Antibody drug conjugates based on metal n-heterocyclic carbene complexes, methods of preparation and use thereof in anti-cancer therapy

Abstract

The present invention provides an antibody drug conjugate, a method of preparing the antibody drug conjugate, and a method of using the antibody drug conjugate for anti-cancer therapy, the antibody drug conjugate is composed of an anti-HER2 trastuzumab monoclonal antibody and an iridium (III) N-heterocyclic carbene (NHC) complex, wherein the anti-HER2 trastuzumab monoclonal antibody and the iridium (III) N-heterocyclic carbene (NHC) complex are connected through a cathepsin B cleavable GGFG tetrapeptide linker. The tumor target specificity and delivery of the provided antibody drug conjugates are improved.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 601,224, filed on November 21, 2023, and the disclosure of the U.S. Provisional Patent Application is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention generally relates to an antibody drug conjugate (ADC), and in particular to an ADC of the type of metal N-heterocyclic carbene (NHC) complex. Background Art

[0003] Cancer is a leading cause of death worldwide. However, due to the non-selective mechanism of action, the chemotherapy drugs used clinically show limited selectivity for cancer cells, thus leading to serious side effects. At present, a lot of efforts have been invested in the development of new anticancer drugs that can be specifically delivered to the tumor site. ADC has attracted a lot of attention in the research and development of anticancer drugs. ADC is composed of monoclonal antibodies targeting tumor-specific antigens, which are covalently conjugated to cytotoxic drugs through chemical linkers, thereby combining the advantages of highly specific targeting ability and highly potent toxicity of both antibodies and drugs. To date, the FDA has approved 14 ADCs, in which the cytotoxic drug load mainly contains potent tubulin inhibitors and DNA damaging agents. However, the former is not very effective against static cancer cells, and the latter is too toxic, which will cause ADCs to cause serious adverse reactions in healthy non-cancerous cells due to inevitable off-target toxicity. Therefore, there is still a need to develop new ADC drug loads that ideally have better therapeutic indexes. On the other hand, metal complexes have unique three-dimensional scaffolds for binding drug target proteins that are different from conventional small molecule drugs. Over the past few decades, significant progress has been made in the development of metal N-heterocyclic carbene (NHC) complexes as anticancer agents. The strong σ-donor properties of NHCs result in stable and strong metal-NHC bonds that are resistant to physiological reducing agents and nucleophiles. In addition to chemical and thermal stability, NHC ligands can be easily modified to impart additional functionalities to the complexes, making the complexes ideal and promising candidates for metallodrugs and, therefore, novel drug payloads for ADCs.

[0004] However, the site of drug conjugation to the antibody may have a significant impact on the potency of the drug, which requires careful consideration. Summary of the invention

[0005] According to a first aspect of the present invention, an antibody drug conjugate (ADC) is provided, which is composed of a tumor-targeting antibody and a metal N-heterocyclic carbene (NHC) complex covalently linked by a linker, wherein the tumor-targeting antibody is selected from but not limited to anti-HER2 trastuzumab, anti-EGFR cetuximab, anti-TROP2 sacituzumab, anti-nectin-4 enfortumab, anti-tissue factor tisotumab and anti-FRα mirvetuximab, and the linker is selected from but not limited to a cathepsin B-cleavable GGFG tetrapeptide linker, an acid-cleavable carbonate linker and a glutathione-cleavable disulfide linker, wherein tumor target specificity and delivery are improved.

[0006] According to a second aspect of the present invention, there is provided a method for preparing the ADC according to the first aspect of the present invention. The method comprises: reacting Fmoc-protected GGFG peptide acetate with a hydroxyl-functionalized iridium (III) N-heterocyclic carbene (NHC) complex to form a first product; deprotecting Fmoc from the first product with DMF containing 20% ​​piperidine to form a second product, and condensing the second product with 6-(maleimido)hexanoic acid succinimidyl ester to obtain a maleimide-bound prodrug; and conjugating the maleimide-bound prodrug with partially reduced trastuzumab to form the ADC.

[0007] The resulting ADC can significantly increase the drug accumulation in tumors by 10-fold and reduce the iridium level in the kidney by 90% compared to the original iridium complex treatment. It is worth noting that the site of conjugation of the drug to the antibody may greatly affect the efficacy of the drug, which requires careful consideration. The present invention successfully demonstrated that the iridium (III) pyridine-NHC complex modified with a hydroxyl group on the pyridine ring for bioconjugation did not significantly affect the half-maximal inhibitory concentration (IC 50 ) value, which provides a useful conjugation site. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Embodiments of the present invention are described in more detail below with reference to the accompanying drawings, in which:

[0009] Figure 1 Shown is a synthetic route for two hydroxyl analogs of a pyridine triflate derivative and an Ir(III)-NHC complex according to an embodiment of the present invention;

[0010] Figure 2 The IC values ​​of the functionalized complexes after different modification stages are shown. 50Comparison of the impact of values;

[0011] Figure 3 An ADC (ADC-Ir) composed of anti-HER2 trastuzumab monoclonal antibody and Ir(III)-NHC according to one embodiment of the present invention is shown;

[0012] Figure 4A and 4B A synthetic route for preparing ADC according to one embodiment of the present invention is shown;

[0013] Figure 5 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of ADC is shown;

[0014] Figure 6 shows the absorption spectra of ADC in the UV region and the visible light region;

[0015] Figure 7 shows mass spectrometry analysis of ADC; and

[0016] Fig. 8A and 8B The in vivo biodistribution of ADC-Ir after 1 day and 7 days of administration is shown in nude mice bearing SKOV-3 tumors, respectively. DETAILED DESCRIPTION

[0017] In the following description, exemplary embodiments of the present invention are described as preferred examples. It will be apparent to those skilled in the art that modifications including additions and / or substitutions may be made without departing from the scope and spirit of the present invention. Specific details may be omitted so as not to obscure the present invention; however, this disclosure is written to enable those skilled in the art to practice the teachings herein without undue experimentation.

[0018] According to one aspect of the present invention, the antibody drug conjugate is composed of an anti-HER2 trastuzumab monoclonal antibody and a metal N-heterocyclic carbene (NHC) complex linked via a maleimide-conjugated prodrug.

[0019] In some embodiments, the metal N-heterocyclic carbene (NHC) complex may be an iridium (III) N-heterocyclic carbene (NHC) complex having the following formula:

[0020] Wherein X is a counter anion selected from CF 3 SO 3 PF 6 , Cl, Br or I.

[0021] The maleimide conjugated prodrug may be a cathepsin B cleavable GGFG tetrapeptide linker having the formula:

[0022]

[0023] The metal center of the NHC complex can be iridium, iron, osmium, ruthenium and rhodium.

[0024] The 2-phenylpyridine and [1,2'-bipyridine]-1-cation ligands of the NHC complex may be derivatized with, including but not limited to, halogen, hydroxy, alkyl, aryl, acyl, alkoxy, acyloxy, amino, nitro, amido, aralkyl, cyano, carboxyl, thio, styryl, aminocarbonyl, carbamoyl, aryloxycarbonyl, phenoxycarbonyl, or alkoxycarbonyl groups.

[0025] To functionalize metal complexes for bioconjugation, hydroxyl groups are introduced into metal ligands with different structures. Figure 1 Synthetic routes to pyridine triflate derivatives and two hydroxy analogs (HA) of the Ir(III)-NHC complex are shown. The pyridine triflate derivative was prepared by heating 4-pyrrolidinylpyridine with methyl 2-(((trifluoromethyl)sulfonyl)oxy)isonicotinate at 150°C under argon. The μ-chloro-bridged dimer was reacted with pyridine triflate in ethylene glycol at reflux to give hydroxy analog 1 (HA1), which was then subjected to reduction with sodium borohydride to give hydroxy analog 2 (HA2).

[0026] The IC values ​​of the functionalized complexes after different modification stages (HA1 and HA2) were examined and compared with those of the original complex. 50 The value of Figure 2 Interestingly, Ir1a (IC 50 =0.20 μM to 0.34 μM), the potency of the functionalized complex HA1 was significantly reduced, and its IC 50 The values ​​ranged from 5.73 μM to 7.46 μM. However, the IC 50 The value shows that the IC of Ir1a 50 This result shows that structural changes in metal ligands can greatly affect the potency of drugs.

[0027] After optimizing the chemical structure for bioconjugation, the submolar anticancer potency and functionalizable sites of Ir1a make it a promising cytotoxic payload for ADCs.

[0028] Figure 3An ADC (ADC-Ir) consisting of an anti-HER2 trastuzumab monoclonal antibody and an Ir1a portion according to one embodiment of the present invention is shown. In some embodiments, the ADC is prepared by conjugating Ir1a-OH to trastuzumab with a GGFG tetrapeptide linker cleavable by cathepsin B with reference to the design of FDA-approved trastuzumab deruxtecan. By mass spectrometry, the drug / antibody ratio (DAR) was determined to be 5.3. In other embodiments, the antibody is selected from, but not limited to, antibodies targeting overexpressed EGFR, TROP2, adhesion protein-4, tissue factor, or FRα on cancer cells. In other embodiments, the linker connecting the antibody to the drug payload may contain any acid, glutathione, enzyme-cleavable unit.

[0029] Figure 4A and 4B A synthetic route for preparing ADC is shown. Figure 4A , an Fmoc-protected peptide acetate (Fmoc-GGFG-OAc) is reacted with a hydroxyl-functionalized iridium complex (Ir1a-OH) to produce a first product (Fmoc-GGFG-Ir), followed by Fmoc deprotection with 20% piperidine in DMF to form a second product, and the second product is subsequently condensed with 6-(maleimido)hexanoic acid succinimidyl ester in DMF containing DIPEA to give a maleimide-conjugated prodrug (i.e., Linker-Ir).

[0030] refer to Figure 4B , the four interchain disulfide bonds of trastuzumab can be conjugated to linker-Ir by Michael addition after reduction with tris(2-carboxyethyl)phosphine hydrochloride to produce ADC-Ir. ADC-Ir was obtained after removing unreacted linker-Ir by Zeba desalting spin column (7kDa MWCO). The protein concentration was determined by standard bicinchoninic acid BCA assay, and the yield was determined to be 85%.

[0031] like Figure 5 As shown, SDS-PAGE analysis of ADC shows that both the light chain fragment and the heavy chain fragment of trastuzumab (mAb) migrate at a larger kDa after conjugation with the metal complex, which can also be observed under UV light. The UV-Vis absorption spectrum of ADC shows the absorption peaks of both Ir1a and trastuzumab ( Figure 6 Further analysis by mass spectrometry showed that the drug / antibody ratio (DAR) was determined to be 5.3 ( Figure 7 ).

[0032] To examine the in vivo tumor targeting effect, ADC-Ir (10 mg / kg) or Ir1a-OH (0.3 mg / kg), both of which had equivalent content of the Ir1a moiety, were intravenously injected into nude mice bearing SKOV-3 tumors. The in vivo biodistribution of the iridium content was analyzed by ICP-MS after 1 and 7 days. Fig. 8A and 8B As shown, administration of ADC-Ir significantly increased iridium content in tumors by 10-fold and reduced iridium levels in the kidney by 90% compared to the effect of injection of Ir1a-OH. These data suggest that Ir1a is suitable for ADC strategies, showing the ability to increase tumor delivery in vivo and reduce drug load distribution in normal tissues throughout the body.

[0033] Synthesis of Hydroxyl-Functionalized Iridium(III) NHC Complexes

[0034] The pyridine trifluoromethanesulfonate is prepared by heating 2-(((trifluoromethyl)sulfonyl)oxy)isonicotinic acid methyl ester and 4-pyrrolidinylpyridine at 150°C. 1 H NMR (600 MHz, CDCl 3 ,298K): δ8.86(d,J=7.8Hz,2H),8.71(d,J=4.8Hz,1H),8.35(s,1H),8.00(d, J=4.8Hz,1H),7.06(d,J=7.8Hz,2H),4.02(s,3H),3.71(m,4H),2.32(m,4H).

[0035] refer to Figure 1 To a solution of pyridine trifluoromethanesulfonate derivative (120 mg, 0.277 mmol) in ethylene glycol (30 mL × 1) was added μ-chloro-bridged iridium dimer Ir III 2 (ppy) 4 (μ-Cl) 2 (200 mg, 0.186 mmol) was added. The mixture was degassed in vacuum for 15 minutes. After that, the mixture was heated at 200°C for 12 hours. The color of the mixture slowly turned orange. After 12 hours, ammonium hexafluorophosphate NH 4 PF 6 (10 equivalents) to obtain an orange precipitate (HA1). The precipitate was collected and redissolved in methanol (10 mL x 1). Sodium borohydride NaBH 4 (3 equivalents), and the resulting mixture was stirred for 6 hours. The color of the mixture turned yellow. After 6 hours, the solvent was removed in vacuo, and the crude compound was extracted with dichloromethane (10 mL×3). Then, column chromatography (SiO 2The crude product was further purified by evaporation with 1% paraformaldehyde (dextrin, dichloromethane / ethyl acetate, v / v=10:1) to obtain HA2 (31 mg, yield: 37%).

[0036] H NMR 1 H NMR (600MHz, CD 3 CN,298K): δ8.59(d,J=7.8Hz,1H),8.12(d,J=7.8Hz,1H),8.05(d,J=7.8Hz,1H),7.94(s,1H),7.88 (t,J=4.8Hz,1H),7.76-7.83(m,3H),7.71(d,J=4.8Hz,1H),7.63(d,J=5.4Hz,1H),7.51(d,J=5.4H z,1H),7.21(d,J=5.4Hz,1H),7.14(t,J=6.0Hz,1H),7.07(t,J=6.0Hz,1H),6.95-6.99(m,2H),6.8 9(t,J=7.2Hz,1H),6.83(t,J=7.2Hz,1H),6.75(d,J=7.2Hz,1H),6.66(d,J=7.2Hz,1H),6.55(dd,J 1 =8.4Hz,J 2 =3.0Hz,1H),5.91(d,J=3.0Hz,1H),4.73(s,2H),3.74(br,1H,OH),3.40-3.47(m,2H),2.76-2.96(m,2H),1.82-1.90(m,4H).

[0037] C NMR 13 C{ 1 H}NMR (150MHz, CD 3 CN,298K): δ177.03,166.50,166.35,157.37,157.23,156.84,156.60,151.6 4,149.39,148.48,148.03,145.29,144.64,139.35,139.01,136.98,136.85 ,136.22,131.35,130.92,125.59,125.49,124.28,124.10,122.62,122.12,120.98,120.54,115.14,110.45,105.78,62.85,48.81,48.24,25.60,25.51.

[0038] NMR fluorine spectrum 19 F{ 1H}NMR (376MHz, CD 3 CN,298K):δ-73.1

[0039] NMR phosphorus spectroscopy 31 P{ 1 H}NMR (162MHz, CD 3 CN,298K):δ-135.1

[0040] High Resolution Electrospray Ionization Mass Spectrometry HR-ESI-MS: C 37 H 33 Ir 5 O + [M-PF 6 ] + Calculated m / z: 756.2314; found: 756.2328.

[0041] Synthesis of Linker-Ir

[0042] Fmoc protected peptide acetate (Fmoc-GGFG-OAc) (4.0 mg, 6.2 μmol) was reacted with hydroxyl functionalized iridium (III) N-heterocyclic carbene (NHC) complex (Ir1a-OH) (4.0 mg, 4.4 μmol) in dichloromethane containing pyridine p-toluenesulfonate (0.12 mg, 0.44 μmol). The reaction mixture was refluxed at 40° C. overnight and the solvent was removed in vacuo. The crude product was purified by semi-preparative high performance liquid chromatography (HPLC) to give Fmoc-GGFG-Ir (2.2 mg, 31%) after lyophilization.

[0043] Subsequently, Fmoc was deprotected from Fmoc-GGFG-Ir with 20% piperidine in DMF, and the resulting product was condensed with 6-(maleimido)hexanoic acid succinimidyl ester in DIPEA in DMF to obtain the maleimide-conjugated prodrug (Linker-Ir).

[0044] H NMR 1 H NMR (600 MHz, DMSO-d 6): δ 8.97 (d, J = 7.9 Hz, 1H), 8.74 (d, J = 4.5 Hz, 1H), 8.48 (s, 1H), 8.43 (d, J = 5.6 Hz, 1H), 8.27 - 8.29 (m, 3H), 8.20 (d, J = 8.1 Hz, 1H), 8.12 - 8.13 (m, 1H), 7.95 (t, J = 6.3 Hz, 1H), 7.83 - 7.89 (m, 5H), 7.72 (d, J = 5.4 Hz, 1H), 7.65 - 7.67 (m, 2H), 7.62 (t, J = 5.9 Hz, 1H), 7.58 (dd, J 1 = 5.7 Hz, J 2 = 1.8 Hz, 1H), 7.37 - 7.41 (m, 3H), 7.28 - 7.32 (m, 3H), 7.14 - 7.25 (m, 6H), 6.91 - 6.95 (m, 2H), 6.85 (t, J = 7.6 Hz, 1H), 6.80 (t, J = 7.6 Hz, 1H), 6.60 - 6.66 (m, 3H), 5.79 (t, J = 3.2 Hz, 1H), 4.69 (d, J = 6.5 Hz, 2H), 4.64 (s, 2H), 4.41 - 4.46 (m, 1H), 4.18 - 4.25 (m, 3H), 3.57 - 3.78 (m, 8H), 2.98 - 3.03 (m, 1H), 2.85 - 2.88 (m, 1H), 2.75 - 2.79 (m, 1H), 2.61 - 2.67 (m, 1H), 1.75 - 1.88 (m, 4H).

[0045] Carbon-13 NMR 13 C NMR (150 MHz, DMSO-d 6): δ175.96,171.99,170.74,170.04,165.69,165.39,165.29,157.05,156.35,156.02,153.08,150.45,148.50,147.35,147.14,144.4 8,144.26,143.79,143.03,141.17,139.88,139.05,138.64,138.32,137.89,136.92,136.05,135.15,130.75,130.20,129.55,129.40 ,128.53,128.10,127.77,127.53,126.72,125.69,125.22,125.01,124.16,123.86,122.57,121.93,121.86,121.76,120.57,120.50,120.17,114.08,110.87,110.25,105.53,69.97,67.66,66.23,48.15,47.40,47.06,45.11,43.99,42.75,42.41,37.58,24.90,24.79.

[0046] High Resolution Electrospray Ionization Mass Spectrometry HR-ESI-MS: C 63 H 65 Ir 11 O 8 + [M-PF 6 ] + Calculated m / z: 1296.4647; found: 1296.4689.

[0047] Agilent 1260 Infinity II LC system equipped with Agilent 1260 quaternary pump and Agilent 1260 DAD WR was used to perform reverse phase HPLC separation on XBridge BEH300 Prep C18 column (5 μm, 10 mm × 250 mm) at a flow rate of 3 ml / min. The conditions for analysis were set as follows: solvent A = deionized water containing 0.1% formic acid (FA), and solvent B = acetonitrile containing 0.1% FA; gradient: 95% A + 5% B in the first 5 minutes, then changed to 85% A + 15% B in 10 minutes, further changed to 5% A + 95% B in 30 minutes, maintained under this condition for 5 minutes, changed to 95% A + 5% B in 5 minutes, and maintained under this condition for another 5 minutes.

[0048] In vitro antiproliferative activity (NBB assay)

[0049] SKOV-3 ovarian cancer cells (5×10 3 Cells / well) were seeded in DMEM in a microtiter plate (96-well) and incubated overnight before treatment. Different concentrations of Ir1a and Ir1a-OH and a solvent control (0.5% DMSO) were added to the wells and then incubated for 72 hours. After removing the culture medium, the cells were fixed with PBS containing formaldehyde (3%; 50 μL) and stained with NBB reagent (0.05%, 0.1 M sodium acetate, 9% acetic acid; 50 μL) overnight. The stained cells in each well were gently washed three times with deionized water and dissolved in sodium hydroxide solution (50 mM; 100 μL). Cell activity was determined by measuring the absorbance at 620 nm in each well with a microplate reader. The experiment was repeated three times. The half-maximal inhibitory concentration (IC 50 Values) are expressed as mean ± standard deviation.

[0050] Preparation and characterization of ADC-Ir

[0051] Trastuzumab (MCE, 2.5 mg / mL) was dissolved in borate buffer (pH 8.0), and tris(2-carbonylethyl)phosphine hydrochloride TCEP (10 equivalents) was added to the reaction mixture for 1.5 hours. After disulfide bond reduction, linker-Ir (16 equivalents) dissolved in DMSO (2% v / v) was added, and the reaction mixture was stirred for 2 hours. The reaction mixture was prepared by using Zeba according to the manufacturer's protocol. TM The ADC-Ir conjugate was purified by desalting spin columns (Thermo Fisher, 7K MWCO). The protein concentration was determined by the BCA assay, and the yield was determined to be 85%. The conjugate was analyzed by ThermoScientific μDrop and reducing SDS-PAGE (12% acrylamide with 4% stacking gel).

[0052] The antibody and conjugate were analyzed on a MAbPac reverse phase HPLC column (4 μm, 3 mm×50 mm) at a flow rate of 0.3 ml / min. Electrospray ionization (ESI) mass spectra were recorded on an Agilent 6546 LC / Q-TOF mass spectrometer. The conditions for analysis were set as follows: solvent A=deionized water containing 0.1% FA, and solvent B=acetonitrile containing 0.1% FA; gradient: 80% A+20% B in the first 0.5 min, then changed to 40% A+60% B in 3 min, maintained under this condition for 5 min, further changed to 80% A+20% B in 0.5 min, maintained under this condition for 1 min.

[0053] The average drug / antibody ratio (DAR) was determined by using the corresponding peak areas of the chromatograms in the deconvoluted mass spectra and was found to be 5.3.

[0054] In vivo biodistribution assay

[0055] All animal experiments were performed according to guidelines approved by the University of Hong Kong Committee CULATRg. Female BALB / cAnN-nu nude mice (5-8 weeks old) were housed in the Hong Kong University Laboratory Animal Center. Mice had free access to food and water. SKOV-3 ovarian cancer cells (1×10 7 ) in PBS (100 μL) was injected subcutaneously into the right groin of each mouse. 3 Afterwards, the mice were divided into 3 groups (n=6 in each group): (A) PBS control group, (B) ADC-Ir (10 mg / kg) and (C) Ir1a-OH (0.3 mg / kg). The mice were sacrificed on the 1st and 7th days after tail vein injection. Tumors and major organs were collected and digested with nitric acid at 70°C overnight, followed by ICP-MS (Agilent 7500A) analysis to quantify the iridium content.

[0056] The foregoing description of the present invention has been provided for the purpose of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations will be apparent to those skilled in the art.

[0057] The embodiment was chosen and described in order to best explain the principles of the invention and its practical application, thereby enabling others skilled in the art to understand the invention for various embodiments and with various modifications as are suited to the particular use contemplated.

Claims

1. An antibody-drug conjugate (ADC), which is composed of a tumor-targeting antibody and a metal N-heterocyclic carbene (NHC) complex connected by a linker; The linker is either non-cleavable or cleavable under stimulation by acid, glutathione or an enzyme.

2. The ADC according to claim 1, wherein the tumor-targeting antibody targets a tumor-associated receptor selected from HER2, TROP2, adhesion protein 4, folate receptor alpha or EGFR.

3. The ADC according to claim 1, wherein the tumor targeting antibody is anti-HER2 trastuzumab.

4. The ADC according to claim 1, wherein the metal N-heterocyclic carbene (NHC) complex is an iridium (III) N-heterocyclic carbene (NHC) complex having the following formula: wherein X is a counter anion selected from CF3SO3, PF6, Cl, Br or I.

5. The ADC according to claim 1, wherein the linker is a GGFG tetrapeptide linker cleavable by cathepsin B.

6. A method for preparing the antibody drug conjugate according to claim 1, the method comprising: reacting an Fmoc-protected GGFG peptide acetate with a hydroxyl-functionalized iridium(III) N-heterocyclic carbene (NHC) complex to form a first product; Fmoc was deprotected from the first product with 20% piperidine in DMF to form a second product, and the second product was condensed with 6-(maleimido)hexanoic acid succinimidyl ester to obtain an iridium (III) NHC complex-loaded cathepsin B-cleavable GGFG tetrapeptide linker having the following formula: as well as The cathepsin B-cleavable GGFG tetrapeptide linker loaded with the iridium (III) NHC complex is conjugated to reduced trastuzumab to form the antibody drug conjugate.

7. The method of claim 6, wherein the hydroxy-functionalized iridium (III) NHC complex is prepared by: reacting a μ-chloro-bridged iridium dimer with pyridinium triflate in an ethylene glycol solution under reflux to obtain a first mixture; precipitating the first mixture with ammonium hexafluorophosphate to obtain a first product; dissolving the first product in methanol; reducing the dissolved precipitate with sodium borohydride to form a second product; as well as The hydroxy-functionalized iridium(III) NHC complex is extracted from the second product with dichloromethane.

8. The method of claim 7, wherein the pyridine triflate derivative is prepared by heating 4-pyrrolidinylpyridine with methyl 2-(((trifluoromethyl)sulfonyl)oxy)isonicotinate at 150°C.

9. The method of claim 6, wherein the cathepsin B-cleavable GGFG tetrapeptide linker loaded with the iridium (III) NHC complex is conjugated to the reduced trastuzumab by Michael addition.

10. The method of claim 6, wherein the reduced trastuzumab is prepared by reducing four interchain disulfide bonds of trastuzumab with tris(2-carboxyethyl)phosphine hydrochloride.

11. A method of using the antibody drug conjugate according to claim 1 in anti-cancer treatment.

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