Preparation and application of anti-her2 nanobody conjugated photosensitizer

Abstract

The application discloses a nano-antibody (also known as single-domain antibody) conjugated photosensitizer targeting HER2, a preparation method of the photosensitizer and application of the photosensitizer in tumor photodynamic therapy. The photosensitizer is shown in a general formula (I). In the photosensitizer, the nano-antibody (N HER2 ) serving as a targeting ligand is connected with a photosensitive moiety (PS) through a catalytic reaction mediated by a transglutaminase (MTGase). Research finds that the anti-HER2 nano-antibody conjugated photosensitizer molecule has good cell killing selectivity and tumor enrichment capacity, and can remove a subcutaneous tumor of a mouse with high HER2 expression in one administration. HER2 -PEG 1K -PS (I).

Description

Technical Field

[0001] This application relates to tumor-targeting photosensitizers based on nanobodies (also known as single-domain antibodies), their preparation methods, and their application in tumor photodynamic therapy. In the photosensitizer, the nanobodies, acting as targeting ligands, are site-specifically linked to the photosensitizer via a catalytic reaction mediated by transglutaminase (MTGase). Background Technology

[0002] Photodynamic therapy is a non-invasive tumor treatment, but the lagging development of photosensitizers currently limits its widespread clinical application. Monoclonal antibody-conjugated photosensitizers... (ASP-1929, cetuximab and) The antibody-drug conjugate (ADC) was approved for marketing in Japan in September 2020. This drug exhibits high tumor enrichment and selective killing capabilities, which is of great significance for the clinical development of photodynamic therapy for tumors. However, this strategy relies on large, full-length antibody molecules as targeting ligands, resulting in weak tumor tissue penetration and relatively low drug loading, thus limiting its tumor-killing ability. Furthermore, the long half-life of this molecule leads to prolonged phototoxicity after treatment, requiring patients to avoid light for extended periods. Additionally, the conjugation of antibodies with photosensitizer molecules is complex, and random conjugation results in heterogeneous conjugate structures, making quality analysis and monitoring difficult. Against this backdrop, we aim to develop novel miniaturized antibody-conjugated photosensitizers based on smaller nanobodies (also known as single-domain antibodies) to enhance the potential of this system for photodynamic tumor therapy.

[0003] In addition, the photosensitive group of this molecule Due to their complex structure, relatively difficult preparation, and poor stability, chlorophyll molecules are very expensive to purchase commercially. In contrast, the photosensitizing molecule pyrophyllin a (Pyro) can be easily prepared from chlorophyll molecules through several simple conversion steps, and its molecular structure is relatively stable. Therefore, this invention selects the Pyro molecule as the photosensitizing part to construct a nanobody-based targeted photosensitizer molecule, which will have greater advantages in clinical applications and in its preparation and synthesis.

[0004] In this invention, we propose an anti-human epidermal growth factor receptor 2 (HER2) nanobody (N HER2 The photosensitizer molecule was designed by coupling the photosensitizing group pyrophyllin a (Pyro), and polyethylene glycol (PEG) was introduced between the nanobody and the photosensitizing group to reduce the influence of the photosensitizing group on the nanobody molecule.

[0005] In molecular preparation and conjugation, site-specific ligation is crucial for antibody-drug conjugates (ADCs), improving drug purity and quality control, and minimizing uncontrollable toxicity caused by heterogeneity. Most ADCs utilize chemical conjugation methods, such as the interaction of active esters with amino groups or the interaction of cysteine ​​side chains with maleimide. However, because nanobodies contain multiple amino and thiol side chains, site-specific ligation cannot be achieved through chemical conjugation. Enzyme-mediated ligation offers a more advantageous protein modification method, characterized by high selectivity, high specificity, rapid reaction speed, mild reaction conditions, and maximum preservation of the substrate macromolecule's structure and function. However, to date, there are no reports of enzyme-mediated conjugation between antibodies and photosensitizer molecules.

[0006] In this invention, we utilize transglutaminase (MTGase) to catalyze the site-specific coupling of photosensitive groups with nanobodies, thereby preparing structurally uniform and stable anti-HER2 nanobodies-coupled photosensitive molecules. HER2 -PEG 1K -Pyro was used, and its photodynamic activity was evaluated in vitro and in vivo. The study found that the anti-HER2 nanobody-conjugated photosensitizer molecule has good cell-killing selectivity and tumor enrichment ability, and can clear subcutaneous tumors of mice with high HER2 expression with a single dose. Summary of the Invention

[0007] The purpose of this invention is to provide an anti-HER2 nanobody-conjugated photosensitizer.

[0008] Another object of the present invention is to provide a method for preparing an anti-HER2 nanobody-conjugated photosensitizer.

[0009] The technical solution of this invention is as follows:

[0010] An anti-HER2 nanobody-conjugated photosensitizer is shown in general formula (I):

[0011] N HER2 -PEG 1K -PS

[0012] (I)

[0013] The PEG mentioned 1K This can be represented by general formula (II).

[0014] (Gly)3-(L) m -Lys

[0015] (II)

[0016] Gly said Lys said

[0017] L represents a connecting chain, which is independently selected from the following structures:

[0018] -NH-(CH2CH2O)6-CH2CH2-C(O)-

[0019] m is an integer between 4 and 6, with the optimal value being 6.

[0020] Wherein N HER2 It has the following structure: the C-terminus contains an MTGase-specific tag sequence LLQGA; the N-terminus contains a 6-histidine (His) tag.

[0021] The photosensitive portion PS is independently selected from the Pyro group, and the Pyro group has the following structure.

[0022]

[0023] The method for preparing the photosensitizer includes:

[0024] 1) Connect m links L to Lys to obtain segment (L) m -Lys;

[0025] 2) Optionally use segment (L) m -Lys is linked with 3 Gly to obtain the PEG-linked portion. 1K :(Gly)3-(L) m -Lys;

[0026] 3) Connect the PEG portion 1K :(Gly)3-(L) m -Lys is linked to a photosensitive compound to obtain fragment PEG. 1K -PS (i.e., the control compound molecule Pyro-PEG) 1K -NH2);

[0027] 4) PEG fragment 1K -PS reacts with nanobody N via an MTGase enzyme-catalyzed reaction. HER2 Connect to obtain a photosensitizer of general formula (I);

[0028] Among them Lys, Gly, L, m, N HER2 PS is defined as above.

[0029] The use of the photosensitizer in the preparation of a drug for treating tumors.

[0030] Abbreviated as N HER2 -PEG 1K -Pyro, and the control compound Pyro-PEG 1KThe structural formula of -NH2,Pyro is as follows:

[0031]

[0032] This invention utilizes a combination of solid-phase and liquid-phase synthesis to obtain nanobody-conjugated photosensitizer molecules in a very simple and practical manner. We synthesized a PEG fragment containing a molecular weight of approximately 1K and a linker chain consisting of one lysine (Lys) and three glycine (Gly) residues using a triphenyl dichloro resin and an Fmoc solid-phase peptide synthesis method. 1K Then, it is coupled with the photosensitive group Pyro to obtain the compound Pyro-PEG. 1K -NH2. Finally, the product N was obtained by conjugation of nanobodies using an MTGase-mediated catalytic reaction. HER2 -PEG 1K -Pyro, the product was separated and purified by nickel column affinity chromatography.

[0033] This invention provides a method for preparing the photosensitizer, the synthetic route of which is attached. Figure 1 As shown.

[0034] N HER2 -PEG 1K -Pyro purity was analyzed using HPLC and SDS-PAGE, and molecular weight was characterized using MALDI-TOF-MS. We further investigated its optical properties by introducing PEG. 1K It can significantly improve the water solubility of Pyro and reduce the optical aggregation phenomenon of Pyro itself; with N HER2 Coupling did not affect its optical properties. Cellular and animal experiments were conducted to investigate the effects of N... HER2 -PEG 1K -Pyro's photodynamic activity was evaluated in vitro and in vivo. In vitro cell MTT assays and in vivo animal imaging experiments demonstrated that this molecule possesses ideal cytotoxic selectivity (approximately a 10-fold difference between cells expressing high and low HER2 receptor levels), photocytotoxicity, and excellent specific tumor accumulation ability. Due to its small molecular weight, the nanobody exhibits good tumor penetration, facilitating drug accumulation in deeper tumor tissues and increasing drug concentration at the tumor site. Therefore, even with low-dose administration, this molecule effectively eliminated subcutaneous tumors in nude mice with high HER2 expression. Thus, nanobody-conjugated photosensitizer molecules have good potential for clinical application. Attached Figure Description

[0035] Figure 1 It is Pyro-PEG 1K Schematic diagram of the synthesis process of -NH2;

[0036] Figure 2 It is Pyro-PEG 1K -NH2( Figure 2 A) and N HER2 -PEG 1K -Pyro( Figure 2 B) High performance liquid chromatography analysis chromatogram;

[0037] Figure 3 It is N HER2 -PEG 1K -Pyro and N HER2 SDS-PAGE analysis plot, in which Figure 3 A is an SDS-PAGE image stained with Coomassie Brilliant Blue. Figure 3 B is the fluorescence imaging image from SDS-PAGE;

[0038] Figure 4 It is Pyro, Pyro-PEG 1K -NH2 and N HER2 -PEG 1K -Emission spectrum of Pyro in PBS solution ( Figure 4 A) and its singlet oxygen quantum yield in PBS solution ( Figure 4 B, C, D);

[0039] Figure 5 It is Pyro, Pyro-PEG 1K -NH2 and N HER2 -PEG 1K - Evaluation of Pyro's drug activity in different cell lines in vitro;

[0040] Figure 6 It is Pyro, Pyro-PEG 1K -NH2 and N HER2 -PEG 1K -Selective binding assays of Pyro to NCI-N87 and PC3 cells, respectively;

[0041] Figure 7 It is Pyro, Pyro-PEG 1K -NH2 and N HER2 -PEG 1K -Pyro in NCI-N87 ( Figure 7 A) and PC3( Figure 7 B) Tumor enrichment in transplanted tumor mice;

[0042] Figure 8 It is N HER2 -PEG 1K - Pharmacokinetic curve of Pyro in mice;

[0043] Figure 9 It is N HER2 -PEG 1K -Pyro's photodynamic therapy effect on NCI-N87 xenograft mice, among which Figure 9 A represents a photograph of one representative mouse selected from each group after photodynamic therapy. Figure 9 B is a photograph of the tumor curve in mice after photodynamic therapy. Figure 9 C represents the mouse body weight curve. Detailed Implementation

[0044] Those skilled in the art will understand that the following embodiments are used to further illustrate the present invention but are not intended to limit the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained from the market.

[0045] abbreviations

[0046] PDT: Photodynamic therapy; Pyro: Phosphoric acid a; Lys: Lysine; Gly: Glycine; His: Histidine; DMF: PIP: Piperidine; HBTU: O-benzotriazole-tetramethylurea hexafluorophosphate; HOBt: 1-hydroxybenzotriazole; N,N-dimethylformamide; DIPEA: N,N-diisopropylethylamine; DCM: Dichloromethane; TFA: Trifluoroacetic acid; Tis: Triisopropylsilane; EDC: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; IPTG: Isopropyl-beta-D-thiogalactopyranoside; SDS-PA GE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; HPLC: High performance liquid chromatography; HRMS: High resolution mass spectrometry; MALDI-TOF-MS: Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry; PBS: Phosphate-buffered saline; MTT: Thiazol blue; IC50: Half-inhibitory dose; NCI-N87: Human gastric cancer cells; BT474: Human breast ductoma cells; SK-BR-3: Human breast cancer cells; SK-OV-3: Human ovarian cancer cells; MCF7: Human breast cancer cells; PC3: Human prostate cancer cells; H-CC70: Human triple-negative breast cancer cells; SPF: Specific pathogen-free.

[0047] Example 1. Chemical Synthesis

[0048] Abbreviated as Pyro-PEG 1K Synthesis of -NH2 compounds

[0049] We first synthesized Pyro-PEG using the Fmoc solid-state method. 1K -NH2.

[0050] (1) Weigh out the amine resin and add it into the solid synthesis tube. After swelling the resin with DMF, remove the Fmoc protecting group with 20% PIP / DMF solution.

[0051] (2) Fmoc-Lys(MTT)-OH (1.5 eq), HBTU (2.0 eq), HOBt (1.5 eq), and DIPEA (4.0 eq) were dissolved in DMF and added to a solid-phase synthesizer. The mixture was reacted at room temperature for 5 hours, followed by washing with DMF 5 times. Then, a blocking solution (DMF / acetic anhydride / DIPEA = 10 / 0.25 / 0.25) was added to the solid-phase synthesizer to block unreacted sites on the resin.

[0052] (3) After removing the Fmoc protecting group on Lys again with 20% PIP / DMF solution, Fmoc-PEG6-CH2CH2COOH (1.5 eq), HBTU (2.0 eq), HOBt (2.0 eq), and DIPEA (4.0 eq) were dissolved in DMF and added to the solid-phase synthesizer. The reaction was carried out at room temperature for 5 hours and washed 5 times with DMF.

[0053] (4) Next, repeat step (3) to connect 6 Fmoc-PEG6-CH2CH2COOH and 3 Fmoc-Gly-OH to the resin in sequence.

[0054] (5) After removing the MTT protecting group of Fmoc-Lys(MTT)-OH with 1% TFA / DCM, Pyro (1.5 eq), EDC (2.0 eq), HOBt (2.0 eq), and DIPEA (4.0 eq) were dissolved in DMF and added to the solid-phase synthesizer. The reaction was carried out at room temperature in the dark for 5 hours, and the mixture was washed 5 times with DMF.

[0055] (6) Finally, after removing the Fmoc protecting group on Gly again with 20% PIP / DMF solution, the product was cut off from the resin by adding cutting fluid (TFA / Tis / H2O = 95 / 2.5 / 2.5) and precipitated with diethyl ether to obtain the final product Pyro-PEG. 1K -NH2. The purity of each reaction product was determined by HPLC, and the purity was above 90%.

[0056] Example 2.N HER2 Expression and Coupling

[0057] 1. Protein expression

[0058] Expression N HER2The plasmid has a His (6 histidine) tag at the N-terminus and an MTG enzyme recognition sequence LLQGA at the C-terminus. Remove competent BL21 cells from the refrigerator and place them on ice. Mix 1-2 μL of the successfully constructed plasmid with the competent cells and incubate on ice for half an hour. Immediately afterwards, heat shock the cells in a 42°C water bath for 45 seconds. Add 450 μL of LB liquid medium in a clean bench. Tilt the EP tube in a shaker and incubate at 37°C, 150 rpm for 45 minutes. After the bacterial suspension is slightly mixed, centrifuge at 12000 rpm for 1 minute. Remove most of the supernatant with a pipette, resuspending the bacterial suspension in approximately 50 μL of medium. Add the resuspended bacterial suspension to a plate containing ampicillin antibiotic. Spread the bacterial suspension evenly on the plate using a heated and cooled spreader. Seal the plate opening with sealing film, label it, and incubate it upside down in an incubator. The next morning, single colonies were picked and placed in shake tubes containing culture medium and ampicillin antibiotic (1‰). The tubes were tilted and placed in a shaker at 37°C, 220 rpm for 12 hours. 1L of LB liquid culture medium was prepared and sterilized beforehand. After observing the bacterial growth in the shake tubes until the liquid became cloudy, the bacterial culture was poured into 1L of culture medium, and 1mL of ampicillin antibiotic was added. The tubes were then placed in a shaker at 37°C, 220 rpm for 12 hours. When the bacterial density reached its maximum logarithmic growth phase, the shaker temperature was lowered to 20°C, and 700μL of IPTG (isopropyl-β-D-thiogalactopyranoside) protein expression inducer was added. After approximately 4-6 hours, the bacterial culture was collected into a centrifuge tube, balanced, and centrifuged at 4000 rpm for 20 minutes. After centrifugation, most of the supernatant was discarded, and the precipitated bacteria were mixed with a small amount of liquid. Sterilize at 800 bar using an autoclave for approximately 5-10 minutes, stopping the process once the effluent gradually becomes clear. Collect the resulting liquid into an ultracentrifuge tube, balance the liquid, and centrifuge at 18,000 rpm for 40 minutes. Obtain the supernatant and purify the protein using nickel affinity chromatography. Pass the supernatant through the nickel column slowly to ensure sufficient binding between the protein and nickel powder. Prepare imidazole solutions of different concentrations (5 mM, 10 mM, 20 mM, 40 mM, 50 mM, 100 mM, and 300 mM) with PBS and elute sequentially. The His tag on the protein binds tightly to nickel ions; low concentrations of imidazole wash away impurities and weakly bound proteins, while high concentrations elute the target protein. Collect the eluted protein flow-through and determine the concentration using a Nanodrop micro-spectrophotometer. Collect the proteins with higher concentrations and place them in a dialysis bag. Dialyze with PBS solution to replace the imidazole approximately 3-4 times. Subsequently, determine the purity of the extracted proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Label the proteins with their names and concentrations and store them at -80°C for long-term storage.

[0059] 2. Coupling

[0060] First, the MTG enzyme catalyzes the nanobody N HER2 With photosensitizer molecule Pyro-PEG 1K The conditions for the -NH2 coupling reaction were explored, including protein concentration, molecular weight equivalent, enzyme concentration, reaction time, and reaction temperature, to determine the optimal reaction conditions. Large-scale coupling reactions were conducted under these optimal conditions. After purification by nickel column affinity chromatography, the conjugates were stored at -80°C for subsequent cell and animal experiments. Since the conjugates contain a His tag that can bind to nickel ions, excess unreacted photosensitizer molecules were first removed with a PBS solution containing 5‰ Triton X-100. Then, the photosensitizer molecules conjugated to the nanobody were eluted with high concentrations of imidazole (300mM, 500mM), followed by dialysis with PBS to finally obtain the nanobody photosensitizer conjugate N. HER2 -PEG 1K -Pyro. HPLC was used for purity testing, and the purity was above 90% (see attached). Figure 2 Molecular weight characterization: N HER2 , MALDI-TOF m / z, calculated 14000 Da, found 14290.195 Da; N HER2 -PEG 1K -Pyro, MALDI-TOF m / z, calculated 16843.0 Da, found 17093.22 Da.

[0061] Purity of the coupled product was determined by SDS-PAGE (see attached image). Figure 3 A) Detection of antibody-photosensitizer conjugation by fluorescence imaging (see attached image). Figure 3 B). SDS-PAGE can be used to observe the positional changes of the bands before and after nanobody conjugation with photosensitizer molecules. The first band represents the unpurified band, and the second band represents the conjugated product after purification. Fluorescence imaging shows that there was residual fluorescence signal from small molecules before purification, while after purification, only the conjugated product band showed fluorescence, indicating higher purity.

[0062] Example 3. UV-Vis spectroscopy and singlet oxygen detection

[0063] We used a Shimadzu UV-1900 ultraviolet spectrophotometer to study N. HER2 -PEG 1K -Pyro and control photosensitizer Pyro-PEG 1K -NH2 and Pyro were subjected to ultraviolet absorption spectroscopy measurements. The scanning wavelength range was from 400 nm to 800 nm, with a resolution of 0.5 nm and a scan rate of 600 nm per minute. Compared with Pyro, NHER2 -PEG 1K -Pyro and Pyro-PEG 1K Both -NH2 compounds exhibited good water solubility. Therefore, we further investigated the optical properties of these two compounds in PBS solution, based on the attached... Figure 4 As can be seen from Figure A, due to the poor water solubility of Pyro, it easily aggregates in PBS solution, thus resulting in a broad and weak absorption peak spectrum. Pyro-PEG modified with PEG... 1K -NH2 exhibits improved water solubility and exists in a non-aggregated state in PBS solution, thus a strong and sharp absorption peak was detected at 680 nm. When Pyro-PEG... 1K When -NH2 is coupled with nanobody, a strong and sharp absorption peak also appears at λ = 680 nm.

[0064] We tested the photosensitizing drugs Pyro and Pyro-PEG. 1K -NH2 and N HER2 -PEG 1K -Pyro singlet oxygen quantum yield in PBS solution (with appendix) Figure 4 (BD). When irradiated with white light, ABDA captured a decrease in the absorption peak of singlet oxygen generated by the photosensitizer in the 300-500 nm wavelength range, and the decrease in the absorption band area integral was proportional to the irradiation time. Because Pyro aggregates in PBS solution, the singlet oxygen quantum yield is very low, only 0.03; Pyro-PEG 1K -NH2 and N after conjugation of nanobody HER2 -PEG 1K -Pyro has good water solubility and can exist in a non-aggregated state in PBS solution, thus generating strong singlet oxygen quantum yields of 0.28 and 0.31, respectively.

[0065] Example 4. Evaluation of drug activity at the cellular level

[0066] Pyro, Pyro-PEG 1K -NH2 and N HER2 -PEG 1K - Evaluation of Pyro's photodynamic activity in different cell lines (with appendix) Figure 5 (Table 1)

[0067] Table 1 lists Pyro and Pyro-PEG. 1K -NH2 and N HER2 -PEG 1K Data on the photodynamic activity of Pyro in different cell lines (nM)

[0068]

[0069] Seven cell lines with different HER2 receptor expression levels were divided into groups of 1×10⁻⁶ cells per well. 4 Cells were seeded into 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator. After removing the supernatant, different concentrations of Pyro and Pyro-PEG were added to each well using fresh culture medium. 1K -NH2 and N HER2 -PEG 1K -Pyro, place the culture plate in a cell culture incubator and incubate for 4 hours. Remove the culture plate and place it under a 660nm light source, applying 40mW / cm² light at a distance of approximately 1cm. 2 After a 10-minute light radiation dose, cells were cultured overnight in a cell culture incubator. 10 μL of MTT (5 mg / mL PBS solution) was added to each well, and the cells were co-incubated for 4 hours. The cell culture medium was then removed, and 100 μL of dimethyl sulfoxide was used to replace the medium in each well. The absorbance at 490 nm was measured using a multifunctional enzyme-linked immunosorbent assay (ELISA) reader, and cell viability was calculated based on the absorbance. A drug activity inhibition curve was then plotted to show the relationship between cell viability and drug concentration, and the corresponding half-inhibitory concentration (IC50) was calculated. 50 Values ​​(Table 1). Five wells were run for each experimental group, and the data are expressed as mean ± SEM. Five wells were run for each experimental group, and the data are expressed as mean ± SEM.

[0070] The experimental results are attached. Figure 5 As shown in Table 1, Pyro has good lipid solubility and no cell selectivity. It adheres to the surface of all cell membranes. After light exposure, the photosensitizers adhering to the cell membrane surface release singlet oxygen, causing cell damage. Pyro exhibits strong killing ability against all cell lines, with an IC50 value of [missing value]. 50 The value is around 50 nM, with no selective killing effect. Pyro-PEG modified with PEG... 1K -NH2 exhibits improved water solubility and weakened cell membrane adhesion, but it does not selectively kill different cell lines. IC50 50 The value is around 300 nM. N is linked to an antibody-targeted N... HER2 -PEG 1K -Pyro, in nanobody N HER2 Mediated by N, it binds to corresponding receptors on the cell surface, thereby attaching the drug to the cell surface. HER2 -PEG 1K -Pyro exhibits strong killing activity only against HER2-highly expressing cell lines NCI-N87, BT-474, SK-BR-3, and SK-OV-3, with corresponding IC50 values. 50The values ​​were 141.4 nM (NCI-N87 cells), 145.8 nM (BT-474 cells), 138.3 nM (SK-BR-3 cells), and 144.4 nM (SK-OV-3 cells), respectively; while the PC3, MCF-7, and H-CC70 cell lines with low HER2 expression showed almost no killing ability. This indicates that N HER2 -PEG 1K -Pyro exhibits excellent cell-killing selectivity against cell lines with varying HER2 receptor expression levels.

[0071] 2. Pyro, Pyro-PEG 1K -NH2 and N HER2 -PEG 1K Selective binding assays of Pyro in NCI-N87 and PC3 cell lines (attached) Figure 6 )

[0072] Pyro and Pyro-PEG can also be detected using flow cytometry. 1K -NH2 and N HER2 -PEG 1K -Pyro's targeting selectivity for the HER2 receptor. HER2-highly expressing NCI-N87 cells and HER2-lowly expressing PC3 cells were divided into groups of 1×10⁻⁶ cells per well. 5 Cells were seeded into 12-well plates and incubated overnight at 37°C in a 5% CO2 incubator. After removing the supernatant, Pyro and Pyro-PEG were added to each well in fresh culture medium containing different drug concentrations (0 nM, 10 nM, 100 nM, 1 μM). 1K -NH2 and N HER2 -PEG 1K -Pyro cells were cultured in a cell culture incubator in the dark for 4 hours. The drug-containing culture medium was removed, and the cells were washed twice with PBS. 500 μL of trypsin was added to each well, and the cells were digested at 37°C for 3-5 min. The cells were then transferred to flow cytometry tubes, centrifuged at 1000 rpm / min for 3 min at 4°C, and resuspended in 200 μL of PBS. The fluorescence intensity of each tube of cells was detected using a BD FACSCalibur flow cytometer (λex = 635 nm; λem = 645-677 nm).

[0073] The experimental results are attached. Figure 6 As shown, due to Pyro and Pyro-PEG 1K-NH2 has good lipid solubility and adheres to the cell surface in a dose-dependent manner. The higher the drug concentration, the more molecules adhere to the cell membrane surface, and the stronger the detected fluorescence signal. It also has no selectivity for cell binding; therefore, strong fluorescence signals were detected on the surface of both NCI-N87 and PC3 cells, with the signal intensity increasing with increasing drug concentration. N2 conjugated with nanobodies... HER2 -PEG 1K -Pyro binds to the cell surface through antigen-antibody interactions. Fluorescence signals were detected in the HER2-overexpressing NCI-N87 cell line at low concentrations, and a very strong fluorescence signal was detected at a high concentration of 1 μM. However, for the HER2-low-expressing PC3 cell line, fluorescence signals similar to background values ​​were detected at both low and medium concentrations, indicating that N... HER2 -PEG 1K -Pyro exhibits excellent binding selectivity among cells with different HER2 expression levels.

[0074] Example 5. Pyro, Pyro-PEG 1K -NH2 and N HER2 -PEG 1K - Distribution characteristics of Pyro in mice and evaluation of its therapeutic effect on tumors.

[0075] 1. Establishment of a mouse subcutaneous tumor model

[0076] The mice used in this invention were 6-8 week old female BALB / c nude mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and were housed in an SPF-grade environment. Subcutaneous tumor models in mice were established using the HER2-highly expressing NCI-N87 cell line and the HER2-lowly expressing PC3 cell line, respectively. The specific procedures are as follows: First, the required number of tumor cells was calculated based on the number of mice, and the corresponding tumor cells were cultured in large quantities appropriately. In this experiment, each mouse in each tumor model required 3 × 10⁶ cells. 6 Cells were collected. Once the cells reached approximately 80% of the space in the culture dish, they were digested and centrifuged at 1000 rpm for 3 minutes in a centrifuge pre-cooled to 4°C. The supernatant was discarded, and the cells were resuspended in PBS. 10 μL of the suspension was then placed in a cell counting chamber for counting. Based on the counting results, a cell suspension was prepared, calculated at 100 μL per mouse for subcutaneous injection.

[0077] Subcutaneous tumor inoculation method in mice: After anesthetizing mice with 15% isoflurane, disinfect the right side of the mouse's back with 75% alcohol, then draw 100 μL (3 × 10⁻⁶) into a syringe. 6 After suspending the tumor cells (in 1 cell), the cells were injected subcutaneously into the mouse. The needle was then inverted and pulled out of the mouse, and the cells were marked.

[0078] 2. Pyro, Pyro-PEG 1K -NH2 and N HER2 -PEG 1K - Tumor enrichment of Pyro in NCI-N87 xenograft mice (attached) Figure 7 )

[0079] Due to the fluorescent properties of photosensitizing drugs, we can easily use the Xenogen IVIS small animal in vivo imaging system to study the distribution of photosensitizing drugs in mice. Subcutaneous tumor models were constructed in NCI-N87 mice with high HER2 expression and PC3 mice with low HER2 expression, respectively. The tumors were allowed to grow to 200 mm. 3 In the initial stages, mice were first briefly anesthetized with 15% isoflurane and then placed in the Xenogen IVIS small animal in vivo imaging system. Each group of mice was scanned using parameters of λex = 640 nm; λem = 695-770 nm, and an exposure time of 5 seconds. The autofluorescence values ​​of the tumor sites and other body parts at this time were used as background subtraction. Then, the mice were injected via tail vein with 20 nmol Pyro and Pyro-PEG, respectively. 1KK -NH2 and N HER2 -PEG 1K -Pyro PBS, and the drug distribution was scanned using an in vivo imaging system at 0h, 2h, 4h, 6h, 8h, 10h, and 24h after administration, starting from the injection time point.

[0080] Experimental results: as attached Figure 7 It was observed that at different time points, Pyro only settled in the liver and kidney regions of both mouse models, with obvious fluorescent signals observed only in the left and right abdominal areas. The fluorescence intensity gradually weakened over time, and by approximately 24 hours, no obvious yellow fluorescent signal was observed. Similarly, Pyro-PEG... 1K -NH2 did not show a significant difference in enrichment between the two mouse models. The fluorescent signal mostly accumulated in the liver and kidneys, with weak fluorescence intensity detected at the tumor site, which gradually decreased in intensity over time. In contrast, N... HER2 -PEG 1K -Pyro showed significant enrichment at tumor sites in vivo. In the NCI-N87 mouse model, after injection of the nanobody-conjugated photosensitizing drug N... HER2 -PEG 1KTwo hours after injection of Pyro, strong yellow fluorescence was observed at the tumor site, but at this time, there was also significant drug accumulation in the liver and kidneys. As time progressed, the fluorescence signal in the liver and kidneys gradually weakened, while the signal at the tumor site increased, reaching its peak at 8-10 hours. At 10 hours, the tumor site was the only location in the body producing fluorescence. The fluorescence signal weakened after 24 hours, indicating that the drug accumulated at the tumor site began to be metabolized. In contrast, in the PC3 mouse model, after injection of N... HER2 -PEG 1K At different time points after Pyro, the drug accumulated only in the liver and kidneys of mice, and the fluorescence signal began to weaken after 6 hours, indicating that the drug was gradually metabolized and excreted from the body. This experimental result strongly confirms the N... HER2 -PEG 1K Pyro's tumor targeting and accumulation capabilities in vivo demonstrate its ability to bind to the HER2-overexpressing NCI-N87 tumor cell surface receptor, delivering photosensitizing drugs to the tumor surface. This experiment can also determine the optimal targeting and accumulation time of photosensitizing drugs at the tumor site, providing a foundation for subsequent in vivo therapeutic experiments.

[0081] 3.N HER2 -PEG 1K - Metabolism of Pyro in the blood over time in mice (with appendix) Figure 8 )

[0082] We further evaluated the metabolic clearance properties of this molecule in the mouse circulatory system. 20 nmol N... HER2 -PEG 1K -Pyro was administered to female BALB / c mice (n=3 mice) via tail vein injection. Blood was collected periodically (3 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 24 h, 48 h) and the N2 bound to the serum was measured using a BioTek Cytation / 5 microplate reader via quantitative fluorescence measurement. HER2 -PEG 1K -Pyro. Three minutes after drug injection, the drug concentration in the blood is considered to be 100%. Serum binding N at different time points... HER2 -PEG 1K - Plot the percentage of fluorescence of Pyro against time and calculate the half-life of the drug in the blood using GraphPad Prism 5 software.

[0083] Experimental results: The half-life of full-length antibodies in the blood can be as long as one to two weeks, and their complete metabolism from the circulatory system takes a long time. Long-term retention of photosensitive molecules in the body may cause phototoxic side effects on the skin and eyes. In contrast, N... HER2 -PEG 1K- The blood half-life of Pyro molecules is only 49.55 minutes, and excess photosensitizer molecules can be rapidly metabolized from the blood. This helps to greatly reduce phototoxic side effects on the skin and eyes and reduce the time patients need to avoid light.

[0084] 4.N HER2 -PEG 1K - Pyro's photodynamic therapy effect on NCI-N87 xenograft mice (with appendix) Figure 9 )

[0085] We used the NCI-N87 mouse subcutaneous tumor model to investigate N HER2 -PEG 1K The therapeutic effects of Pyro were studied. 3×10 6 One NCI-N87 cell was subcutaneously injected into the right back of a BALB / c nude mouse. After approximately 14 days, the tumor volume in the mouse reached about 100 mm. 3 Mice were randomly divided into four groups (n=5): PBS group, 50mW / cm² group, and 50mW / cm² group. 2 Treatment group, 100mW / cm 2 Treatment group, 200mW / cm 2 Treatment group. PBS and 20 nmol N2 were injected via tail vein, respectively. HER2 -PEG 1K -Pyro, after 8 hours, illuminated the tumor sites in mice with a light power of 50 mW / cm². 2 100mW / cm 2 200mW / cm 2 Following light therapy, mice were photographed and observed every three days. Tumor volume changes were recorded using calipers, along with changes in mouse weight. The treatment continued until the tumor volume reached 1500 mm². 3 At this point, the mouse was considered dead and data recording was stopped; the mice were observed for up to 40 days. The formula for calculating the tumor volume in mice is: Tumor volume = length × width 2 ×0.5.

[0086] Experimental results: as attached Figure 9 In mice A and B, one day after treatment, all treatment groups except the PBS group developed extensive edema at the tumor site. The edema gradually decreased in size and darkened, eventually forming scabs that fell off, restoring the mouse's skin to normal and eliminating the tumor. In the PBS group, the tumors gradually increased in size over time, reaching approximately 1500 mm² in volume around day 18. 3 No further data measurement or recording was conducted. In the treatment group mice, a power of 50 mW / cm² was used. 2 100mW / cm 2The survival time of the treated mice was longer than that of the PBS group, but recurrence began three weeks after treatment, with tumor growth starting at the original site, although the growth rate was slow; while the mice treated with 200mW / cm 2 Mice treated with this method showed tumor recurrence six weeks after treatment, significantly extending their survival time. Meanwhile, as shown in the attached... Figure 9 Based on the recorded body weights of mice in different groups, it can be seen that the body weight of mice in the PBS group showed a continuous increasing trend, until the tumor reached 1500 mm. 3 Data recording was then stopped. The mice in the treatment group experienced weight loss in the first few days after treatment, possibly due to drug accumulation causing some inflammation, resulting in slight weight loss. However, the mice subsequently regained normal weight and gradually gained it back.

[0087] In summary, N HER2 -PEG 1K Pyro has shown great promise for clinical application in the field of photodynamic therapy for tumors.

[0088] The foregoing general description and specific embodiments of this invention should not be construed as limiting the scope of the invention. Those skilled in the art, based on this invention, may add, reduce, or combine the technical features disclosed in the foregoing general description and / or specific embodiments (including examples) without departing from the essential elements of the invention, forming other technical solutions belonging to the invention, which are also within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents.

Claims

1. Photosensitizers of general formula (I) or pharmaceutically acceptable salts thereof: N HER2 -PEG 1K -PS (I) The PEG mentioned 1K This can be represented by general formula (II). (Gly)3-(L) m -Lys (II) Gly said Lys said L represents a connecting chain, which is independently selected from the following structures: -NH-(CH2CH2O)6-CH2CH2-C(O)- m is an integer between 4 and 6, with the optimal value being 6.

2. The photosensitizer as described in claim 1, characterized in that, Wherein N HER2 It has the following structure: the C-terminus contains an MTGase-specific tag sequence LLQGA; the N-terminus contains a 6-histidine (His) tag.

3. The photosensitizer as described in claim 1, characterized in that, The photosensitive portion PS is independently selected from the Pyro group, which has the following structure:

4. A method for preparing the photosensitizer according to any one of claims 1-3, comprising: 1) Connect m links L to Lys to obtain segment (L) m -Lys; 2) Optionally use segment (L) m -Lys is linked with 3 Gly to obtain the PEG-linked portion. 1K :(Gly)3-(L) m -Lys; 3) Connect the PEG portion 1K :(Gly)3-(L) m -Lys is linked to a photosensitive compound to obtain fragment PEG. 1K -PS (i.e., the control compound molecule Pyro-PEG) 1K -NH2); 4) PEG fragment 1K -PS reacts with nanobody N via an MTGase enzyme-catalyzed reaction. HER2 Connect to obtain a photosensitizer of general formula (I); Among them Lys, Gly, L, m, N HER2 And PS as defined in any one of claims 1-3.

5. Use of the photosensitizer according to any one of claims 1-3 in the preparation of a medicament for treating tumors.

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