SCLC-21H (small cell lung cancer cells).

Experimental design

Cells or tumor tissue mass were obtained and subcutaneously inoculated into the anterior right lateral region of athymic BALB/c mice. When the tumor volume increased to 80-160 mm ³ , randomization was performed into groups and included a control group and a treatment group in relation to the model. Mice were administered from the first day of group assignment, with the dose adjusted according to the last weight measured. Administration to mice was performed by tail vein injection at an injected volume of 10 μl/g. Following group assignment and administration, the effects of tumor growth and treatment were monitored with respect to normal animal behavior parameters, particularly including experimental animal activity, food and fluid intake status, weight gain or loss status, and other abnormal conditions associated with with eyes, hair, etc. After group assignment and administration, the weight of the mice was measured twice a week to calculate the rate of weight change; at the same time, the long and short axis size of the tumor was measured with a caliper, and the tumor volume, the relative proliferation rate of tumor cells, the degree of suppression of tumor volume and other indicators were calculated. The formula for calculating tumor volume is TV=0.5 a × b ² where a is the long axis size of the tumor and b is the short axis size of the tumor.

Table 24. Suppressive effects of CB-1020, CB-1320 and CB-1820 on growth in CDX models and receptor gene expression levels in each model (citing data obtained from Crown Bioscience Inc.)

Model TRPV6 gene expression level GNRHR gene expression level SSTR2 gene expression level FOLH1 gene expression level TGI (%)
(CB-1020) TGI (%)
(CB-1320) TGI (%)
(CB-1820) P-value Dose HPAF-II + - +/- +/- 0.283 0.461 3 mg/kg 0.445 0.153 5 mg/kg 0.899 0.023 10 mg/kg NCIH226 +/- +/- +/- - 0.823 0.011 3 mg/kg 0.962 0.006 3 mg/kg 0.992 0.005 10 mg/kg SCLC-21H 2+ +/- 3+ - 0.995 0.131 3 mg/kg 0.361 0.610 3 mg/kg 0.985 0.134 10 mg/kg

CB-1020 has an obvious tumor growth suppressive effect in subcutaneous xenograft models based on humanized HPAF-II cell lines; CB-1320 has an obvious tumor growth suppressive effect in subcutaneous xenograft models based on the NCI-H226 and SCLC-21H cell lines; and CB-1820 has an obvious tumor growth suppressive effect in subcutaneous xenograft models based on SCLC-21H cell lines.

8. Tumor suppression experiment for conjugate CR19428 conducted in a subcutaneous xenograft (CDX) model based on humanized cell lines CALU-3, SCLC-21H and SPC-A1

Experimental design. A mass of tumor tissue was obtained and subcutaneously inoculated into the anterior right lateral region of athymic BALB/c mice. When the tumor volume increased to 80-160 mm ³ , randomization was performed into groups and included a control group and a treatment group in relation to the model. Mice were administered from the first day of group assignment, with the dose adjusted according to the last weight measured. Administration to mice was performed by tail vein injection at an injection volume of 10 μl/g, and administration was performed twice a week for 3 consecutive weeks. Following group assignment and administration, the effects of tumor growth and treatment were monitored with respect to normal animal behavior parameters, particularly including experimental animal activity, food and fluid intake status, weight gain or loss status, and other abnormal conditions associated with with eyes, hair, etc. After group assignment and administration, the weight of the mice was measured twice a week to calculate the rate of weight change; at the same time, the long and short axis size of the tumor was measured with a caliper, and the tumor volume, the relative proliferation rate of tumor cells, the degree of suppression of tumor volume and other indicators were calculated. The formula for calculating tumor volume is TV=0.5 a × b ² where a is the long axis size of the tumor and b is the short axis size of the tumor.

Table 25. Suppressive effects of CR19428 in PDX models of lung cancer

Model TGI (%) Dose/time of administration CALU-3 51 50 mg/kg q2/w*3 SCLC-21H 66 50 mg/kg q2/w*3 SPC-A1 75 50 mg/kg q2/w*3 SPC-A1 91 100 mg/kg q2/w*3

CR-19428 is a drug conjugate based on the FOLR1 and FOLH1 ligands and the Dxd payload. From the above table, it can be seen that the drug conjugate has an obvious tumor growth suppression effect in subcutaneous xenograft models based on the humanized cell lines CALU-3, SCLC-21H and SPC-A1.

9. Study of the effectiveness of the conjugated compound CBP-1018 conducted on the lung cancer model based on LU2505 cells (PDX models)

This experiment used a 3rd generation LU2505-based lung cancer PDX model (from female Asian patients), which is a fast-growing tumor model. Nude BALB/c mice were treated with tumors subcutaneously, and when the tumor volume reached approximately 150 mm ³ , the following groups were allocated: groups receiving the test sample at low, medium and high doses, control group receiving low molecular weight MMAE, control group receiving targeting polypeptide 20BK-SM09, and a blank control group (6 groups in total, 8 mice per group); administration to mice was performed on day 1, day 8 and day 15 after assignment to groups; and observation was continued for 14 days after the last administration. The active ingredient of the CBP-1018 test sample is CB-20BK, and the sample is prepared by mixing CB-20BK with auxiliary materials and then lyophilizing them.

Table 26: Pharmacodynamic Results for CBP-1018 Injection in LU2505 Lung Cancer Models (First Experiment)

Test sample
/control sample Dose
(mg/kg) Weight
(G) Tumor volume
(mm ³ ) The degree of suppression of tumor growth,
TGI% Tumor weight
(mg) Idle control group / 24.0 2219.62 / 2031.4 (D22) CBP-1018 4.5 22.5 0.00 100.00 0.0 (D29) 1.5 22.1 386.16 82.60 400.7 (D29) 0.5 23.6 1634.50 26.36 2084.7 (D26) 20BK-SM09 10 23.8 2024.75 8.78 1811.5 (D22) MMAE 0.375 23.5 1132.61 48.97 1597.1 (D29)

Notes. 1) Due to the requirements of humane treatment of animals, animals in a group must be euthanized when the average tumor volume in the group reaches 2000 mm ³ , which causes different execution times for each group. 2) Data regarding tumor weight, tumor volume, and tumor growth suppression rate are data obtained on day 22 (D22).

As shown in Table 26 and FIG. 8A,

animals in all groups were not characterized by the presence of abnormal clinical manifestations and cases of death after administration; and at the same time the weight of the animals in each group slowly increased.

Animals in the blank control group, 20BK-SM09-treated group, and low-dose CBP-1018-treated group were euthanized on day 22, day 22, and day 26 (D26) because the average tumor volume exceeded 2000 mm ³ . Therefore, the data regarding tumor weight, tumor volume and tumor growth suppression rate in Table 26 are data obtained in D22.

The effectiveness of CBP-1018 injection showed an obvious correlation with dose, with CBP-1018 injection being ineffective in the low dose group and effective in the medium dose group and high dose group. The tumor in the high dose group was completely cured on day 19 (D19), and no signs of tumor growth were observed on day 29 (D29).

The group treated with the 20BK-SM09 polypeptide did not demonstrate an obvious tumor suppression effect, suggesting that the targeting polypeptide alone is not sufficient to produce a clear antitumor effect.

The group treated with MMAE showed a clear tumor growth suppression effect. An equimolar amount of MMAE is contained in the MMAE dose of 0.375 mg/kg and the CBP-1018 dose of 1.5 mg/kg. From the comparison, it can be seen that the tumor growth suppression effect of CBP-1018 at a dose of 1.5 mg/kg is significantly superior to that of the MMAE-treated group, indicating the advantage of ligand targeting (tumor growth suppression rate: 82.60% by compared to 48.97%).

10. Study of the effectiveness of the conjugated compound CBP-1018 conducted on the lung cancer model based on LU1206 cells (PDX models)

This experiment used a 5th generation LU1206-based lung cancer PDX model (from female Asian patients), which is a fast-growing tumor model. Nude BALB/c mice were treated with tumors subcutaneously, and when the tumor volume reached approximately 150 mm ³ , the following groups were allocated: groups receiving the test sample at low, medium and high doses, control group receiving low molecular weight MMAE, control group receiving targeting polypeptide 20BK-SM09, and a blank control group (6 groups in total, 8 mice per group); administration to mice was performed on day 1, day 8 and day 15 after assignment to groups; and observation was continued for 14 days after the last administration.

Table 27: Pharmacodynamic Results for CBP-1018 Injection in LU1206 Lung Cancer Models (First Experiment)

Groups Dose
(mg/kg) Weight
(G) Tumor volume
(mm ³ ) Tumor volume
Degree of tumor growth suppression
(%) Tumor weight
(mg) Tumor weight
Suppression degree
(%) Idle control group / 22.7 1216.67 / 856.14 / CBP-1018 4.5 23.4 31.44 97.42 13.55 98.42 1.5 23.7 301.64 75.21 185.10 78.38 0.5 22.7 1118.30 8.08 852.19 0.46 20BK-SM09 10 23.3 1158.80 4.76 914.36 -6.80 MMAE 0.375 23.6 778.32 36.03 437.73 54.53

As shown in Table 27 and FIG. 8B,

animals in all groups showed no abnormal clinical manifestations or deaths after administration, and all animals were euthanized on day 29. The weight of animals in each group essentially remained unchanged and was slightly higher than the weight on the first day of administration.

The effectiveness of CBP-1018 injection showed an apparent correlation with dose, with CBP-1018 injection being ineffective in the low-dose group (with a tumor growth suppression rate of 8.08%), but effective in the medium-dose group and in group receiving the high dose, with tumor growth suppression rates of 75.21% and 97.42%, respectively. The difference in effectiveness between the low-dose group and the medium-dose group is relatively large.

The group treated with the 20BK-SM09 polypeptide did not demonstrate an obvious tumor suppression effect, suggesting that the targeting polypeptide alone is not sufficient to produce a clear antitumor effect in this model.

The group treated with MMAE showed a clear tumor growth suppression effect. An equimolar amount of MMAE is contained in the MMAE dose of 0.375 mg/kg and the CBP-1018 dose of 1.5 mg/kg. From the comparison, it can be seen that the tumor growth suppression effect of CBP-1018 at a dose of 1.5 mg/kg is significantly superior to that of the MMAE-treated group, indicating the advantage of ligand targeting (tumor volume suppression rate: 75.21% by compared with 36.03%; degree of tumor weight suppression: 78.38% compared with 54.53%).

eleven. Tissue distribution in tumor-bearing mice (LU2505-based PDX model)

Twelve female tumor-bearing mice inoculated with the tumor mass of the LU2505 ^HuPrime® cell-based lung cancer model and 12 healthy male BALB/c nude mice were administered 1.5 mg/140 μCi/kg [ ³ H]CBP-1018 (isotopically labeled for MMAE) by a single injection into the tail vein. After 0.5 hours, 2 hours, 6 hours and 24 hours, respectively, 3 male mice and 3 female mice were placed in an induction chamber and anesthetized by inhalation of an appropriate amount of carbon dioxide; and blood was collected by cardiac puncture followed by immediate euthanasia for sample collection.

Table 28. Total radioactivity in various tissues at different time points after a single intravenous administration of [ ³ H]CBP-1018

Textile Radioactivity concentration (ng eq/g) 0.5 hours 2 hours 6 hours 24 hours (% _Cmax ) ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ Tumor 763 / 643 / 566 / 413 / Esophagus 915 784 628 508 521 172 286 91.7 Adipose tissue 341 479 288 276 231 177 67.2 43.7 Skeletal muscle 321 311 207 137 188 95.5 90.8 61.5 Spleen 744 693 701 524 671 421 253 163 Stomach wall 658 623 414 370 460 259 211 123 Whole brain 66.3 67.9 53.8 39.5 82.0 48.2 76.4 68.3 Epididymis 350 494 279 298 183 248 92.3 64.8 Heart 785 795 415 326 219 143 114 69.2 Lung 1493 1807 997 920 416 392 109 93.7 Bud 5682 6118 3300 4197 1227 1391 241 238 Liver 1423 1306 1257 865 1138 599 712 518 Small intestinal wall 726 778 641 816 490 334 210 90.9 Colon wall 550 520 672 823 1074 490 215 128 Whole blood 2087 3194 684 691 211 132 86.5 64.6 Blood plasma 7372 7610 1844 1760 495 306 214 140

From the above table it can be seen that the distribution in animal tissues does not show any difference between males and females; wherein after administration the drug is mainly distributed in the kidneys, whole blood (mainly distributed in blood plasma), liver and lungs; in all tissues, the highest concentration is reached after 0.5 hours, and then the drug is quickly eliminated, with the slowest elimination occurring in the liver and tumor; and slow clearance in the tumor may explain the benefits of CBP-1018 in terms of efficacy.

12. Excretion experiment in rats

Six normal SD rats, half male and half female, were administered 0.75 mg/70 μCi/kg [ ³ H]CBP-1018 by a single tail vein injection. At specified intervals, samples such as urine, feces, cell wash/cleaning solution and whole rat carcasses were collected before administration and 0-168 hours after administration, and samples such as bile, urine, feces and wash solution/ purification of BDC rat cells, collected before administration and 0-72 hours after administration. The above samples were cryopreserved in a low-temperature refrigerator (-10°C to -30°C).

An appropriate amount of scintillation liquid was added to each sample and mixed uniformly, and then the level of radioactivity was measured using a liquid scintillation counter. The level of radioactivity measured in samples such as bile, urine, feces, cell washing/cleaning solution and carcasses was used to calculate the percentage at a given dose. The level of radioactivity in the blood plasma sample was used to calculate the total level of radioactivity in each gram of sample. The main pharmacokinetic parameters of the total level of plasma radioactivity were calculated using WinNonLin software (version 7.0, Pharsight) in accordance with the non-compartmental model.

Table 29. Results of mass balance studies in whole rats after administration

Sex (number of rats) Urine
(%) Feces
(%) Cell washing/cleaning solution
(%) Carcass
(%) Total extraction
(%) Females (n=3) 50.26 ± 6.11 23.93 ± 0.98 5.70 ± 2.63 4.51 ± 1.19 84.40 ± 4.02 Males (n=3) 53.60 ± 3.68 20.61 ± 1.74 4.24 ± 2.16 4.83 ± 0.42 83.28 ± 0.71 Females and males
(n=3 females and 3 males) 51.93 ± 4.87 22.27 ± 2.21 4.97 ± 2.30 4.67 ± 0.82 83.84 ± 2.65

From the table above, it can be seen that CBP-1018 is primarily excreted in urine, which is approximately 57% of the total dose administered, and the amount excreted in feces is less than 25%. Our result demonstrating that excretion occurs primarily in urine via the kidneys is consistent with the finding of high levels of renal distribution in tissue distribution in nude mice.

13. Stability in blood plasma

CBP-1008 (its active ingredient is LDC10B, described in WO 2017025047 A1, and CBP-1008 is prepared by mixing LDC10B with auxiliary materials and lyophilizing them; WO 2017025047A1 is incorporated by reference in its entirety) and CBP-1018 at a concentration of 1 μg /ml, 10 μg/ml and 100 μg/ml were incubated with blood plasma from different species at 37°C for 2 hours to monitor the stability of these two compounds. The results demonstrate (in Table 30) that CBP-1018 is stable in the plasma of different species, and its residual percentage after 2 hours of incubation is more than 90% of the concentration at 0 hours. However, CBP-1008 is only stable in rat plasma (with residual levels of 87.92% - 91.82%) and is not stable in plasma of other species, with residual levels of only 0.751% - 24.52%.

Experimental results indicate that the stability of CBP-1018 in blood plasma is higher than that of CBP-1008.

Table 30. Summary of Plasma Stability Data for CBP-1008 and CBP-1018 at Various Concentrations in Species

(% relative to 0 hours)

Compound Concentration Mouse Rat Cynomolgus macaque Human CBP-1008 1 µg/ml 0.805 87.92 0.961 0.751 10 µg/ml 1.27 91.47 0.988 0.807 100 µg/ml 24.52 91.82 1.10 0.953 CBP-1018 1 µg/ml 94.4 92.2 97.3 99.1 10 µg/ml 96.5 95.0 95.8 102 100 µg/ml 98.1 94.8 101 103

Notes.

1) The data in the table represents the percentage of the concentration of the compound in the blood plasma after incubation of the blood plasma for 2 hours relative to 0 hours.

2) Since the data in the table is obtained from two experiments, the actual numbers after the decimal point are not consistent. For traceability and authenticity, the data in the report is mentioned directly without ensuring uniformity.

14. Half-life in PK study

Consistent with plasma stability comparisons, the pharmacokinetic half-life of CBP-1018 (approximately 1 hour) is significantly longer than that of CBP-1008 (approximately 20 minutes) in rats and cynomolgus monkeys, suggesting that CBP-1018 is more more stable than CBP-1008.

Table 31. Comparison of half-life (in hours) of CBP-1008 and CBP-1018 in a PK study in rats

0.15 mg/kg 0.5 mg/kg 1.5 mg/kg Male Female Male Female Male Female CBP-1008 0.245 0.215 0.249 0.202 0.222 0.368 CBP-1018 0.928 0.833 1.37 0.952 1.49 1.51

Claims (79)

1. A conjugated compound or a pharmaceutically acceptable salt thereof for delivering a payload to a subject in need thereof, comprising said payload and two targeting molecules, wherein said payload is conjugated to at least one of the targeting molecules, where the two targeting molecules are respectively a fragment, being a synergistic molecule, and a fragment being a ligand to a prostate-specific membrane antigen, wherein said synergistic molecule is a small molecule compound or polypeptide that binds to a molecule selected from the group consisting of FOLR1, TRPV6, SSTR2 and GNRHR, wherein the payload is selected from the group consisting of a small molecule compound, a nucleotide, a peptide and a protein; A prostate-specific membrane antigen ligand is an antibody, aptamer, or small molecule compound that is capable of specifically recognizing and binding to a prostate-specific membrane antigen. 2. A conjugated compound or a pharmaceutically acceptable salt thereof according to claim 1, where the ligand to the prostate-specific membrane antigen has the following structure: . 3. The conjugated compound or its pharmaceutically acceptable salt according to claim 1, where the ligand to the prostate-specific membrane antigen has the following structure: . 4. A conjugated compound or a pharmaceutically acceptable salt thereof according to claim 1, where the ligand to the prostate-specific membrane antigen has the following structure: . 5. The conjugated compound or its pharmaceutically acceptable salt according to claim 1, where the ligand to the prostate-specific membrane antigen has the following structure: . 6. A conjugated compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-5, wherein the synergistic molecule is P10, a ligand moiety represented by formula (I): , or a fragment representing a ligand having the structure: . 7. A conjugated compound or a pharmaceutically acceptable salt thereof for delivering a payload to a subject in need thereof, comprising said payload and two targeting molecules, wherein said payload is conjugated to at least one of the targeting molecules, where the two targeting molecules are respectively a fragment, being a synergistic molecule, and a moiety being a ligand represented by formula (I), and wherein said synergistic molecule is a low molecular weight compound or polypeptide that binds to a FOLR1 molecule, wherein the payload is selected from the group consisting of a small molecule compound, a nucleotide, a peptide and a protein, 8. A conjugated compound or a pharmaceutically acceptable salt thereof for delivering a payload to a subject in need thereof, comprising said payload and two targeting molecules, wherein said payload is conjugated to at least one of the targeting molecules, wherein the two targeting molecules are respectively a fragment, being a synergistic molecule, and P10, wherein P10 has the amino acid sequence Cys-Lys-Glu-Phe-Leu-His-Pro-Ser-Lys-Val-Asp-Leu-Pro-Arg, and wherein said synergistic molecule is a low molecular weight compound or polypeptide that binds to the FOLR1 molecule, and the payload is camptothecin or any derivative thereof. 9. A conjugated compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-8, wherein the synergistic molecule is folate or an analog thereof. 10. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 9, wherein the folate analogue is selected from the group consisting of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate and 5,10-methylenetetrahydrofolate. 11. A conjugate compound or a pharmaceutically acceptable salt thereof according to claim 1 or 7, containing one, two, three, four or more payloads. 12. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 1 or 7, wherein the payload is a low molecular weight compound. 13. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 12, where the low molecular weight compound is selected from the group consisting of camptothecin and any derivative thereof, dactinomycin and any derivative thereof, auristatin and any derivative thereof, maytansine and any derivative thereof, radionuclide complex, cyclooxygenase-2 inhibitor, paclitaxel and any derivative thereof, epothilone and any derivative thereof, bleomycin and any derivative thereof, plicamycin and any derivative thereof and mitomycin C. 14. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 13, wherein the low molecular weight compound is camptothecin or any derivative thereof, auristatin or any derivative thereof, a radionuclide complex or a cyclooxygenase-2 inhibitor. 15. A conjugated compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-14, wherein the payload is linked to at least one of the targeting molecules via a linker. 16. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 15, wherein the linker is a peptide linker, a disulfide linker, a pH-dependent linker, or a combination of the above linkers. 17. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 16, wherein the peptide linker is cleavable when exposed to a particular physiological environment by protease cleavage or reduction. 18. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 16 or 17, where 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. 19. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 16, wherein the disulfide linker is selected from the group consisting of DMDS, MDS, DSDM and NDMDS. 20. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 16, wherein the pH-dependent linker is cis-aconitic anhydride. 21. The conjugated compound or its pharmaceutically acceptable salt according to claim 15, where the linker is characterized by the following structures: , , , , , , , , , , , , or the linker is a combination of the above structures and a peptide linker. 22. A conjugated compound or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-21, where two targeting molecules are linked to each other via a spacer. 23. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 22, where the spacer contains 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. 24. The conjugated compound or a pharmaceutically acceptable salt thereof according to claim 1, where the conjugated compound is selected from the group consisting of the following compounds: (20R-SM09) and (CB-20R), where M represents a radionuclide. 25. The conjugated compound or a pharmaceutically acceptable salt thereof according to claim 6, where the conjugated compound is 26. The conjugated compound or a pharmaceutically acceptable salt thereof according to claim 7, where the conjugated compound is 27. A pharmaceutical composition for delivering a payload to a subject in need thereof, comprising a therapeutically effective amount of a conjugated compound or a pharmaceutically acceptable salt thereof according to any one of claims. 1-26 and a pharmaceutically acceptable carrier. 28. The pharmaceutical composition according to claim 27, wherein the composition is administered intravenously, subcutaneously, orally, intramuscularly or intraventricularly. 29. A method of delivering a payload to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate compound or a pharmaceutically acceptable salt thereof according to any one of claims. 1-26 or the pharmaceutical composition according to claim 27 or 28. 30. A method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate compound or a pharmaceutically acceptable salt thereof according to any one of claims. 1-26 or the pharmaceutical composition of claim 27 or 28, wherein the disease is selected from the group consisting of cancer, immune system disease, cardiovascular disease, metabolic disease and neurological disease. 31. The method according to claim 30, where the cancer is selected from the group consisting of prostate cancer, breast cancer, lung cancer, kidney cancer, leukemia, ovarian cancer, gastrointestinal cancer, uterine cancer, endometrial carcinoma, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, testicular cancer, skin cancer, lymphoma and multiple myeloma. 32. The method of claim 29, wherein the immune system disease is an autoimmune disease. 33. The method of claim 32, wherein the autoimmune disease is selected from the group consisting of connective tissue disease, systemic sclerosis, rheumatoid arthritis and systemic lupus erythematosus. 34. The method of claim 30, wherein the cardiovascular disease is selected from the group consisting of angina, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia and congenital heart disease. 35. The method of claim 30, wherein the metabolic disease is selected from the group consisting of diabetes, gout, obesity, hypoglycemia, hyperglycemia and dyslipidemia. 36. The method of claim 30, 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. 37. Method according to any one of paragraphs. 30-36, wherein the method further comprises administering one or more therapeutic agents in combination with a conjugate compound or a pharmaceutically acceptable salt or pharmaceutical composition thereof.

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