A HER2-targeted platinum-based antibody conjugate and its synthesis and application in tumor treatment

Abstract

The present invention relates to a kind of HER2 targeting platinum-based antibody conjugate and its synthesis method and tumor treatment application, the HER2 targeting platinum-based antibody conjugate of the present invention is formed by connecting an anti-HER2 monoclonal antibody and a cyclometallated N-heterocyclic carbene platinum (II) complex via a linker, and the linker is a self-eliminating linker including a maleimide linker connecting the antibody and the cyclometallated N-heterocyclic carbene platinum (II) complex. The present invention combines a cyclometallated N-heterocyclic carbene platinum (II) complex with antiproliferative activity with a monoclonal antibody with a specific recognition function as a carrier to improve the tumor targeting of the platinum complex, improve its distribution in the body, and achieve efficient delivery of platinum drugs to tumor tissue, thereby improving cancer treatment effect; can effectively reduce the accumulation of drugs in non-tumor sites, reduce toxic and side effects; can effectively reduce the amount of platinum metal used, reduce toxic and side effects; can also achieve efficient, low-toxic tumor treatment effect by effectively killing tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals, and specifically relates to a HER2-targeted platinum-based antibody conjugate and its synthesis and application in tumor treatment. Background Art

[0002] The number of new cancer cases worldwide continues to rise annually, from 17.3 million in 2016 to 20 million in 2022. The global cancer burden is projected to reach 35 million cases by 2050, a 77% increase from 2022. In 2022, China will have approximately 4.825 million new cancer cases and 2.574 million cancer deaths, accounting for 24.1% and 26.4% of the global total, respectively, both ranking first in the world. The burden this places on the nation and families is significant, and society urgently needs more effective anti-cancer treatments. From cisplatin derivatives that act on nucleic acids to physiologically stable platinum(II) complexes with non-nucleic acid targets, platinum-based drugs have made significant progress in both therapeutic efficacy and combating drug resistance. However, due to their lack of selectivity for tumor cells, platinum-based drugs indiscriminately kill tumor cells, leading to significant toxic side effects after long-term use, hindering further therapeutic progress.

[0003] Antibody-drug conjugates (ADCs) consist of monoclonal antibodies that specifically recognize antigens on the surface of cancer cells, a payload responsible for killing cancer cells, and a linker that connects the antibody and payload. They combine the powerful lethality of traditional small molecule chemotherapy with the tumor-targeting properties of antibody drugs. After recognizing and binding to the antigen, the ADC enters the cell through the endocytosis pathway and is then degraded by the lysosome. The payload is released in a bioactive form, killing tumor cells. The concentration of the intracellular payload is determined by the number of cell-surface antigens, the amount of payload in each ADC (also known as the drug-antibody ratio, DAR), and the time required for the antigen to return to the cell surface. Compared with traditional non-targeted and non-specific chemotherapy methods, the targeted strategy of ADCs can improve the therapeutic index of cell-killing agents used to treat cancer and improve safety by reducing off-target side effects.

[0004] Currently, cytotoxins used in ADCs still face challenges such as a limited variety and a single mechanism of action. Research on metal-based complexes as cytotoxins for ADCs is far less extensive than on organic compounds, and most studies are based on clinical platinum drugs such as cisplatin and oxaliplatin. There is still significant potential for the development of other highly effective and low-toxic metal-based complexes for ADC applications.

[0005] Over the past two decades, physiologically stable cyclometallated N-heterocyclic carbene platinum(II) complexes have demonstrated promising potential for photoimaging and photodynamic therapy, while their subnanomolar cytotoxicity has also laid the foundation for their further development. Monoclonal antibodies are excellent targeting vehicles. Chemically conjugating monoclonal antibodies with platinum anticancer drugs to create antibody-platinum drug conjugates not only enables efficient delivery of platinum drugs to tumor tissues through the targeting effect of monoclonal antibodies, but also effectively reduces drug accumulation in non-tumor areas, minimizing toxic side effects. Simultaneously, they effectively kill tumor cells, achieving highly effective, low-toxicity treatment.

[0006] Existing non-metallic ADCs still suffer from numerous potentially fatal side effects in clinical applications, including the inability to differentiate between tumor and normal cells. These effects can lead to therapeutic side effects such as interstitial pneumonitis, bone marrow suppression, and neurotoxicity. The complex manufacturing process, including drug-antibody conjugation and purification, can increase production costs and time. Some metal-based ADCs have demonstrated cytotoxicity against non-target cells in in vitro testing and lack specificity for cancer cells. For example, NHC*-Au-Thiomab exhibited similar cytotoxicity in triple-negative breast cancer cell lines and normal breast epithelial cells. The preparation process for metal-based ADCs can alter the antibody structure, affecting its antigen-binding capacity and specificity. Currently, the primary method for conjugating classic clinical platinum drugs such as cisplatin and oxaliplatin to antibodies is chemical conjugation to produce antibody-drug conjugates. However, this approach suffers from poor conjugate stability and a low drug-antibody ratio (DAR). Using complex natural products, such as dolastatin, as toxin molecules also presents drawbacks such as complex toxin modification and high synthesis costs. Summary of the Invention

[0007] The purpose of the present invention is to provide a cyclometallated N-heterocyclic carbene platinum (II) complex with anti-tumor cell proliferation activity, which is combined with trastuzumab as a carrier to prepare a trastuzumab-platinum (II) complex conjugate, thereby improving the tumor targeting of the platinum complex and its distribution in the body, achieving the same or even better tumor treatment effect with a lower amount of platinum metal, thereby achieving the goal of high-efficiency, low-toxicity tumor treatment.

[0008] A cyclometallated N-heterocyclic carbene platinum (II) complex having the following structural formula:

[0009]

[0010] This invention leverages the potential for photoimaging and photodynamic therapy of physiologically stable cyclometallated N-heterocyclic carbene platinum (II) complexes, as well as their subnanomolar cytotoxicity, providing a foundation for further development. By chemically coupling a monoclonal antibody with specific recognition to the platinum anticancer drug to form an antibody-platinum drug conjugate, the monoclonal antibody's targeted action enables efficient delivery of the platinum drug to tumor tissue, thereby improving therapeutic efficacy. It also effectively reduces drug accumulation in non-tumor areas, minimizing toxic side effects and significantly reducing the amount of platinum metal used. Ultimately, the therapeutic goal is achieved through the effective killing of tumor cells.

[0011] Furthermore, the synthesis method of the cyclometalated N-heterocyclic carbene platinum (II) complex comprises: firstly reacting a tridentate ligand with a platinum salt K2PtCl4 to generate [Pt(CNN)Cl], and then reacting with an N-heterocyclic imidazole salt containing a secondary amine alkyl chain to form a cyclometalated N-heterocyclic carbene platinum (II) complex.

[0012] A HER2-targeted platinum-based antibody conjugate is formed by connecting an antibody and the above-mentioned cyclometallated N-heterocyclic carbene platinum (II) complex via a linker.

[0013] Furthermore, the antibody is an anti-HER2 monoclonal antibody; the linker comprises a maleimide linker for connecting the antibody and a self-immolative linker for connecting the cyclometallated N-heterocyclic carbene platinum (II) complex; the dipeptide is valine-alanine; and each antibody conjugate carries approximately 4.5 drug molecules (cyclometallated N-heterocyclic carbene platinum (II) complex).

[0014] Furthermore, the linker is maleimide-amide-polyethylene glycol-valine alanine-amino carbonate.

[0015] The present invention provides an antibody-drug conjugate targeting HER2, which is composed of an antibody, a cytotoxic drug as a warhead molecule, and a linker. The antibody is trastuzumab monoclonal antibody, the cytotoxic drug is a cyclometallated N-heterocyclic carbene platinum (II) complex, and the linker is a maleimide-amide-polyethylene glycol-valine alanine-amino carbonate. The linker is used to achieve the connection between the antibody and the platinum (II) complex. The platinum (II) complex is converted into a linker-platinum (II) complex form, and the antibody is then coupled to the linker-platinum (II) complex by a chemical method that reduces the amide bond between the antibody chains. The linker-platinum (II) complex has a high reactivity with the amines of the trastuzumab monoclonal antibody.

[0016] Previous experiments conducted by the present invention explored the potential for combining platinum (II) complexes with trastuzumab. The experiments revealed that cyclometallated N-heterocyclic carbene platinum (II) complexes possess highly effective anti-tumor properties, acting on tumor cell mitochondria, causing mitochondrial damage and killing tumor cells. The present invention leverages the respective advantages of platinum (II) complexes and trastuzumab. The antibody-drug conjugate synthesized from the platinum (II) complex and trastuzumab enhances the platinum (II) complex's inhibitory effect on tumor cells and improves its targeting, reducing the amount of platinum metal used and, in turn, reducing the platinum (II) complex's toxicity to surrounding healthy cells, allowing the cytotoxic drug to act on tumor cells at a targeted location.

[0017] The method for synthesizing the above-mentioned HER2-targeting platinum-based antibody conjugate comprises the following steps:

[0018] (1) connecting a cyclometallated N-heterocyclic carbene platinum (II) complex with a linker to obtain Linker-Pt1;

[0019] (2) dissolving Linker-Pt1 in a solution; the solution is preferably DMSO;

[0020] (3) Dissolve tris(2-carbonylethyl)phosphine hydrochloride (TCEP) in PBS buffer and add dropwise to the PBS solution containing trastuzumab. Stir at room temperature for 2-4 hours to break the interchain disulfide bonds.

[0021] (4) adding the product obtained in step (3) to the solution obtained in step (2) and stirring at room temperature for 1-5 hours;

[0022] (5) Dilute the mixture with PBS buffer and use Zeba TM Purification by desalting centrifugal columns;

[0023] (6) The sample was then concentrated using an ultrafiltration centrifuge tube and washed with PBS buffer.

[0024] Furthermore, the connection method of step (1) is as follows: dissolving the linker in ultra-dry N,N-dimethylformamide, stirring at 0°C, then adding N,N-diisopropylethylamine, and then slowly adding the cyclometalated N-heterocyclic carbene platinum (II) complex dropwise. After the reaction is complete, the solvent is removed under reduced pressure to obtain a crude product; the crude product is purified by reverse phase liquid phase preparation. Preferably, the molar ratio of the linker to the cyclometalated N-heterocyclic carbene platinum (II) complex is 1:1-2.

[0025] Furthermore, the molar ratio of the added PBS solution of trastuzumab to the added Linker-Pt1 is 1:10-20.

[0026] An antibody-drug conjugate comprises the above-mentioned HER2-targeting platinum-based antibody conjugate, and the antibody-drug conjugate has a good anti-tumor effect.

[0027] Application of the above-mentioned HER2-targeted platinum-based antibody conjugate in the preparation of anti-tumor drugs.

[0028] Compared with existing technologies, it has the following advantages:

[0029] (1) The present invention combines a cyclometallated N-heterocyclic carbene platinum (II) complex with antiproliferative activity with a monoclonal antibody with specific recognition function as a carrier to prepare a trastuzumab-platinum (II) complex conjugate, thereby improving the tumor targeting of the platinum complex and its distribution in the body, thereby achieving efficient delivery of platinum drugs to tumor tissues, thereby improving the therapeutic effect; effectively reducing the accumulation of drugs in non-tumor areas and reducing toxic side effects; and achieving efficient and low-toxic tumor treatment effects by effectively killing tumor cells.

[0030] (2) The method of the present invention uses a more toxic and stable platinum (II) complex, avoiding the shortcomings of the current conjugates such as poor stability and low drug-antibody ratio, and obtains antibody-drug conjugates with stronger anti-tumor activity and high uniformity.

[0031] (3) The method of the present invention adopts a simple synthesis and selects platinum (II) complexes that are time-consuming and energy-saving, which can avoid the disadvantages of complex toxin modification and high synthesis cost, and obtain antibody-drug conjugates with stronger anti-tumor activity and high uniformity. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the structure of the HER2-targeting platinum-based antibody conjugate of the present invention;

[0033] Figure 2 It is the imidazole salt synthesis route diagram of Example 1;

[0034] Figure 3 is a synthetic route diagram of the cyclometallated N-heterocyclic carbene platinum (II) complex of Example 2;

[0035] Figure 4 The figure is a preparation scheme for the connection between the linker and the cyclometallated N-heterocyclic carbene platinum (II) complex, i.e., Linker-Pt1;

[0036] Figure 5 This is a synthetic route for the HER2-targeted platinum-based antibody conjugate of the present invention;

[0037] Figure 6 The UV spectrum (left), SDS-PAGE and drug-antibody ratio analysis (right) of the HER2-targeting platinum-based antibody conjugate of the present invention are shown;

[0038] Figure 7 This is a diagram showing the binding of the HER2-targeted platinum-based antibody conjugate of the present invention to different tumor cell lines;

[0039] Figure 8 This is a graph showing the endocytosis efficiency of the HER2-targeted platinum-based antibody conjugate of the present invention;

[0040] Figure 9 The in vitro enzymatic drug release of the HER2-targeting platinum-based antibody conjugate of the present invention;

[0041] Figure 10 The in vitro anti-tumor activity of the HER2-targeting platinum-based antibody conjugate of the present invention;

[0042] Figure 11 This is an in vivo anti-tumor activity evaluation of the HER2-targeting platinum-based antibody conjugate of the present invention, wherein Figure A is a graph showing changes in tumor volume during treatment, Figure B is a graph showing tumor weight and inhibition rate after treatment, Figure C is a graph showing changes in mouse body weight during treatment, and Figure D is a tumor anatomy diagram; compared with the control group, *p<0.05, **p<0.01, ***p<0.001;

[0043] Figure 12 This is the in vivo distribution of the HER2-targeting platinum-based antibody conjugate of the present invention. DETAILED DESCRIPTION

[0044] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be described in further detail below with reference to the accompanying drawings.

[0045] Example 1

[0046] A synthetic method of imidazole salts, such as Figure 2 As shown, 4-[tert-butyloxycarbonyl(methyl)amino]butyl p-toluenesulfonate reacts with imidazole to generate intermediate 1, which is then reacted with p-tert-butylbenzene bromide to prepare imidazole salt 2; specifically comprising the following steps:

[0047] (1) Under argon protection, imidazole (0.2 mmol, 2.0 eq.), sodium hydride (0.3 mmol, 3.0 eq.), and ultra-dry tetrahydrofuran were added and stirred at 0°C.

[0048] (2) Then, 4-[tert-Butyloxycarbonyl(methyl)amino]butyl p-toluenesulfonate (0.1 mmol, 1.0 eq.) was added and refluxed for 8 hours. After the reaction was completed by TLC monitoring, the reaction was quenched with water and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by alumina column chromatography to obtain intermediate 1 as a colorless transparent oil with a yield of 61%.

[0049] (3) Intermediate 1 (1.0 mmol, 1.0 eq.) and p-tert-butylbenzyl bromide (2.0 mmol, 2.0 eq.) were weighed and added to 5 mL of dry tetrahydrofuran and stirred at 75°C overnight. The solvent was concentrated and removed, and the reaction solution was ultrasonically washed with diethyl ether (3 × 25 mL) to obtain a colorless oil with a yield of 40%.

[0050] The obtained intermediate 1 has a H NMR spectrum: 1 H NMR (500 MHz, Chloroform-d) δ 7.56 (s, 1H), 7.00 (s, 1H), 6.87 (s, 1H), 3.94 (s, 2H), 3.19 (s, 2H), 2.74 (s, 3H), 1.73–1.67 (m, 2H), 1.47–1.41 (m, 2H), 1.38 (s, 9H). Intermediate 1 Mass Spectrum HR-ESI-MS: m / z Calcd. for C 13 H 23 N3O2([M+H] + ):254.1863,found:254.1860.

[0051] The obtained imidazole salt 2 has a H NMR spectrum: 1 H NMR (400 MHz, Methanol-d4) δ 9.17 (s, 1H), 7.70 (s, 1H), 7.66 (s, 1H), 7.48 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H), 5.42 (s, 2H), 4.29 (t, J = 7.3 Hz, 2H), 3.27 (t, J = 7.0 Hz, 2H), 2.83 (s, 3H), 1.91-1.83 (m, 2H), 1.59-1.52 (m, 2H), 1.43 (s, 9H), 1.31 (s, 9H). Imidazolium salt 2 mass spectrum: HR-ESI-MS: m / z Calcd. for C 24 H 38 N3O2([M-Br] + ):400.2959,found:400.2960.

[0052] Example 2

[0053] A method for synthesizing a cyclometallated N-heterocyclic carbene platinum (II) complex Pt-1, such as Figure 3 As shown, the tridentate ligand first reacts with the platinum salt K2PtCl4 to form [Pt(CNN)Cl], and then potassium tert-butoxide reacts with the imidazole salt 2 prepared in Example 1 to form a carbene, which then attacks the metal platinum center to generate the target product Pt-1. The process mainly includes the following steps:

[0054] Weigh 6-(thiophen-2-yl)-2,2′-bipyridine (0.21 mmol, 1.0 eq.) and K2PtC l4 To the mixture of 1,2-dimethyl-1-oxo-2-nitropropene (0.23 mmol, 1.1 eq.), acetic acid (5 ml) was added and the mixture was refluxed overnight to give an orange suspension. The solid was filtered and washed with n-hexane, methanol, deionized water and ether, and dried to give the product as an orange powder with a yield of 41%.

[0055] [Pt(CNN)Cl] (0.2 mmol, 1.0 eq.) and the imidazole salt 2 prepared in Example 1 (0.3 mmol, 1.5 eq.) were placed in a two-necked flask under argon protection. 10 ml of acetonitrile was added and heated at 85°C. Potassium tert-butoxide (0.3 mmol, 1.5 eq.) was added in three portions. After cooling to room temperature, an excess of ammonium hexafluorophosphate (2.0 mmol, 10.0 eq.) was added to the reaction mixture and stirred for 1 hour. After filtering out the insoluble matter, the filtrate was concentrated to dryness. The resulting solid was redissolved in dry DCM (2 ml), TFA (0.4 ml) was added, and stirring was continued for 1 hour. After completion of the reaction monitored by TLC, the solvent was removed by spin drying and purified by acid-base extraction to obtain an orange-yellow solid powder in a yield of 52%.

[0056] The obtained complex Pt-1 H NMR spectrum: 1 H NMR (400MHz, Methanol-d4) δ8.24–8.10(m,2H),7.95–7.81(m,2H),7.77(d,J=7.9Hz,1 H),7.53(d,J=4.7Hz,1H),7.52–7.44(m,3H),7.40(d,J=7.9Hz,1H),7.16–7.05(m,4H), 6.37(d,J=4.7Hz,1H),5.33(d,J=14.7Hz,1H),5.25(d,J=14.8Hz,1H),4.36–4.15(m,2 H),2.54–2.35(m,2H),2.20(s,3H),1.92–1.76(m,2H),1.46–1.37(m,2H),1.09(s,9H).

[0057] Pt-1 NMR spectrum of the complex: 13C NMR(126MHz,Methanol-d4)δ160.42,159.59,157.70,154.96,152.27,151.03,142.38,141.87,140.75,140.75,140.48,133.88,1 30.35,128.31,127.76,124.96,124.12,122.41,121.43,116.96,116.21,54.07,50.42,50.27,34.51,33.87,30.26,27.84,25.42.

[0058] Pt-1 NMR fluorine spectrum of the complex: 19 F NMR (376MHz, Methanoli-d4) δ-73.66 (d, J = 709.3Hz).

[0059] Pt-1 NMR phosphorus spectrum of the complex: 31 P NMR (162MHz, Methanol-d4) δ-144.41 (hept, J = 709.3Hz).

[0060] Mass spectrum of complex Pt-1: HR-ESI-MS: m / z Calcd.for C 33 H 38 N5PtS([M-PF6] + ):731.2490,found:731.2481.

[0061] Example 3

[0062] A method of targeting HER2 platinum-based antibody conjugates, such as Figure 4 and Figure 5 As shown, the following steps are included:

[0063] (1) Preparation of the complex Pt-1 and the linker, i.e., Linker-Pt1. Figure 4As shown, Pt-1 and a linker (maleimide-amide-polyethylene glycol-valine-alanine-aminocarbonate) were reacted in DMF to produce Linker-Pt1. The following steps were performed: Linker (0.1 mmol, 1.0 eq.) was weighed and dissolved in ultra-dry N,N-dimethylformamide (2 ml). N,N-diisopropylethylamine (10.0 mmol, 10.0 eq.) was added at 0°C, and Pt-1 (0.12 mmol, 1.2 eq., dissolved in ultra-dry dichloromethane) was slowly added dropwise over 2 hours. The reaction was monitored by thin-layer chromatography. Upon completion, the solvent was removed under reduced pressure to yield the crude product. The crude product was purified by reverse-phase preparative liquid chromatography (C18, 10% acetonitrile / water to 100% acetonitrile, v / v) to obtain a red powder in 30% yield.

[0064] (2) TCEP (0.06 mg, 0.22 μmol, 3.0 eq.) was dissolved in PBS buffer (50 μl) and added dropwise to a PBS solution containing trastuzumab (11 mg, 0.07 μmol, 4.7 ml). The mixture was stirred at room temperature for 2 h to disrupt the interchain disulfide bonds.

[0065] (3) Then, Linker-Pt1 (0.7 μmol, 10.0 eq.) dissolved in DMSO was added and stirred at room temperature for 1 hour;

[0066] (4) Dilute the mixture with 5.0 ml of PBS buffer and use Zeba TM Purification was performed using a desalting spin column (Thermo, 40KMWCO, 2 mL) to remove uncoupled Linker-Pt1;

[0067] (5)) The sample was then concentrated using an ultrafiltration centrifuge tube (Amicon Ultra-15 10K NMWL), washed with PBS buffer (pH = 7.2), and diluted to a volume of 2 ml to obtain a trastuzumab-platinum (II) complex conjugate with an appropriate concentration.

[0068] Linker-Pt1 H NMR spectrum: 11H NMR (500 MHz, Methanol-d4) δ 8.28–8.14 (m, 2H), 8.14–7.68 (m, 4H), 7.67–7.54 (m, 2H), 7.54–7.46 (m, 3H), 7.44–7.41 (m, 2H), 7.27 (d, J = 7.0 Hz, 1H), 7.18 (d, J = 8.1 Hz, 2H), 7.14–7.08 (m, 3H), 6.80 (s, 2H), 6.31–6.22 (m, 1H), 5.37–5.22 (m, 2H), 5.01–4.94 (m, 1H), 4.54–4.44 (m, 1H), 4.40–4.26 (m, 1H), 4.26–4.15 (m, 2H), 3.75 (t, J = 6.9 Hz, 4H), 3.61–3.54 (m, 12H), 3.47 (t, J = 5.4 Hz, 2H), 3.29–3.26 (m, 2H), 3.24–3.14 (m, 2H), 3.14–3.05 (m, 2H), 2.73–2.51 (m, 5H), 2.45 (t, J = 6.9 Hz, 2H), 2.15–2.05 (m, 1H), 1.94–1.83 (m, 1H), 1.84–1.69 (m, 3H), 1.65–1.53 (m, 2H), 1.50–1.40 (m, 2H), 1.13 (s, 9H), 0.89 (t, J = 6.0 Hz, 6H).

[0069] 13C NMR of Linker-Pt1: 13 13C NMR (126 MHz, Methanol-d4) δ 172.54, 171.73, 171.64, 170.95, 170.75, 160.88, 157.60, 156.24, 152.04, 150.94, 142.33, 141.99, 140.63, 140.48, 138.05, 134.09, 133.97, 133.83, 130.36, 128.29, 128.06, 127.76, 124.93, 124.18, 119.77, 119.56, 116.92, 116.25, 70.13, 70.09, 70.07, 70.00, 69.91, 69.80, 69.02, 66.86, 66.13, 54.04, 53.68, 39.11, 38.99, 35.99, 35.94, 34.41, 34.07, 33.86, 33.26, 32.78, 30.30, 28.87, 27.15, 26.72, 24.47, 18.44, 17.43. 19F NMR (376MHz, Methanol-d4) δ-74.52 (d, J=706.9Hz). 31 P NMR (202MHz, Methanol-d4) δ-144.58 (hept, J = 706.2Hz).

[0070] Linker-Pt1 mass spectrum: HR-ESI-MS: m / z Calcd.for C 70 H 91 N 12 O 13 PtS([M-PF6] + ):1534.6192,found:1534.6208.

[0071] Performance testing

[0072] 1. UV spectrum, SDS-PAGE and drug-antibody ratio analysis of HER2-targeted platinum-based antibody conjugates

[0073] The platinum complex was successfully coupled to the antibody by UV spectrophotometry, gel electrophoresis, and bicinchoninic acid method. Figure 6 As shown. The conjugate has a characteristic peak for the antibody at 280 nanometers and a characteristic peak for the complex at 350 nanometers. A distinct fluorescent band appears in lane 1, and the band position overlaps with lane 1'; since trastuzumab does not produce fluorescence under ultraviolet light, no band is observed in lane 3; the combination of the imaging results of these two sets of lanes indicates that the platinum (II) complex can be successfully conjugated to the light and heavy chains of trastuzumab. Furthermore, by comparing the positions of the light and heavy chain bands of the antibody conjugate and the naked monoclonal antibody under white light, it was found that both the light and heavy chain bands of the conjugate shifted upward, which corresponds to the increase in molecular weight and the slowing of migration speed after conjugation. Based on the absorbance of the conjugate at the maximum absorption wavelength of the complex, the concentration of the complex in the conjugate was calculated. Combined with the antibody concentration determined by the BCA method, the DAR value of the antibody conjugate was calculated to be 4.34.

[0074] 2. Detection of affinity of HER2-targeted platinum-based antibody conjugates for HER-2 protein

[0075] When the antibody is conjugated with a platinum (II) complex, its affinity for the antigen may be affected. Therefore, the binding of the conjugate to different cell lines was detected by flow cytometry. Among them, the breast cancer cell lines SK-BR-3 and BT-474, and the ovarian cancer cell line SK-OV-3 are HER-2 high-expressing cell lines, while the breast cancer cell line MDA-MB-231 and the lung cancer cell line NCI-H460 are HER-2 low-expressing cell lines. Figure 7As shown in the results, the antibody conjugate Trastuzumab-Pt1 has almost the same ability to recognize HER-2 antigens as naked monoclonal antibodies for three HER-2 high-expressing cell lines SK-BR-3, SK-OV-3, and BT-474, but has almost no binding to HER-2 low-expressing cell lines MDA-MB-231 and NCI-H460. This indicates that the antibody part of the antibody conjugate Trastuzumab-Pt1 almost completely retains the antigen recognition ability, can specifically recognize the HER-2 antigen on the cell surface, and bind to the surface of HER-2 high-expressing cells, but does not bind to HER-2 low-expressing cells.

[0076] 3. Targeted HER2 platinum-based antibody conjugate endocytosis detection

[0077] Before antibody conjugates can exert their activity in tumor cells, they must first be internalized by the cells. Therefore, for ADCs, endocytosis is particularly important.

[0078] BT-474 and SKOV-3 cells were harvested and digested, and 0.5 ml of the test compound was added to each cell. The cells were incubated on ice for 30 minutes to allow the antibody to bind to the antigen on the cell surface. After incubation, unbound antibody or antibody conjugate was removed by centrifugation at 4°C (300 g) for 5 minutes, and the cells were washed twice with 1 ml of FACS buffer. 0.5 ml of FACS solution was added to each centrifuge tube, and the cells were incubated in a 37°C incubator for 3 hours to allow internalization of the antibody or antibody conjugate bound to the cell surface. A separate tube incubated at 4°C for the same time served as a negative control for no internalization. After 3 hours, all tubes were transferred to an ice bath, and 1 ml of pre-chilled FACS solution was added to terminate internalization. The cells were then pelleted by centrifugation at 4°C (300 g) for 5 minutes. The supernatant was discarded, and a fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG secondary antibody was added in the dark, and incubation continued on ice for 60 minutes. After incubation, cells were washed twice with 1 ml of FACS buffer to remove any unbound secondary antibody. Resuspend the cells in 300 μl of PBS and analyze the fluorescence intensity using a BDC6 flow cytometer. FlowJo software was used for data analysis. % Internalization = [Total Surface MFI (4°C) - Total Surface MFI (37°C)] / Total Surface MFI (4°C) × 100%. Figure 8As shown, compared to the 4°C incubation group, the surface fluorescence intensity of BT-474 cells incubated for three hours at 37°C with trastzuzumab-1 decreased by approximately 42%, while that of trastzuzumab decreased by approximately 46%. The surface fluorescence intensity of SKOV-3 cells incubated with trastzuzumab-1 decreased by approximately 25%, while that of trastzuzumab decreased by approximately 12%. This indicates that the Pt-1-conjugated antibody retains comparable internalization efficiency to the naked mAb, and even exhibits higher internalization efficiency in SK-OV-3 cells.

[0079] 4. In vitro enzymatic analysis of HER2-targeted platinum-based antibody conjugates

[0080] To investigate drug release from the conjugate, a nucleophilic addition reaction between acetylcysteine ​​(NAC) and the maleimide of Linker-Pt1 was used to simulate enzymatic release of the trastuzumab-gold(III) complex conjugate by cathepsin B. MES buffer (10mM MES, 1mM EDTA, 1mM DTT, pH 5.5) was prepared and set aside. NAC was dissolved in 1× PBS to a 30mM solution. Linker-Pt1 was dissolved in DMF to a 2mM solution. 50μl of each solution was mixed and incubated at room temperature for 1 hour to prepare NAC-L-Pt1, which shielded the maleimide active site and simulated the enzymatic degradation of the antibody conjugate. A certain amount of NAC-L-Pt1 and activated cathepsin B solution were mixed evenly with MES buffer and incubated at 37°C. Samples were taken at different time points, quickly added to pre-cooled ACN, and stored in a -80°C refrigerator until the sample was analyzed. The sample was removed and centrifuged at 13000 rpm for 15 minutes. The supernatant was transferred to an injection vial and analyzed by UPLC (Accucore C18 analytical column (100×3mm; Thermo)). Figure 9 In the ELISA, the peak of Pt-1 was observed at 1 hour. By 6 hours, the dipeptide had been completely hydrolyzed, and the peak area of ​​Pt-1 had stopped increasing, indicating that the enzymatic hydrolysis was complete. This indicates that the platinum complex was successfully released from the conjugate.

[0081] 5. In vitro antitumor activity of HER2-targeted platinum-based antibody conjugates

[0082] The MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay was used to detect the in vitro anti-proliferative activity of the antibody conjugate against different cell lines. Due to the limited concentration of the antibody stock solution, it was impossible to determine its accurate IC50 value. Figure 10After treating cells with the antibody conjugate Trastuzumab-Pt1 for 96 hours, it was obvious that the cell survival rate was about 50% at a concentration of 5μM for HER-2 high-expressing cancer cell lines SK-OV-3, BT-474, and SK-BR-3. For HER-2 low-expressing cancer cell lines MDA-MB-231 and NCI-H460, it was observed that the cell survival rate was about 95% at a concentration of 2.5μM, and about 90% at a concentration of 5μM. For normal human breast cells MCF-10A, the cell survival rate was also about 80% at a concentration of 5μM. This shows that Trastuzumab-Pt1 can target HER-2 high-expressing cell lines and kill cells, while having almost no cytotoxicity to low-expressing cells and weak toxicity to normal human cells.

[0083] 6. In vivo evaluation of anti-tumor activity of HER2-targeted platinum-based antibody conjugates

[0084] SK-OV-3 cells (1.0×10 7 The cells were resuspended in 0.15 ml of serum-free McCoy's 5A medium and injected subcutaneously into the right inguinal region of BALB / c nude mice (5 weeks old, weighing 15-17 g, female). After the tumor was formed, the size of the tumor was measured with a digital caliper and the tumor volume (mm 3 )=1 / 2×L×W 2 (L represents length, W represents width) Calculate the tumor size. When the tumor grows to about 50 mm 3 At the same time, the nude mice with successful tumors were randomly divided into 4 different treatment groups (4 mice in each group). Trastuzumab-Pt1 (10 mg / kg and 15 mg / kg, diluted in PBS) and trastuzumab (10 mg / kg, diluted in PBS) were injected into the tail vein once every 7 days, and the control group was injected with an equal amount of PBS. The tumor volume and weight of the mice were measured every 2 days. On day 28, the tumor volume of the mice in the blank group reached 1200 mm 3 All mice were killed according to the animal treatment guidelines, and the tumors were dissected and weighed.

[0085] The inhibition rate of tumor growth was calculated using the following formula:

[0086] Tumor growth inhibition rate (%) = (1-average tumor weight of the treatment group / average tumor weight of the control group) × 100%.

[0087] All measurements are expressed as mean ± standard deviation; statistical analysis was performed using one-way analysis of variance and least significant difference (LSD) test. All data were analyzed by Graphpad Prism8. Figure 11As shown, compared to the blank control group, the tumor inhibition rates in the 10mg / kg and 15mg / kg trastuzumab-Pt1 treatment groups reached 75.93% and 86.53%, respectively, which is superior to the trastuzumab group (57.66%). Furthermore, the nude mice in the trastuzumab-Pt1 treatment group grew steadily during treatment, with no weight loss. This demonstrates that trastuzumab-Pt1 has excellent anti-tumor activity in vivo.

[0088] Table 1 Tumor weight and tumor inhibition rate after 28 days of administration

[0089]

[0090] 7. In vivo distribution detection of HER2-targeted platinum-based antibody conjugates

[0091] First, a double tumor-bearing mouse model (n=3) was established. 1×10 7 SK-OV-3 cells were injected subcutaneously into the right flank of BALB / c nude mice. 6 MDA-MB-231 cells were injected subcutaneously into the left lateral region. When the xenograft tumor volume reached approximately 150 mm 3 At 15 mg / kg of trastuzumab-Pt was administered intravenously. The animals were sacrificed 72 hours later, the tumors were excised, and the platinum concentration was determined by ICP-MS. 7 SK-OV-3 ovarian cancer cells were suspended in 0.15 ml serum-free medium and injected subcutaneously into the right ventral region of BALB / c-nu mice to establish a xenograft model. When the xenograft tumor volume reached 150 mm 3 Mice were randomly divided into three groups (n=4). Trastuzumab-Pt1 15 mg / kg was administered via intravenous tail injection. Mice were sacrificed 12, 48, or 96 hours later. The heart, liver, spleen, lung, kidney, and tumor tissues of the mice were removed, and the platinum concentration was measured by ICP-MS to explore the distribution of platinum in major organs. Figure 12As shown, in the dual-tumor model, platinum concentrations in SK-OV-3 tumors were significantly higher than in MDA-MB-231 tumors. In the single-tumor model, platinum concentrations in the heart, liver, spleen, lungs, kidneys, and tumors were assessed by ICP-MS at 12, 48, and 96 hours after intravenous injection. The study found that the liver and spleen were the primary sites of platinum accumulation (12 hours), with concentrations of 351 and 437 ng / g, respectively. Notably, tumor platinum concentrations increased significantly from 96 ng / g to 635 ng / g at 48 hours and remained at a high concentration of 766 ng / g at 96 hours. Combining the results from the dual-tumor model and the single-tumor model, it can be seen that the trastuzumab-platinum(II) complex conjugate, Trastuzumab-Pt1, is able to target and bind to HER-2-highly expressing tumors and accumulates significantly in the tumor over time. This shows that the trastuzumab-platinum (II) complex conjugate Trastuzumab-Pt1 has excellent tumor targeting ability.

Claims

1. A cyclometallated N-heterocyclic carbene platinum (II) complex, characterized in that The structural formula is as follows:

2. The method for synthesizing the cyclometallated N-heterocyclic carbene platinum (II) complex according to claim 1, characterized in that: include: First, the tridentate ligand reacts with the platinum salt K2PtCl4 to generate [Pt(CNN)Cl], which then reacts with an N-heterocyclic imidazole salt containing a secondary amine alkyl chain.

3. A HER2-targeted platinum-based antibody conjugate, characterized in that: The antibody is formed by connecting the cyclometallated N-heterocyclic carbene platinum (II) complex according to claim 1 via a linker.

4. The HER2-targeting platinum-based antibody conjugate according to claim 3, characterized in that: The antibody is an anti-HER2 monoclonal antibody; the linker comprises a maleimide linker connected to the antibody and a self-eliminating linker connected to the cyclometallated N-heterocyclic carbene platinum (II) complex.

5. The HER2-targeting platinum-based antibody conjugate according to claim 3, characterized in that: The linker is maleimide-amide-polyethylene glycol-valine alanine-amino carbonate.

6. The method for synthesizing a HER2-targeting platinum-based antibody conjugate according to claim 3, wherein: The following steps are involved: (1) connecting a cyclometallated N-heterocyclic carbene platinum (II) complex with a linker to obtain Linker-Pt1; (2) Dissolve Linker-Pt1 in the solution; (3) Dissolve tris(2-carbonylethyl)phosphine hydrochloride in PBS buffer and add dropwise to the PBS solution containing trastuzumab. Stir at room temperature for 2-4 hours to break the interchain disulfide bonds. (4) adding the product obtained in step (3) to the solution obtained in step (2) and stirring at room temperature for 1-5 hours; (5) diluting the mixture with PBS buffer and then purifying it using a desalting spin column; (6) The sample was then concentrated using an ultrafiltration centrifuge tube and washed with PBS buffer.

7. The synthesis method according to claim 6, characterized in that The connection method of step (1) is as follows: dissolving the linker in ultra-dry N,N-dimethylformamide, stirring at 0°C, then adding N,N-diisopropylethylamine, and then slowly adding the cyclometalated N-heterocyclic carbene platinum (II) complex dropwise. After the reaction is complete, the solvent is removed under reduced pressure to obtain a crude product; the crude product is purified by reverse phase liquid phase preparation.

8. The method for synthesizing a HER2-targeting platinum-based antibody conjugate according to claim 6, wherein: The molar ratio of the added PBS solution of trastuzumab to the added Linker-Pt1 is 1:10-20.

9. An antibody-drug conjugate, characterized in that: Comprising the HER2-targeting platinum-based antibody conjugate according to claim 3.

10. Use of the HER2-targeting platinum-based antibody conjugate according to claim 3 in the preparation of anti-tumor drugs.

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