Preparation and anti-tumor application of target CXCR4-based polypeptide drug conjugate

Abstract

The present invention belongs to the field of cancer treatment and diagnostic drug development, and relates to a conjugate compound or a pharmaceutically acceptable salt thereof, the conjugate compound or the pharmaceutically acceptable salt thereof comprising a payload and two or more cell interacting molecules. The cell interaction molecule is a ligand capable of specifically binding to a cell surface receptor or a non-cell surface receptor. The payload of the present disclosure includes a cytotoxic drug capable of causing cancer cell death, inhibiting tumor growth, or a group capable of chelating with a radionuclide. The disclosure also provides conjugate compounds, markers of conjugate compounds and radionuclides, pharmaceutical compositions and their use in disease diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of cancer treatment and diagnostic drug development, and particularly relates to the application of a polypeptide drug conjugate based on chemokine receptor 4 (CXCR4) in the treatment and diagnosis of breast cancer. Background Art

[0002] Breast cancer is a common tumor among female tumors, and the incidence rate of breast cancer shows an increasing trend year by year worldwide. There are 1.2 million to 1.4 million newly diagnosed breast cancer cases globally every year, and approximately 500,000 people die from the disease every year. Once breast cancer metastasizes to the bone, the median survival period of patients is 2 years, and the 5-year survival rate is about 10%. Tumor cells express a variety of specific receptor proteins on their surfaces. As a member of the chemokine receptor family, chemokine receptor 4 (CXCR4) is highly expressed on the surfaces of various tumor cells, and its expression level is directly related to the malignancy of the tumor and the prognosis of the patient. The earliest research on CXCR4 mainly explored its role in the pathological process of acquired immunodeficiency syndrome. Müller et al. reported that CXCR4 was highly expressed in human breast cancer cell lines and tissues, and the role of CXCR4 in tumor pathological occurrence and development was studied. The results showed that the high expression of CXCR4 was related to tumor proliferation, angiogenesis, etc. Some studies have found that at least 23 different types of tumors, including hematological malignancies and other solid tumors, highly express CXCR4. Therefore, blocking the binding of CXCR4 and CXCL12 and inhibiting its biological activity can inhibit the progression of tumors.

[0003] In recent years, tumor-targeted therapy targeting CXCR4 has developed rapidly, and a variety of CXCR4 antagonists have been developed, including antibodies, polypeptides, and small molecule compounds. Among them, the drug with relatively significant therapeutic effect is AMD3100, which was approved by the FDA for marketing in 2008 and approved for marketing in China in 2018 for the treatment of multiple myeloma, non-Hodgkin lymphoma, hematopoietic stem cell transplantation, and lymphoma. However, except for this, other CXCR4 antagonists have not been approved for clinical cancer treatment. The main reason is that the killing power of these CXCR4 antagonists against tumors is limited, and antagonizing CXCR4 is not sufficient to achieve significant clinical treatment effects.

[0004] Due to the good affinity between CXCR4 antagonists and CXCR4, the radionuclide conjugates formed by coupling them with radionuclides have great potential in the field of PET imaging agents. Currently, the most clinically studied radionuclide drug for CXCR4-related diseases is Pentixafor. Pentixafor is the first PET imaging agent targeting CXCR4. It has high CXCR4 affinity, thus having high and persistent tumor uptake and appropriate overall pharmacokinetics. Pentixafor shows obvious lesion marking effects in the diagnosis of various diseases, including malignant tumors, atherosclerosis, fungal infections, systemic mastocytosis, etc. Among them, the imaging application of Pentixafor for tumors is the most extensive. Currently, it has successfully achieved imaging of tumors such as non-small cell lung cancer, lymphoma, breast cancer, esophageal cancer, and adrenocortical carcinoma in clinical trials. Compared with the traditional imaging agent FDG, Pentixafor can more accurately locate tumor tissues with high CXCR4 expression. In addition to being used for the imaging diagnosis of cancer, Pentixather, which has a similar structure to Pentixafor, also shows significant effects in the treatment of cancer. In a recent clinical study, 3 patients with advanced T-cell lymphoma underwent stem cell transplantation treatment after injection of 90Y-Pentixather, and their survival periods all exceeded 54 months, indicating that CXCR4-targeted radiotherapy plays an important role in the treatment of cancers with high CXCR4 expression.

[0005] Generally speaking, the binding force between CXCR4 antagonists and CXCR4 can well promote the drug to target the lesion area with high CXCR4 expression. Therefore, the research on targeted tumor treatment and diagnostic drugs based on CXCR4 antagonists is a very promising research direction, and polypeptide drug conjugates targeting CXCR4 will be applied to the precise and personalized targeted treatment of tumors. Summary of the Invention

[0006] This application relates to conjugate compounds or pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof, and methods of use thereof. More specifically, this application relates to multi-ligand drug conjugates, particularly multi-ligand drug conjugates capable of inducing endocytosis and pharmaceutical compositions thereof, methods of using the drug conjugates to treat and diagnose diseases, including but not limited to cancer.

[0007] One aspect of the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, the conjugate compound or the pharmaceutically acceptable salt thereof comprising a payload and two or more cell interaction molecules, wherein the payload is conjugated to at least one of the cell interaction molecules. The cell interaction molecule comprises a first ligand capable of specifically binding to a first cell surface receptor, and a second ligand capable of specifically binding to a second cell surface receptor or a third ligand capable of specifically binding to a third non-cell surface receptor.

[0008] In some embodiments, the first cell surface receptor, the second cell surface receptor, and the third non-cell surface receptor are different from each other.

[0009] In some embodiments, the first cell surface receptor, the second cell surface receptor, and the third cell surface receptor provided by the present application are independently selected from the group consisting of: chemokine receptor 4 (CXCR4), folate receptor (FR), integrin receptor (α v β3), fibroblast activation protein (FAP), albumin.

[0010] In some embodiments, the first ligand, the second ligand, and the third ligand are independently selected from the group consisting of: cyclic peptide T140 and its analogs, folic acid, cyclic peptide RGD and its analogs, FAPI-04, albumin ligand.

[0011] In some embodiments, the amino acid sequence of the cyclic peptide T140 and its analogs comprised by the ligand is: NH2-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-(D-Lys)-Pro-Tyr-Arg-Cit-Cys-Arg-COOH (two cysteines in the sequence form a disulfide bond), or an analog with substitution, deletion, or insertion of 1-3 amino acids, including substitution of Lys with Arg in the sequence, acetylation at the amino terminus, amidation at the carboxyl terminus, and incorporation of L-Lys into the side chain of D-Lys.

[0012] In some embodiments, at least one cell interaction molecule as described in the present application is an endocytosis molecule capable of mediating endocytosis. In some embodiments, the endocytosis molecule is further capable of specifically binding to a cell surface receptor.

[0013] In some embodiments, the ligand is selected from the group consisting of: cyclic peptide T140 and its analogs, folic acid, FAPI-04, cyclic peptide RGD and its analogs.

[0014] In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof comprises one or more payloads.

[0015] In some embodiments, the payload is a cytotoxic drug MMAE or a radionuclide chelating group, and the structure is as follows:

[0016]

[0017] In some embodiments, the radionuclide chelated by the chelating group is selected from 64 Cu.

[0018] Another aspect of the present application discloses a method for diagnosing or treating a disease characterized by overexpression of chemokine receptor 4 (CXCR4) in a subject, the method comprising administering to the subject a diagnostically effective amount of the conjugate compound provided by the present application or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition provided by the present application. In some embodiments, the disease is cancer.

[0019] In some embodiments, the cancer is selected from the group consisting of: breast cancer, lung cancer, prostate cancer, kidney cancer, ovarian cancer, gastric cancer, uterine cancer, endometrial cancer, liver cancer, thyroid cancer, pancreatic cancer, colon cancer, colorectal cancer, esophageal cancer, skin cancer, lymphoma, leukemia, and multiple myeloma.

[0020] In some embodiments, the conjugate compound is selected from the following structures:

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032] In the above structure, the linker C portion may be substituted with corresponding substituents, and the substituents may be selected from the following non-hydrogen substituents: at least one non-hydrogen substituent-substituted C1-12 alkyl, C3-10 cycloalkyl, C1-12 alkoxy, C5-12 aryl, C5-12 heteroaryl, hydroxy, halogen, and C1-4 alkoxycarbonyl, C3-10 cycloalkyl, C1-12 alkoxy, C5-12 aryl, C5-12 heteroaryl, hydroxy, and halogen, and the aryl and heteroaryl are unsubstituted or substituted with at least one non-hydrogen substituent selected from C1-4 alkyl, C1-4 alkoxy, hydroxy, and halogen.

[0033] As used herein, a "substituted" group refers to a group in which at least one hydrogen atom is replaced by at least one non-hydrogen atom group, provided that the group must meet the valence requirements and result in a chemically stable compound due to the substitution. In this specification, unless specifically described as "unsubstituted", it should be understood that all substituents may be substituted or unsubstituted.

[0034] "Alkyl" refers to straight-chain and branched-chain saturated hydrocarbon groups, usually having a specific number of carbon atoms (e.g., 1 to 12 carbon atoms). Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, and n-heptyl. The alkyl can be attached to the parent group or matrix through any ring atom, unless such attachment would violate the valence requirements. Similarly, the alkyl or alkenyl can contain at least one non-hydrogen substituent, unless such substitution would violate the valence requirements.

[0035] "Cycloalkyl" refers to saturated monocyclic and polycyclic hydrocarbon rings, usually having a specific number of carbon atoms including the ring (e.g., C3-10 cycloalkyl refers to a cycloalkyl having 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms as ring members). The cycloalkyl can be attached to the parent or matrix through any ring atom, unless such attachment would violate the valence requirements. Similarly, the cycloalkyl can contain at least one non-hydrogen substituent, unless such substitution would violate the valence requirements.

[0036] "Aryl" refers to monovalent and divalent aromatic groups, including 5- and 6-membered monocyclic aromatic groups respectively. "Heteroaryl" refers to monovalent and divalent aromatic groups, including 5- and 6-membered monocyclic aromatic groups containing 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Examples of monocyclic aryl and heteroaryl groups include, but are not limited to, phenyl, pyridyl, furyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrazinyl, pyridazinyl, pyrimidinyl, etc. Aryl and heteroaryl groups also include bicyclic groups, tricyclic groups, etc., including the above-mentioned fused 5- and 6-membered rings. Examples of polycyclic aryl and heteroaryl groups include, but are not limited to, isoquinolyl, naphthyl, biphenyl, anthracenyl, pyrenyl, carbazolyl, benzoxazolyl, benzodioxazolyl, benzothiazolyl, benzimidazolyl, benzothienyl, quinolinyl, indolyl, benzofuryl, purinyl, indolizinyl, etc. Aryl and heteroaryl groups can be attached to the parent group or matrix through any ring atom, unless such attachment would violate the valence requirements. Similarly, aryl and heteroaryl groups can contain at least one non-hydrogen substituent, unless such substitution would violate the valence requirements. The non-hydrogen substituents of aryl and heteroaryl groups can also be substituted by additional non-hydrogen substituents.

[0037] "Carbonyl" refers to -C(O)R'. As used herein, "(O)" refers to an oxygen atom connected by a double bond to an atom such as carbon or sulfur. Here, "R" refers to a non-hydrogen substituent such as a lower alkyl group, a lower alkoxy group, etc. Examples of carbonyl groups include, but are not limited to, 2-methoxyoxoethyl, 3-methoxyoxopropyl, etc. The carbonyl group can be attached to the parent group or matrix through any ring atom, unless such attachment would violate the valence requirements. Similarly, the carbonyl group can contain at least one non-hydrogen substituent, unless such substitution would violate the valence requirements.

[0038] "Alkoxy" refers to alkyl-O-. Here, the alkyl group is the same as defined above. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, etc. The alkoxy group can be attached to the parent group or matrix through any ring atom, unless such attachment would violate the valence requirements. Similarly, the alkoxy group can contain at least one non-hydrogen substituent, unless such substitution would violate the valence requirements.

[0039] "Hydroxy" refers to -OH, "halogen" refers to fluorine, chlorine, bromine, and iodine, and "oxo" refers to =O.

[0040] All amino acids used in this patent are L-amino acids except D-Lys.

[0041] The preparation method of the tumor-targeting polypeptide drug conjugate of the present invention includes the following steps:

[0042] a) Synthesize the T140 linear peptide and the T140 linear peptide linked with the second ligand or the third ligand by using the Fmoc solid-phase synthesis strategy;

[0043] b) Cyclize the T140 linear peptide in a) through a disulfide bond cyclization reaction to obtain a T140 cyclic peptide, and then connect it with a second ligand or a third ligand through a liquid-phase condensation reaction or a click reaction to generate a coupling intermediate; or directly cyclize the T140 linear peptide connected with a second ligand or a third ligand in a) through a disulfide bond cyclization reaction to obtain a coupling intermediate; or connect the T140 linear peptide or the T140 linear peptide connected with a second ligand or a third ligand with MMAE through a click reaction to generate a coupling intermediate;

[0044] c) For the coupling intermediate obtained in b), connect MMAE through a click reaction to generate a coupling intermediate to obtain a conjugate compound; or connect the coupling intermediate connected with MMAE obtained in b) with a radionuclide chelating group through a condensation reaction to obtain a conjugate compound.

[0045] In the present invention, in vivo activity evaluation was carried out on tumor-bearing mice with different types of cancer cells, and a polypeptide-drug conjugate with good in vivo anti-tumor activity was obtained. In addition, in the present invention, PET imaging tests were carried out on tumor-bearing mice, and a polypeptide-radionuclide conjugate with good tumor uptake effect was obtained. Therefore, the present invention provides an application of a polypeptide conjugate based on the chemokine receptor protein CXCR4 in the preparation of tumor-targeted therapeutic and diagnostic drugs. Among them, the tumors preferably include breast cancer and other tumors with abnormal CXCR4 expression.

[0046] On the other hand, the present invention provides a drug comprising the above-mentioned tumor-targeting polypeptide drug conjugate or a pharmaceutically acceptable salt thereof as an active ingredient.

[0047] The pharmaceutical composition of the present invention further comprises at least one pharmaceutically acceptable carrier in addition to the active ingredient. The "pharmaceutically acceptable carrier" used in the present invention refers to known pharmaceutically acceptable excipients, which can be used to formulate a medicinally active compound for administration to a subject and are substantially non-toxic and non-irritating under the conditions of use. The exact amount of the excipient is determined by standard pharmaceutical practice and the solubility, chemical properties of the active compound, and the selected route of administration.

[0048] The pharmaceutical composition of the present invention can be formulated into an appropriate form for the desired method of administration using suitable and physiologically acceptable adjuvants (such as excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, brighteners, flavoring agents, etc.).

[0049] The preparations of the pharmaceutical composition with the polypeptide drug conjugate as the active ingredient include various forms such as freeze-dried powder injections, injections, tablets, liposomes, and nano-formulations.

[0050] Advantages of the present invention: The polypeptide drug conjugate provided by the present invention connects a cytotoxic drug or a radionuclide group with a CXCR4-targeting polypeptide, achieving high-concentration enrichment and long-term retention of the cytotoxic drug or radionuclide at the tumor tissue site, enabling the cytotoxic drug or radionuclide to precisely target tumor cells, thereby realizing a more precise treatment and diagnosis function for CXCR4-highly expressed tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0052] Figure 1 It is the HRMS analysis spectrum of HLK-001.

[0053] Figure 2 It is the HRMS analysis spectrum of HLK-002.

[0054] Figure 3 It is the HRMS analysis spectrum of HLK-003.

[0055] Figure 4 It is the HRMS analysis spectrum of HLK-004.

[0056] Figure 5 It is the HRMS analysis spectrum of HLK-005

[0057] Figure 6 It is the HRMS analysis spectrum of HLK-006

[0058] Figure 7 It is the HRMS analysis spectrum of HLK-007.

[0059] Figure 8 It is the HRMS analysis spectrum of HLK-008.

[0060] Figure 9 It is the HRMS analysis spectrum of HLK-009.

[0061] Figure 10 It is the HRMS analysis spectrum of HLK-010.

[0062] Figure 11 It is the HRMS analysis spectrum of HLK-011.

[0063] Figure 12 It is the HRMS analysis spectrum of HLK-012.

[0064] Figure 13 It is the HLK-013 HRMS analysis spectrum.

[0065] Figure 14 It is the HLK-014 HRMS analysis spectrum.

[0066] Figure 15 It is the HLK-015 HRMS analysis spectrum

[0067] Figure 16 It is the HLK-016 HRMS analysis spectrum

[0068] Figure 17 It is the HLK-017 HRMS analysis spectrum.

[0069] Figure 18 It is the HLK-018 HRMS analysis spectrum.

[0070] Figure 19 It is the HLK-019 HRMS analysis spectrum.

[0071] Figure 20 It is the HLK-05 HRMS analysis spectrum.

[0072] Figure 21 It is the HLK-16 HRMS analysis spectrum.

[0073] Figure 22 It is the inhibitory activity graph of HLK-001 and HLK-007 against human breast cancer cell MCF-7 mouse xenograft tumors, including the effects of the drugs on the changes in the volume of the xenograft tumors and the body weight of the mice.

[0074] Figure 23 It is the inhibitory activity graph of HLK-007, HLK-008, HLK-009, HLK-010 and HLK-012 against human breast cancer cell MCF-7 mouse xenograft tumors, including the effects of the drugs on the changes in the volume of the xenograft tumors and the body weight of the mice.

[0075] Figure 24 It is the inhibitory activity graph of HLK-007, HLK-009, HLK-010, HLK-011, HLK-012, HLK-015, HLK-016, HLK-017 and HLK-018 against murine breast cancer cell 4T1 mouse xenograft tumors, including the effects of the drugs on the changes in the volume of the xenograft tumors and the body weight of the mice.

[0076] Figure 25 It is 64 Cu]HLK-05, 64 Cu]HLK-16 and 64 Cu]T140-DOTA PET-CT imaging results of human lymphoma cell Daudi mouse xenograft tumor model.

[0077] Figure 26 For 64 Cu]HLK-05, 64 Cu]HLK-16 and 64 Analysis of the organ uptake results of Cu]T140-DOTA in a xenograft tumor model of human lymphoma cell Daudi in mice. Detailed implementation manners

[0078] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative efforts shall fall within the scope protected by the present invention.

[0079] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative efforts shall fall within the scope protected by the present invention.

[0080] In some embodiments of the present invention, the cells that can be used include:

[0081] Human breast cancer cells: MCF-7;

[0082] Mouse breast cancer cells: 4T1;

[0083] Human lymphoma cells: Daudi;

[0084] The above cells can all be obtained commercially, for example, purchased from the Cell Bank of the Chinese Academy of Sciences..

[0085] Example 1 Chemical synthesis

[0086] (1) Synthesize the T140 linear peptide and the T140 linear peptide with functional groups (the R groups in the structure include folic acid, ibuprofen, 4-methylphenylbutyric acid, 4-methoxyphenylbutyric acid, 4-chlorophenylbutyric acid, truncated Evans blue, dodecanoic acid, hexadecanoic acid, 4-iodophenyl terminal group), and the structure is as follows:

[0087]

[0088]

[0089] The synthesis route is as shown below:

[0090]

[0091]

[0092] The specific method is as follows:

[0093] Peptide washing method: DMF (3 × 10 mL × 2 min).

[0094] Method for removing Fmoc protecting group: 3% piperidine + 3% DBU + 94% DMF (3 × 10 mL × 5 min).

[0095] Method for removing Dde protecting group: 2% hydrazine hydrate + 98% DMF (3 × 10 mL × 3 min)

[0096] Ninhydrin test: Add prepared ninhydrin, phenol, and pyridine, dosage = 1 drop: 1 drop: 2 drops, heat at 100 °C for 30 s - 90 s. If there is an exposed amino group, the system turns blue or light brown; if there is no exposed amino group, the system is colorless.

[0097] Method for introducing the next amino acid: 3 eq amino acid + 3 eq HBTU (different condensing agents can be selected according to different situations) + 4 eq HOBT + 6 eq DIEA, 10 mL of DMF, shake at room temperature for 1 - 4 h.

[0098] Peptide cleavage: 95% TFA + 2% H2O + 2% TIPs + 1% 2 - Mercaptoethanol, shake for 3 h.

[0099] Peptide extraction, HPLC preparation, and lyophilization: 10 mL of ice - cold diethyl ether, 2 × 10 mL of water, collect the aqueous phase, HPLC preparation: acetonitrile / water, acetonitrile: 20% - 100%. Lyophilize.

[0100] Synthesis of compound a: Swell (434 mg, 0.2 mmol) MBHA resin in 10 mL of DCM for 30 min, wash with DMF 3 × 10 ml × 2 min. Introduce Fmoc - Arg(pbf) - OH, wash the peptide, remove Fmoc, wash the peptide, perform ninhydrin test, and then successively introduce Cys - Cit - Arg - Tyr - Pro - (D - Lys) - Lys - Cit - Tyr - Cys - Nal - Arg - Arg - azidoacetic acid (D - Lys is Fmoc - D - Lys(Boc) - OH) in the order of (introduce amino acid → ninhydrin test → remove Fmoc → wash peptide → ninhydrin test → introduce the next amino acid), cleave the peptide, extract the peptide, perform HPLC preparation, and lyophilize to obtain product a (0.09 mmol, 200 mg, yield: 47%).

[0101] Synthesis of intermediate compounds of series b: (434 mg, 0.2 mmol) MBHA resin was swollen in 10 mL of DCM for 30 min and washed with DMF 3×10 ml×2 min. Fmoc-Arg(pbf)-OH was introduced, the peptide was washed, the Fmoc group was removed, the peptide was washed, and ninhydrin test was performed. Subsequently, Cys-Cit-Arg-Tyr-Pro-(D-Lys)-Lys-Cit-Tyr-Cys-Nal-Arg-Arg (D-Lys is Fmoc-D-Lys(Boc)-OH) was introduced successively according to the sequence of (introducing amino acid → ninhydrin test → removing Fmoc → washing peptide → ninhydrin test → introducing the next amino acid). The terminal Fmoc protecting group was removed, Fmoc-L-Lys(Dde)-OH was introduced, the Fmoc protecting group was removed again, azidoacetic acid was introduced, and finally the Dde protecting group was removed. Different functional groups (folic acid, ibuprofen, 4-methylphenylbutyric acid, 4-methoxyphenylbutyric acid, 4-chlorophenylbutyric acid) were introduced, the peptide was cleaved, the peptide was extracted, prepared by HPLC, and lyophilized to obtain products b-1 (0.060 mmol, 160 mg, yield: 30%), b-2 (0.074 mmol, 180 mg, yield: 37%), b-3 (0.073 mmol, 175 mg, yield: 37%), b-4 (0.078 mmol, 188 mg, yield: 39%), b-5 (0.076 mmol, 184 mg, yield: 38%).

[0102] The structures of compound a and intermediate compounds of series b are as follows:

[0103]

[0104] Synthesis of compound c: (434 mg, 0.2 mmol) MBHA resin was swollen in 10 mL of DCM for 30 min and washed with DMF 3×10 ml×2 min. Fmoc-Arg(pbf)-OH was introduced, the peptide was washed, the Fmoc group was removed, the peptide was washed, and ninhydrin test was performed. Subsequently, Cys-Cit-Arg-Tyr-Pro-(D-Lys)-Lys-Cit-Tyr-Cys-Nal-Arg-Arg (D-Lys is Fmoc-D-Lys(Dde)-OH) was introduced successively according to the sequence of (introducing amino acid → ninhydrin test → removing Fmoc → washing peptide → ninhydrin test → introducing the next amino acid). The Dde protecting group was removed, azidoacetic acid was introduced, and finally the terminal Fmoc protecting group was removed. The peptide was cleaved, the peptide was extracted, prepared by HPLC, and lyophilized to obtain product c (0.09 mmol, 190 mg, yield: 45%).

[0105] Synthesis of intermediate compounds of series d: (434 mg, 0.2 mmol) MBHA resin was swollen in 10 mL of DCM for 30 min and washed with DMF three times (10 mL each time, 2 min each time). Fmoc-Arg(pbf)-OH was introduced, the peptide was washed, the Fmoc group was removed, the peptide was washed, and indan detection was carried out. Subsequently, Cys-Cit-Arg-Tyr-Pro-(D-Lys)-Lys-Cit-Tyr-Cys-Nal-Arg-Arg (where (D-Lys) is Fmoc-D-Lys(Dde)-OH) was introduced successively according to the sequence of (introducing amino acid → indan detection → removing Fmoc → washing peptide → indan detection → introducing the next amino acid). The Dde protecting group was removed, Fmoc-L-Lys(Dde)-OH was introduced, the Fmoc protecting group was removed again, azidoacetic acid was introduced, and finally the Dde protecting group was removed. Different functional groups (folic acid, ibuprofen, 4-methylphenylbutyric acid, 4-methoxyphenylbutyric acid, 4-chlorophenylbutyric acid) were introduced, the peptide was cleaved, the peptide was extracted, prepared by HPLC, and freeze-dried to obtain products d-1 (0.062 mmol, 165 mg, yield: 31%), d-2 (0.062 mmol, 150 mg, yield: 31%), d-3 (0.069 mmol, 166 mg, yield: 34%), d-4 (0.069 mmol, 167 mg, yield: 34%), d-5 (0.073 mmol, 176 mg, yield: 36%).

[0106] The structures of compound c and intermediate compounds of series d are as follows:

[0107]

[0108] Synthesis of compound e: (434 mg, 0.2 mmol) MBHA resin was swollen in 10 mL of DCM for 30 min and washed with DMF three times (10 mL each time, 2 min each time). Fmoc-Arg(pbf)-OH was introduced, the peptide was washed, the Fmoc group was removed, the peptide was washed, and indan detection was carried out. Subsequently, Cys-Cit-Arg-Tyr-Pro-(D-Lys)-Arg-Cit-Tyr-Cys-Nal-Arg-Arg-Ac (where (D-Lys) is Fmoc-D-Lys(Dde)-OH) was introduced successively according to the sequence of (introducing amino acid → indan detection → removing Fmoc → washing peptide → indan detection → introducing the next amino acid). The Dde protecting group was removed, Fmoc-L-Lys(Dde)-OH was introduced, the Fmoc protecting group was removed again, azidoacetic acid was introduced, and finally the Dde protecting group was removed. The peptide was cleaved, the peptide was extracted, prepared by HPLC, and freeze-dried to obtain product e (0.08 mmol, 186 mg, yield: 40%).

[0109] Synthesis of f-series intermediate compounds: (434 mg, 0.2 mmol) MBHA resin was swollen in 10 mL of DCM for 30 min and washed with DMF 3×10 ml×2 min. Fmoc-Arg(pbf)-OH was introduced, the peptide was washed, Fmoc was removed, the peptide was washed, and ninhydrin test was performed. Subsequently, Cys-Cit-Arg-Tyr-Pro-(D-Lys)-Arg-Cit-Tyr-Cys-Nal-Arg-Arg-Ac was introduced in sequence according to the sequence of (introducing amino acid → ninhydrin test → removing Fmoc → washing peptide → ninhydrin test → introducing the next amino acid) (D-Lys is Fmoc-D-Lys(Dde)-OH). The Dde protecting group was removed, Fmoc-L-Lys(Dde)-OH was introduced, the Fmoc protecting group was removed again, azidoacetic acid was introduced, and finally the Dde protecting group was removed, and different functional groups (dodecanoic acid, hexadecanoic acid) were introduced. The peptide was cleaved, extracted, prepared by HPLC, and lyophilized to obtain product f-1 (0.062 mmol, 172 mg, yield: 31%), f-2 (0.062 mmol, 150 mg, yield: 31%).

[0110] Synthesis of compound g: (434 mg, 0.2 mmol) MBHA resin was swollen in 10 mL of DCM for 30 min and washed with DMF 3×10 ml×2 min. Fmoc-Arg(pbf)-OH was introduced, the peptide was washed, Fmoc was removed, the peptide was washed, and ninhydrin test was performed. Subsequently, Cys-Cit-Arg-Tyr-Pro-(D-Lys)-Arg-Cit-Tyr-Cys-Nal-Arg-Arg-Ac was introduced in sequence according to the sequence of (introducing amino acid → ninhydrin test → removing Fmoc → washing peptide → ninhydrin test → introducing the next amino acid) (D-Lys is Fmoc-D-Lys(Dde)-OH). The Dde protecting group was removed, Fmoc-L-Lys(Boc)-OH was introduced, the Fmoc protecting group was removed, Fmoc-L-Lys(Dde)-OH was introduced, the Fmoc protecting group was removed again, azidoacetic acid was introduced, and finally the Dde protecting group was removed, and folic acid (FA) was introduced. The peptide was cleaved, extracted, prepared by HPLC, and lyophilized to obtain product g (0.042 mmol, 120 mg, yield: 21%).

[0111] The structures of the f-series intermediate compounds of the compound are as follows:

[0112]

[0113] (2) Synthesis of HA-Val-Cit-PAB-MMAE(5), Glu-HA-Val-Cit-PAB-MMAE(9), Gly-Pro-MMAE(12) fragments:

[0114]

[0115]

[0116] The specific synthesis method is as follows:

[0117] Synthesis of HA-Val-Cit-PAB-MMAE(5):

[0118] Dissolve compound 1 (500 mg, 1.05 mmol) and compound 2 (384 mg, 3.15 mmol) in 10 mL of DMF, then add DIEA (30 mg, 0.231 mmol), and stir at room temperature for 5 h. Concentrate under reduced pressure, add 40 mL of ethyl acetate, filter, and wash with ethyl acetate 3×30 mL to obtain compound 3 (620 mg, 0.97 mmol, yield 92%).

[0119] Dissolve compound 3 (267 mg, 0.42 mmol), compound 4 (300 mg, 0.42 mmol) and HOBT (25 mg, 0.1878 mmol) in 6 mL of DMF, then add (883 mg, 11.18 mmol) pyridine, and stir overnight. Concentrate under reduced pressure, dissolve in acetonitrile / water, and prepare by HPLC: 0.1% TFA acetonitrile / 0.1% TFA water, 20%-100% acetonitrile from 0 to 80 min, flow rate: 8 ml / min, Rt = 40 min, and dry under reduced pressure to obtain compound 5 (280 mg, 0.23 mmol, yield 55%).

[0120] Synthesis of HA-Glu-Val-Cit-PAB-MMAE(9):

[0121] Dissolve compound 6 (691 mg, 1.05 mmol) and compound 2 (384 mg, 3.15 mmol) in 10 mL of DMF, then add DIEA (30 mg, 0.231 mmol), and stir at room temperature for 5 h. Concentrate under reduced pressure, add 40 mL of ethyl acetate, filter, and wash with ethyl acetate 3×30 mL to obtain compound 7 (749 mg, 0.91 mmol, yield 87%).

[0122] Dissolve compound 7 (344 mg, 0.42 mmol), compound 4 (300 mg, 0.42 mmol) and HOBT (25 mg, 0.1878 mmol) in 6 mL of DMF, then add (883 mg, 11.18 mmol) pyridine, and stir overnight. Concentrate under reduced pressure, dissolve in acetonitrile / water, and prepare by HPLC: 0.1% TFA acetonitrile / 0.1% TFA water, 20%-100% acetonitrile from 0 to 80 min, flow rate: 8 ml / min, Rt = 45 min, and dry under reduced pressure to obtain compound 8 (305 mg, 0.22 mmol, yield 53%).

[0123] Compound 8 (305 mg, 0.22 mmol) was dissolved in 1 mL of 20% TFA / TCM, stirred at room temperature for 1 h, concentrated under reduced pressure, dissolved in acetonitrile / water, and prepared by HPLC: 0.1% TFA acetonitrile / 0.1% TFA water, acetonitrile 20%-100% from 0 to 80 min, flow rate: 8 ml / min, Rt = 42 min. After drying under reduced pressure, compound 9 (195 mg, 0.14 mmol, yield 66%) was obtained.

[0124] Synthesis of HA-Gly-Pro-PAB-MMAE (12):

[0125] Compound 10 (390 mg, 1.05 mmol) and compound 2 (384 mg, 3.15 mmol) were dissolved in 10 mL of DMF, then DIEA (30 mg, 0.231 mmol) was added, and the mixture was stirred at room temperature for 5 h. Concentrated under reduced pressure, 40 mL of ethyl acetate was added, filtered, and washed with ethyl acetate 3×30 mL to obtain compound 11 (335 mg, 0.62 mmol, yield 59%).

[0126] Compound 11 (224 mg, 0.42 mmol), compound 4 (300 mg, 0.42 mmol) and HOBT (25 mg, 0.1878 mmol) were dissolved in 6 mL of DMF, then pyridine (883 mg, 11.18 mmol) was added, and the mixture was stirred overnight. Concentrated under reduced pressure, dissolved in acetonitrile / water, and prepared by HPLC: 0.1% TFA acetonitrile / 0.1% TFA water, acetonitrile 20%-100% from 0 to 80 min, flow rate: 8 ml / min, Rt = 40 min. After drying under reduced pressure, compound 12 (215 mg, 0.19 mmol, yield 45%) was obtained.

[0127] (2) Synthesis of HLK-001:

[0128]

[0129] The specific method is as follows:

[0130] The intermediate compound a (100 mg, 0.047 mmol) was added to a round-bottom flask, 50 mL of an aqueous solution of 20% DMSO was added, then NH4HCO3 (40 mg, 0.5 mmol) was added. The round-bottom flask was left open and stirred at room temperature for 24 h, then freeze-dried, prepared, and freeze-dried to obtain the disulfide-bond cyclized product T140-a (60 mg, 0.028 mmol, yield 60%).

[0131] (19 mg, 0.009 mmol) of T140-a, (11 mg, 0.009 mmol) of HA-Val-Cit-PAB-MMAE (5), (2.3 mg, 0.009 mmol) of CuSO4·5H2O, and (3.6 mg, 0.018 mmol) of VcNa were added to a round-bottom flask. The solvent H2O / DMF (0.4 mL: 1.6 mL) was added, and the mixture was stirred overnight at room temperature. It was concentrated under reduced pressure. For HPLC preparation: acetonitrile / water, acetonitrile: 20% - 100%. After lyophilization, HLK-001 (20 mg, 0.006 mmol, yield 64%) was obtained. HRMS (ESI): C 156 H 242 N 46 O 32 S2[M+H] 4+ calcd: 834.9614, found: 834.9620. Purity: >99.00%.

[0132] (3) Synthesis of HLK-002:

[0133]

[0134] The specific method is as follows:

[0135] (100 mg, 0.037 mmol) of intermediate compound b-1 was added to a round-bottom flask. 50 mL of an aqueous solution of 20% DMSO was added, and then (40 mg, 0.5 mmol) of NH4HCO3 was added. The round-bottom flask was left open, and the mixture was stirred at room temperature for 24 h. After lyophilization and preparation, the disulfide-bond cyclized product T140-b-1 (65 mg, 0.024 mmol, yield 65%) was obtained.

[0136] (24 mg, 0.009 mmol) of T140-b-1, (11 mg, 0.009 mmol) of HA-Val-Cit-PAB-MMAE, (2.3 mg, 0.009 mmol) of CuSO4·5H2O, and (3.6 mg, 0.018 mmol) of VcNa were added to a round-bottom flask. The solvent H2O / DMF (0.4 mL: 1.6 mL) was added, and the mixture was stirred overnight at room temperature. It was concentrated under reduced pressure. For HPLC preparation: acetonitrile / water, acetonitrile: 20% - 100%. After lyophilization, HLK-002 (19.5 mg, 0.005 mmol, yield 56%) was obtained. HRMS (ESI): C 181 H 271 N 55 O 38 S2[M+H] 3+ calcd: 1296.6875, found: 1296.6861. Purity: >99.00%.

[0137] (4) Synthesis of HLK-003:

[0138]

[0139] The specific method is as follows:

[0140] Add the intermediate compound b-2 (100 mg, 0.041 mmol) to a round-bottom flask, add 50 mL of an aqueous solution of 20% DMSO, then add NH4HCO3 (40 mg, 0.5 mmol). Keep the round-bottom flask open and stir at room temperature for 24 h. Freeze-dry to prepare, and freeze-dry to obtain the disulfide bond cyclized product T140-b-2 (70 mg, 0.029 mmol, yield 70%).

[0141] Add T140-b-2 (22 mg, 0.009 mmol), HA-Val-Cit-PAB-MMAE (11 mg, 0.009 mmol), CuSO4·5H2O (2.3 mg, 0.009 mmol) and VcNa (3.6 mg, 0.018 mmol) to a round-bottom flask, add the solvent H2O / DMF (0.4 mL: 1.6 mL), stir at room temperature overnight, concentrate under reduced pressure, and prepare by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Freeze-dry to obtain HLK-003 (22 mg, 0.006 mmol, yield 67%). HRMS (ESI): C 175 H 270 N 48 O 34 S2[M+H] 4+ calcd: 914.0152, found: 914.0163. Purity: >99.00%.

[0142] (5) Synthesis of HLK-004:

[0143]

[0144] The specific method is as follows:

[0145] Add the intermediate compound b-3 (100 mg, 0.042 mmol) to a round-bottom flask, add 50 mL of an aqueous solution of 20% DMSO, then add NH4HCO3 (40 mg, 0.5 mmol). Keep the round-bottom flask open and stir at room temperature for 24 h. Freeze-dry to prepare, and freeze-dry to obtain the disulfide bond cyclized product T140-b-3 (66 mg, 0.027 mmol, yield 66%).

[0146] (22 mg, 0.009 mmol) of T140-b-3, (11 mg, 0.009 mmol) of HA-Val-Cit-PAB-MMAE, (2.3 mg, 0.009 mmol) of CuSO4·5H2O and (3.6 mg, 0.018 mmol) of VcNa were added to a round-bottom flask, and the solvent H2O / DMF (0.4 mL: 1.6 mL) was added. The mixture was stirred overnight at room temperature, concentrated under reduced pressure, and prepared by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Freeze-drying gave HLK-004 (18 mg, 0.005 mmol, yield 55%). HRMS (ESI): C 173 H 266 N 48 O 34 S2[M+H] 4+ calcd: 907.0073, found: 907.0079. Purity: >99.00%.

[0147] (6) Synthesis of HLK-005:

[0148]

[0149] The specific method is as follows:

[0150] (100 mg, 0.041 mmol) of intermediate compound b-4 was added to a round-bottom flask, 50 mL of an aqueous solution of 20% DMSO was added, and then (40 mg, 0.5 mmol) of NH4HCO3 was added. The round-bottom flask was left open and stirred at room temperature for 24 h, and then freeze-dried. Preparation and freeze-drying gave the disulfide-bond cyclized product T140-b-4 (59 mg, 0.024 mmol, yield 59%).

[0151] (22 mg, 0.009 mmol) of T140-b-4, (11 mg, 0.009 mmol) of HA-Val-Cit-PAB-MMAE, (2.3 mg, 0.009 mmol) of CuSO4·5H2O and (3.6 mg, 0.018 mmol) of VcNa were added to a round-bottom flask, and the solvent H2O / DMF (0.4 mL: 1.6 mL) was added. The mixture was stirred overnight at room temperature, concentrated under reduced pressure, and prepared by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Freeze-drying gave HLK-005 (19 mg, 0.005 mmol, yield 58%). HRMS (ESI): C 173 H 266 N 48 O 35 S2[M+H] 4+ calcd: 911.0061, found: 911.0068. Purity: >99.00%.

[0152] (7) Synthesis of HLK-006:

[0153]

[0154] The specific method is as follows:

[0155] Add the intermediate compound b-5 (100 mg, 0.041 mmol) into a round-bottom flask, add 50 mL of an aqueous solution of 20% DMSO, then add NH4HCO3 (40 mg, 0.5 mmol). The round-bottom flask is left open and stirred at room temperature for 24 h, followed by freeze-drying to prepare the disulfide-bond cyclized product T140-b-5 (70 mg, 0.029 mmol, yield 70%).

[0156] Add T140-b-5 (22 mg, 0.009 mmol), HA-Val-Cit-PAB-MMAE (11 mg, 0.009 mmol), CuSO4·5H2O (2.3 mg, 0.009 mmol) and VcNa (3.6 mg, 0.018 mmol) into a round-bottom flask, add the solvent H2O / DMF (0.4 mL: 1.6 mL), stir at room temperature overnight, concentrate under reduced pressure, and prepare by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Freeze-dry to obtain HLK-006 (15 mg, 0.004 mmol, yield 46%). HRMS (ESI): C 172 H 263 ClN 48 O 34 S2[M + H] 3+ calcd: 1215.6558, found: 1215.6573. Purity: >99.00%.

[0157] (8) Synthesis of HLK-007:

[0158]

[0159] The specific method is as follows:

[0160] Add the intermediate compound c (100 mg, 0.047 mmol) into a round-bottom flask, add 50 mL of an aqueous solution of 20% DMSO, then add NH4HCO3 (40 mg, 0.5 mmol). The round-bottom flask is left open and stirred at room temperature for 24 h, followed by freeze-drying to prepare the disulfide-bond cyclized product T140-c (66 mg, 0.031 mmol, yield 66%).

[0161] (19 mg, 0.009 mmol) of T140-c, (11 mg, 0.009 mmol) of HA-Val-Cit-PAB-MMAE, (2.3 mg, 0.009 mmol) of CuSO4·5H2O and (3.6 mg, 0.018 mmol) of VcNa were added to a round-bottom flask, and the solvent H2O / DMF (0.4 mL: 1.6 mL) was added. The mixture was stirred overnight at room temperature, concentrated under reduced pressure, and prepared by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Lyophilization gave HLK-007 (17 mg, 0.005 mmol, yield 57%). HRMS (ESI): C 156 H 242 N 46 O 32 S2[M+H] 4+ calcd: 834.9614, found: 834.9613. Purity: >99.00%.

[0162] (9) Synthesis of HLK-008:

[0163]

[0164] The specific method is as follows:

[0165] (100 mg, 0.037 mmol) of intermediate compound d-1 was added to a round-bottom flask, 50 mL of an aqueous solution of 20% DMSO was added, and then (40 mg, 0.5 mmol) of NH4HCO3 was added. The round-bottom flask was left open and stirred at room temperature for 24 h, followed by lyophilization. Preparation by lyophilization gave the disulfide cyclized product T140-d-1 (60 mg, 0.022 mmol, yield 60%).

[0166] (24 mg, 0.009 mmol) of T140-d-1, (11 mg, 0.009 mmol) of HA-Val-Cit-PAB-MMAE, (2.3 mg, 0.009 mmol) of CuSO4·5H2O and (3.6 mg, 0.018 mmol) of VcNa were added to a round-bottom flask, and the solvent H2O / DMF (0.4 mL: 1.6 mL) was added. The mixture was stirred overnight at room temperature, concentrated under reduced pressure, and prepared by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Lyophilization gave HLK-008 (23 mg, 0.006 mmol, yield 66%). HRMS (ESI): C 181 H 271 N 55 O 38 S2[M+H] 3+ calcd: 972.7674, found: 972.7685. Purity: >99.00%.

[0167] (10) Synthesis of HLK-009:

[0168]

[0169] The specific method is as follows:

[0170] Add the intermediate compound d-2 (100 mg, 0.041 mmol) to a round-bottom flask, add 50 mL of an aqueous solution of 20% DMSO, then add NH4HCO3 (40 mg, 0.5 mmol). Keep the round-bottom flask open and stir at room temperature for 24 h, then freeze-dry to prepare the disulfide-bond cyclized product T140-d-2 (52 mg, 0.021 mmol, yield 52%).

[0171] Add T140-d-2 (22 mg, 0.009 mmol), HA-Val-Cit-PAB-MMAE (11 mg, 0.009 mmol), CuSO4·5H2O (2.3 mg, 0.009 mmol) and VcNa (3.6 mg, 0.018 mmol) to a round-bottom flask, add the solvent H2O / DMF (0.4 mL: 1.6 mL), stir at room temperature overnight, concentrate under reduced pressure, and prepare by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Freeze-dry to obtain HLK-009 (18 mg, 0.005 mmol, yield 55%). HRMS (ESI): C 175 H 270 N 48 O 34 S2[M+H] 4+ calcd: 914.0152, found: 914.0169. Purity: >99.00%.

[0172] (11) Synthesis of HLK-010:

[0173]

[0174] The specific method is as follows:

[0175] Add the intermediate compound d-3 (100 mg, 0.042 mmol) to a round-bottom flask, add 50 mL of an aqueous solution of 20% DMSO, then add NH4HCO3 (40 mg, 0.5 mmol). Keep the round-bottom flask open and stir at room temperature for 24 h, then freeze-dry to prepare the disulfide-bond cyclized product T140-d-3 (40 mg, 0.017 mmol, yield 40%).

[0176] (22 mg, 0.009 mmol) of T140-d-3, (11 mg, 0.009 mmol) of HA-Val-Cit-PAB-MMAE, (2.3 mg, 0.009 mmol) of CuSO4·5H2O and (3.6 mg, 0.018 mmol) of VcNa were added to a round-bottom flask. The solvent H2O / DMF (0.4 mL: 1.6 mL) was added, and the mixture was stirred overnight at room temperature, concentrated under reduced pressure, and prepared by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Lyophilization gave HKLK-010 (21 mg, 0.006 mmol, yield 64%). HRMS (ESI): C 173 H 266 N 48 O 34 S2[M+H] 4+ calcd: 907.0073, found: 907.0065. Purity: >99.00%.

[0177] (12) Synthesis of HLK-011:

[0178]

[0179] The specific method is as follows:

[0180] (100 mg, 0.041 mmol) of the intermediate compound d-4 was added to a round-bottom flask. 50 mL of an aqueous solution of 20% DMSO was added, and then (40 mg, 0.5 mmol) of NH4HCO3 was added. The round-bottom flask was left open and stirred at room temperature for 24 h, and then lyophilized. The disulfide-bond cyclized product T140-d-4 (53 mg, 0.022 mmol, yield 53%) was obtained by preparation and lyophilization.

[0181] (22 mg, 0.009 mmol) of T140-d-4, (11 mg, 0.009 mmol) of HA-Val-Cit-PAB-MMAE, (2.3 mg, 0.009 mmol) of CuSO4·5H2O and (3.6 mg, 0.018 mmol) of VcNa were added to a round-bottom flask. The solvent H2O / DMF (0.4 mL: 1.6 mL) was added, and the mixture was stirred overnight at room temperature, concentrated under reduced pressure, and prepared by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Lyophilization gave HLK-011 (19 mg, 0.005 mmol, yield 58%). HRMS (ESI): C 173 H 266 N 48 O 35 S2[M+H] 4+ calcd: 911.0061, found: 911.0065. Purity: >99.00%.

[0182] (13) Synthesis of HLK-012:

[0183]

[0184] The specific method is as follows:

[0185] Add the intermediate compound d-5 (100 mg, 0.041 mmol) into a round-bottom flask, add 50 mL of an aqueous solution of 20% DMSO, then add NH4HCO3 (40 mg, 0.5 mmol). Keep the round-bottom flask open and stir at room temperature for 24 h, then lyophilize to prepare. After lyophilization, the disulfide-bond cyclized product T140-d-5 (61 mg, 0.025 mmol, yield 61%) is obtained.

[0186] Add T140-d-5 (22 mg, 0.009 mmol), HA-Val-Cit-PAB-MMAE (11 mg, 0.009 mmol), CuSO4·5H2O (2.3 mg, 0.009 mmol) and VcNa (3.6 mg, 0.018 mmol) into a round-bottom flask, add the solvent H2O / DMF (0.4 mL: 1.6 mL), stir at room temperature overnight, concentrate under reduced pressure, and prepare by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. After lyophilization, HLK-012 (20 mg, 0.005 mmol, yield 61%) is obtained. HRMS (ESI): C 172 H 263 ClN 48 O 34 S2[M+H] 4+ calcd: 911.9937, found: 911.9946. Purity: >99.00%.

[0187] (14) Synthesis of HLK-013:

[0188]

[0189] Add T140-c (19 mg, 0.009 mmol), HA-Gly-Pro-PAB-MMAE (10 mg, 0.009 mmol), CuSO4·5H2O (2.3 mg, 0.009 mmol) and VcNa (3.6 mg, 0.018 mmol) into a round-bottom flask, add the solvent H2O / DMF (0.4 mL: 1.6 mL), stir at room temperature overnight, concentrate under reduced pressure, and prepare by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. After lyophilization, HLK-013 (16 mg, 0.005 mmol, yield 55%) is obtained. HRMS (ESI): C 152 H232 N 44 O 31 S2[M+H] 4+ Calculated: 809.4416, Found: 809.4411. Purity: >99.00%.

[0190] (15) Synthesis of HLK-014:

[0191]

[0192] The specific method is as follows:

[0193] Add (24 mg, 0.009 mmol) T140-d-1, (12 mg, 0.009 mmol) HA-Glu-Val-Cit-PAB-MMAE, (2.3 mg, 0.009 mmol) CuSO4·5H2O and (3.6 mg, 0.018 mmol) VcNa into a round-bottom flask, add the solvent H2O / DMF (0.4 mL: 1.6 mL), stir overnight at room temperature, concentrate under reduced pressure, prepare by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Lyophilize to obtain HLK-014 (16 mg, 0.004 mmol, yield 44%). HRMS (ESI): C 186 H 278 N 56 O 41 S2[M+H] 4+ Calculated: 1005.0281, Found: 1005.0295. Purity: >99.00%.

[0194] (16) Synthesis of HLK-015:

[0195]

[0196] The specific method is as follows:

[0197] Add (100 mg, 0.040 mmol) intermediate compound f-1 into a round-bottom flask, add 50 mL of an aqueous solution of 20% DMSO, then add (40 mg, 0.5 mmol) NH4HCO3, keep the round-bottom flask open, stir at room temperature for 24 h, lyophilize, prepare, and lyophilize to obtain the disulfide-bond cyclized product T140-f-1 (65 mg, 0.026 mmol, yield 65%).

[0198] (22.5 mg, 0.009 mmol) of T140-f-1, (11 mg, 0.009 mmol) of HA-Val-Cit-PAB-MMAE, (2.3 mg, 0.009 mmol) of CuSO4·5H2O and (3.6 mg, 0.018 mmol) of VcNa were added to a round-bottom flask, and the solvent H2O / DMF (0.4 mL: 1.6 mL) was added. The mixture was stirred overnight at room temperature, concentrated under reduced pressure, and prepared by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Lyophilization gave HLK-015 (20 mg, 0.005 mmol, yield 60%). HRMS (ESI): C 176 H 278 N 50 O 35 S2[M+H] 4+ calcd: 930.0311, found: 930.0348. Purity: >99.00%.

[0199] (17) Synthesis of HLK-016:

[0200]

[0201] The specific method is as follows:

[0202] (100 mg, 0.039 mmol) of the intermediate compound f-2 was added to a round-bottom flask, 50 mL of an aqueous solution of 20% DMSO was added, and then (40 mg, 0.5 mmol) of NH4HCO3 was added. The round-bottom flask was left open and stirred at room temperature for 24 h, then lyophilized and prepared. Lyophilization gave the disulfide-bond cyclized product T140-f-2 (56 mg, 0.022 mmol, yield 56%).

[0203] (23 mg, 0.009 mmol) of T140-f-2, (11 mg, 0.009 mmol) of HA-Val-Cit-PAB-MMAE, (2.3 mg, 0.009 mmol) of CuSO4·5H2O and (3.6 mg, 0.018 mmol) of VcNa were added to a round-bottom flask, and the solvent H2O / DMF (0.4 mL: 1.6 mL) was added. The mixture was stirred overnight at room temperature, concentrated under reduced pressure, and prepared by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Lyophilization gave HLK-016 (18 mg, 0.005 mmol, yield 53%). HRMS (ESI): C 180 H 286 N 50 O 35 S2[M+H] 4+ calcd: 944.0467, found: 944.0491. Purity: >99.00%.

[0204] (18) Synthesis of HLK-017:

[0205]

[0206] The specific method is as follows:

[0207] Add the intermediate compound e (100 mg, 0.039 mmol) into a round-bottom flask, add 50 mL of an aqueous solution of 20% DMSO, then add NH4HCO3 (40 mg, 0.5 mmol). Keep the round-bottom flask open and stir at room temperature for 24 h, then freeze-dry to prepare the disulfide-bond cyclized product T140-e (66 mg, 0.028 mmol, yield 66%).

[0208] Dissolve T140-e (30 mg, 0.013 mmol), truncated Evans blue (8.5 mg, 0.013 mmol), HATU (5 mg, 0.013 mmol), and HOAT (1.8 mg, 0.013 mmol) in 2 ml of DMF. Then add DIEA (5 μL, 0.026 mmol) and stir at room temperature for 6 h, followed by concentration under reduced pressure. Dissolve in acetonitrile / water and prepare by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Freeze-dry to obtain compound T140-e-1 (20 mg, 0.007 mmol, yield 52%).

[0209] Add T140-e-1 (13 mg, 0.0045 mmol), HA-Val-Cit-PAB-MMAE (5.5 mg, 0.0045 mmol), CuSO4·5H2O (1 mg, 0.0045 mmol), and VcNa (1 mg, 0.0045 mmol) into a round-bottom flask, add the solvent H2O / DMF (0.4 mL: 1.6 mL), stir at room temperature overnight, and concentrate under reduced pressure. Prepare by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Freeze-dry to obtain HLK-017 (8 mg, 0.002 mmol, yield 43%). HRMS (ESI): C 193 H 282 N 54 O 43 S4[M+H] 4+ calcd: 1044.0178, found: 1044.0193. Purity: >99.00%.

[0210] (19) Synthesis of HLK-018:

[0211]

[0212] The specific method is as follows:

[0213] (30 mg, 0.013 mmol) of T140-e, (7.6 mg, 0.013 mmol) of 4-iodophenyl end group and (5 mg, 0.013 mmol) of HATU, (1.8 mg, 0.013 mmol) of HOAT were dissolved in 2 ml of DMF. Subsequently, (5 μL, 0.026 mmol) of DIEA was added, and the mixture was stirred at room temperature for 6 h and concentrated under reduced pressure. It was dissolved in acetonitrile / water, and prepared by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. After lyophilization, compound T140-e-2 (24 mg, 0.008 mmol, yield 65%) was obtained.

[0214] (24 mg, 0.008 mmol) of T140-e-2 was added to a round-bottom flask, and then 2 mL of TFA / DCM (TFA:DCM = 1:4) was added. The mixture was stirred at room temperature for 1 h and concentrated under reduced pressure. (10 mg, 0.008 mmol) of HA-Val-Cit-PAB-MMAE, (2 mg, 0.008 mmol) of CuSO4·5H2O and (3 mg, 0.008 mmol) of VcNa were added to the round-bottom flask. Solvent H2O / DMF (0.4 mL:1.6 mL) was added, and the mixture was stirred overnight at room temperature and concentrated under reduced pressure. Prepared by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. After lyophilization, HLK-018 (6 mg, 0.0015 mmol, yield 19%) was obtained. HRMS(ESI): C 185 H 283 IN 52 O 39 S2[M+H] 4+ calcd: 1013.0134, found: 1013.0147. Purity: >99.00%.

[0215] (20) Synthesis of HLK-019:

[0216]

[0217]

[0218] The specific method is as follows:

[0219] (50 mg, 0.083 mmol) of RGD-1 and (17 mg, 0.083 mmol) of Alkyne NHS ester (906564-59-8) were added to a round-bottom flask, dissolved in 3 mL of DMF, and then (29 μL, 0.166 mmol) of DIEA was added. The mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. It was dissolved in acetonitrile / water, and prepared by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. After lyophilization, compound RGD-2 (40 mg, 0.057 mmol, yield 69%) was obtained.

[0220] (7 mg, 0.003 mmol) of T140-e, (4 mg, 0.003 mmol) of Val-Cit-PAB-MMAE, (0.8 mg, 0.003 mmol) of CuSO4·5H2O and (0.64 mg, 0.003 mmol) of VcNa were added to a round-bottom flask, and the solvent H2O / DMF (0.4 mL: 1.6 mL) was added. The mixture was stirred overnight at room temperature, concentrated under reduced pressure, and prepared by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Lyophilization gave T140-e-3 (5 mg, 0.0014 mmol, yield 47%).

[0221] (20 mg, 0.006 mmol) of T140-e-3 and (1 mg, 0.006 mmol) of NHS ester of azidoacetic acid were added to a round-bottom flask, dissolved in 3 mL of DMF, and then (2 μL, 0.011 mmol) of DIEA was added. The mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. Prepared by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Lyophilization gave T140-e-4 (15 mg, 0.004 mmol, yield 74%).

[0222] (10 mg, 0.003 mmol) of T140-e-4, (2 mg, 0.003 mmol) of RGD-2, (0.8 mg, 0.003 mmol) of CuSO4·5H2O and (0.64 mg, 0.003 mmol) of VcNa were added to a round-bottom flask, and the solvent H2O / DMF (0.4 mL: 1.6 mL) was added. The mixture was stirred overnight at room temperature, concentrated under reduced pressure, and prepared by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Lyophilization gave HLK-019 (3 mg, 0.0007 mmol, yield 26%). C199H304N62O43S2 [M+H] 5+ calcd: 863.8663, found: 863.8645. Purity: >99.00%.

[0223] (21) Synthesis of HLK-05:

[0224]

[0225]

[0226] The specific method is as follows:

[0227] The intermediate compound g (50 mg, 0.018 mmol) was added to a round-bottom flask, 50 mL of an aqueous solution of 20% DMSO was added, followed by NH4HCO3 (40 mg, 0.5 mmol). The round-bottom flask was left open and stirred at room temperature for 24 h, then lyophilized to prepare the disulfide cyclized product T140-g (28 mg, 0.010 mmol, yield 56%).

[0228] T140-g (20 mg, 0.007 mmol), Val-Cit-PAB-MMAE (8.7 mg, 0.007 mmol), CuSO4·5H2O (1.8 mg, 0.007 mmol) and VcNa (1.4 mg, 0.007 mmol) were added to a round-bottom flask, the solvent H2O / DMF (0.4 mL: 1.6 mL) was added, and the mixture was stirred overnight at room temperature, concentrated under reduced pressure. Preparation by HPLC: acetonitrile / water, acetonitrile: 20% - 100%. Lyophilization gave T140-g-1 (15 mg, 0.004 mmol, yield 51%).

[0229] T140-g-1 (8 mg, 0.002 mmol) and the compound DOTA-NCS (1.1 mg, 0.002 mmol) were added to a round-bottom flask, dissolved in 1 mL of DMF, then (1 μL, 0.004 mmol) DIEA was added, and the mixture was stirred at room temperature for 2 h, concentrated under reduced pressure. Dissolved in acetonitrile / water, preparation by HPLC: acetonitrile / water, acetonitrile: 20% - 100%, lyophilization gave the compound HLK-05 (4 mg, 0.001 mmol, yield 43%). C 213 H 318 N 64 O 48 S3[M+H] 3+ calcd: 1546.4597, found: 1546.4583. Purity: >99.00%.

[0230] (21) Synthesis of HLK-16:

[0231]

[0232]

[0233] The specific method is as follows:

[0234] T140-e (30 mg, 0.013 mmol), Val-Cit-PAB-MMAE (16 mg, 0.013 mmol), CuSO4·5H2O (3.2 mg, 0.013 mmol) and VcNa (2.6 mg, 0.013 mmol) were added into a round-bottom flask, and the solvent H2O / DMF (0.4 mL: 1.6 mL) was added. Stir at room temperature overnight, concentrate under reduced pressure, and concentrate under reduced pressure. HPLC preparation: acetonitrile / water, acetonitrile: 20% - 100%. Lyophilize to obtain T140-e-5 (25 mg, 0.007 mmol, yield 54%).

[0235] T140-e-5 (10 mg, 0.003 mmol) and DOTA-NCS (1.8 mg, 0.003 mmol) were added into a round-bottom flask, dissolved in 1 mL of DMF, and then (1.2 μL, 0.006 mmol) DIEA was added. Stir at room temperature for 2 h and concentrate under reduced pressure. Dissolve in acetonitrile / water, HPLC preparation: acetonitrile / water, acetonitrile: 20% - 100%. Lyophilize to obtain compound HLK-16 (5 mg, 0.001 mmol, yield 43%). C 188 H 289 N 55 O 42 S3[M+H] 3+ calcd: 1022.2901, found: 1022.2923. Purity: >99.00%.

[0236] Example 2 Test the in vivo antitumor activity of the polypeptide-drug conjugate

[0237] (1) Immunodeficient nude mice were used as the objects for cancer cell transplantation, and the mice started to inoculate MCF-7 cells at 6 weeks old. The specific operation process is as follows:

[0238] Digest the cells to prepare a cell suspension, wash with PBS, place on ice, and inoculate 5×10 6 cells / 200 μL / animal subcutaneously into the axilla of the mice. On the 10th day, the average tumor volume reached 90 - 110 mm 3 , divided into 3 groups, 2 drug administration groups (HLK-001, HLK-007), 1 blank control group, and started drug administration on this day. Intraperitoneal injection, drug administration groups (HLK-001, HLK-007): 0.5 mg / kg / 2d, blank control group: 200 μL of normal saline / animal / 2d.

[0239] Record the tumor volume and mouse body weight once every 2 days. The calculation method of the tumor volume: V (mm3) = (L×W2 / 2), L: long diameter; W: short diameter. Wait until the average tumor volume of the blank control group reaches 1500 mm 3Around this time, the mice were sacrificed, and the tumors were excised and weighed.

[0240] (2) Immunodeficient nude mice were used as recipients for cancer cell transplantation. The mice started inoculating MCF-7 cells at 6 weeks of age. The specific operation process is as follows:

[0241] Digest the cells to prepare a cell suspension, wash with PBS, place on ice, and inoculate 5×10 6 cells / 200 μL / mouse subcutaneously into the axilla of the mice. On the 10th day, the average tumor volume reached 90 - 110 mm 3 . The mice were divided into 6 groups, 5 drug treatment groups (HLK-007, HLK-008, HLK-009, HLK-010, HLK-012), and 1 blank control group. Drug administration started on this day. By intraperitoneal injection, the drug treatment groups (HLK-007, HLK-008, HLK-009, HLK-010, HLK-012): 0.5 mg / kg / 2d, and the blank control group: 200 μL of normal saline / mouse / 2d.

[0242] Record the tumor volume and mouse body weight once every 2 days. The calculation method for tumor volume: V(mm3) = (L×W2 / 2), where L is the long diameter and W is the short diameter. When the average tumor volume of the blank control group reached about 1500 mm 3 around this time, the mice were sacrificed, and the tumors were excised and weighed.

[0243] (3) Balb / c mice were used as recipients for cancer cell transplantation. The mice started inoculating 4T1 cells at 6 weeks of age. The specific operation process is as follows:

[0244] Digest the cells to prepare a cell suspension, wash with PBS, place on ice, and inoculate 5×10 5 cells / 200 μL / mouse subcutaneously into the axilla of the mice. On the 10th day, the average tumor volume reached about 50 mm 3 . The mice were divided into 10 groups, 9 drug treatment groups (HLK-007, HLK-008, HLK-009, HLK-010, HLK-012, HLK-013), and 1 blank control group. Drug administration started on this day. By intraperitoneal injection, the drug treatment groups (HLK-007, HLK-009, HLK-010, HLK-011, HLK-012, HLK-015, HLK-016, HLK-017, HLK-018): 0.5 mg / kg / 2d, and the blank control group: 200 μL of normal saline / mouse / 2d.

[0245] Record the tumor volume and mouse body weight once every 2 days. The calculation method for tumor volume: V(mm3) = (L×W2 / 2), where L is the long diameter and W is the short diameter. When the average tumor volume of the blank control group reached 700 mm 3Around this time, the mice were sacrificed, and the tumors were excised and weighed.

[0246] The tumor inhibitory effects of each compound and its impact on the body weight of mice are shown as Figure 22 , Figure 23 , Figure 24 follows.

[0247] Figure 22 It was shown that on the 16th day of drug administration, the average tumor volume of the two drug-administered groups was significantly different from that of the blank control group, indicating that both compounds HLK-001 and HLK-007 had good inhibitory activity against MCF-7 transplanted tumors, with HLK-007 showing the best activity. There were no significant changes in the body weights of the mice in the drug-administered groups, indicating that the compounds had good safety profiles.

[0248] Figure 23 It was shown that on the 16th day of drug administration, the average tumor volume of the five drug-administered groups was significantly different from that of the blank control group, indicating that compounds HLK-007, HLK-008, HLK-009, HLK-010, and HLK-012 all had good inhibitory activity against MCF-7 transplanted tumors, with HLK-008 showing the best activity. There were no significant changes in the body weights of the mice in the drug-administered groups, indicating that the compounds had good safety profiles.

[0249] Figure 24 It was shown that on the 16th day of drug administration, the average tumor volume of the nine drug-administered groups was significantly different from that of the blank control group, indicating that compounds HLK-007, HLK-009, HLK-010, HLK-011, HLK-012, HLK-015, HLK-016, HLK-017, and HLK-018 all had good inhibitory activity against 4T1 transplanted tumors, with HLK-011 showing the best activity. There were no significant changes in the body weights of the mice in the drug-administered groups, indicating that the compounds had good safety profiles.

[0250] Example 3 PET / CT Imaging of Polypeptide-Drug Conjugates in a Tumor-Bearing Mouse Model with High CXCR4 Expression

[0251] 20 μg of HLK-05, HLK-16, and T140-DOTA were separately taken and mixed with 64 CuCl2 (500 - 1000 μCi, 0.1 mol / L NaOAc, pH = 4.1), and the reaction mixture was heated to 80 °C and maintained for 10 min.

[0252] The labeled 64 Cu]HLK-05, 64 Cu]HLK-16, 64The [Cu]T140-DOTA was diluted with normal saline, and each labeled polypeptide-drug conjugate was intravenously injected into the tail vein of mice at 200 μCi. PET / CT scan imaging was performed at 1 h, and reconstruction was carried out using PET / CT image processing software to obtain the PET / CT imaging results. By processing the PET / CT imaging results, the uptake values of the labeled polypeptide-radionuclide conjugate in mouse organs were obtained, with the unit of %ID / cc (the ratio of the radioactivity count in each cubic centimeter of the organ to the total injected radioactivity count).

[0253] Figure 25 and Figure 26 showed 64 [Cu]HLK-05, 64 [Cu]HLK-16 had obvious uptake in Daudi tumors, and 64 [Cu]HLK-05 had significantly higher uptake in tumors than 64 [Cu]HLK-16 and the known compound 64 [Cu]T140-DOTA, indicating that 64 [Cu]HLK-05 had good targeting ability for tumors with high CXCR4 expression.

[0254] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claimed rights.

Claims

1. An antibody conjugate compound or a pharmaceutically acceptable salt thereof, the antibody conjugate compound or the pharmaceutically acceptable salt thereof comprising a payload, a linker, and one or more cell interaction molecules, wherein the payload is conjugated to at least one of the cell interaction molecules, and wherein some of the antibody conjugate compounds simultaneously comprise two different payloads.

2. The antibody conjugate compound or the pharmaceutically acceptable salt thereof according to claim 1, the antibody conjugate compound or the pharmaceutically acceptable salt thereof comprising a first ligand capable of specifically binding to a first cell surface receptor, and a second ligand capable of specifically binding to a second cell surface receptor or a third ligand capable of specifically binding to a third non-cell surface receptor, wherein the first cell surface receptor is chemokine receptor 4 (CXCR4), and the first ligand is selected from cyclopeptide T140 and its analogs; the second ligand is selected from folic acid, cyclopeptide RGD and its analogs, and FAPI-04; the third ligand is selected from albumin ligands.

3. The antibody conjugate compound or the pharmaceutically acceptable salt thereof according to claims 1-2, wherein the amino acid sequence of the first ligand cyclopeptide T140 and its analogs is: NH2-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-(D-Lys)-Pro-Tyr-Arg-Cit-Cys-Arg-COOH (two cysteines in the sequence form a disulfide bond), or analogs with substitution, deletion, or insertion of 1-3 amino acids, including substitution of Lys with Arg in the sequence, acetylation at the amino terminus, amidation at the carboxyl terminus, and incorporation of L-Lys into the side chain of D-Lys.

4. The antibody conjugate compound or the pharmaceutically acceptable salt thereof according to claims 1-2, wherein the structures of the folic acid and FAPI-04 are as follows:

5. The antibody conjugate compound or the pharmaceutically acceptable salt thereof according to claims 1-2, wherein the cyclopeptide RGD and its analogs are selected from:

6. The antibody conjugate compound or the pharmaceutically acceptable salt thereof according to claims 1-2, wherein the albumin ligands are selected from: ibuprofen, 4-methylphenylbutyric acid, 4-methoxyphenylbutyric acid, 4-chlorophenylbutyric acid, truncated Evans blue, dodecanoic acid, hexadecanoic acid, 4-iodophenyl terminal group. The structure of the albumin ligand is as follows:

7. The antibody conjugate compound or the pharmaceutically acceptable salt thereof according to any one of claims 1-6, wherein the payload is a cytotoxic drug, a radionuclide chelating group, and a radionuclide chelated thereby, the cytotoxic drug is MMAE; the chelating group is DOTA. The structure of the payload is as follows:

8. The conjugate compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, wherein the radionuclide is selected from 177 Lu, 64 Cu, 67 Cu, 68 Ga, 67 Ga, 18 F, 51 Cr, 111 In, 99 mTc, 186 Re, 188 Re, 139 La, 140 La, 175 Yb, 15 3Sm, 166 Ho, 86 Y, 88 Y, 90 Y, 149 Pm, 165 Dy, 169 Er, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Ph, 101m Ph, 119 Sb, 128 Ba, 123 I, 124 I, 131 I, 197 Hg, 211 At, 151 Eu, 153 Eu, 169 Eu, 201 T1, 203 Pb, 212 Pb, 188 Re, 186 Re, 198 Au, 225 Ac, 227 Th and 199 Ag.

9. The antibody conjugate compound or the pharmaceutically acceptable salt thereof according to any one of claims 1-8, wherein the linker comprises a cleavable linker and a non-cleavable linker, and the linker is selected from one or more of the following structures:

10. The antibody conjugate compound or the pharmaceutically acceptable salt thereof according to claims 1-9, selected from the following structures:

11. A method for preparing the antibody conjugate compound according to claims 1-10, the steps of which are as follows: a) Synthesize the T140 linear peptide and the T140 linear peptide conjugated with the second ligand or the third ligand using the Fmoc solid-phase synthesis strategy; b) Cyclize the T140 linear peptide in a) through a disulfide bond cyclization reaction to obtain the T140 cyclic peptide, and then connect it with the second ligand or the third ligand through liquid-phase condensation or click reaction to generate a coupling intermediate; or directly cyclize the T140 linear peptide conjugated with the second ligand or the third ligand in a) through a disulfide bond cyclization reaction to obtain a coupling intermediate; or connect the T140 linear peptide or the T140 linear peptide conjugated with the second ligand or the third ligand with MMAE through click reaction to generate a coupling intermediate; c) For the coupling intermediate obtained in b), connect MMAE through click reaction to generate a coupling intermediate to obtain a conjugate compound; or connect the coupling intermediate conjugated with MMAE obtained in b) with a radionuclide chelating group through a condensation reaction to obtain a conjugate compound.

12. A pharmaceutical composition, comprising the conjugate compound according to any one of claims 1-10 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

13. Use of the conjugate according to any one of claims 1-10 or the pharmaceutical composition according to claim 12 in the preparation of a reagent for diagnosing or treating a disease characterized by overexpression of chemokine receptor 4 (CXCR4) in a subject.

14. The use according to claim 13, wherein the disease is selected from cancers.

15. The use according to claim 14, wherein the cancer is selected from: breast cancer, lung cancer, prostate cancer, kidney cancer, leukemia, ovarian cancer, gastric cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, skin cancer, lymphoma, multiple myeloma.

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