Photosensitizer chimera targeting PD-l1 protein, and preparation method therefor and use thereof

Abstract

A photosensitizer chimera targeting PD-L1 protein, and a preparation method therefor and a use thereof. The photosensitizer chimera is of an X-R-G structure, wherein X is a photosensitizer unit, R is a linker, G is a polypeptide unit targeting PD-L1 protein, the photosensitizer is a porphyrin photosensitizer, and the excitation light wavelength of the photosensitizer is longer than 550 nm; the molar absorption coefficient at the maximum absorption wavelength longer than the wavelength of 550 nm is greater than 1000 M -1cm-1; the quantum yield of a triplet state exceeds 0.01; the linker is selected from the following structural formulas, wherein the amino side of the linker is connected to the photosensitizer unit, the carboxyl side of the linker is connected to the polypeptide unit, and n is an integer of 0-50; the binding constant of the polypeptide and h-PD-L1 is less than 20 µm. Combination therapy of immune checkpoint blockade and cytotoxicity induced by the photosensitive chimera based on photodegradation of PD-L1 protein in PD-1 / PD-L1 can cause stronger immune response than monotherapy, and thus the photosensitive chimera has great potential in enhancing antitumor therapy.

Description

Photosensitizer chimera targeting PD-L1 protein and preparation method and application thereof Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a photosensitizer chimera targeting PD-L1 protein, and a preparation method and application thereof. Background Art

[0002] Tumor immunotherapy has become a hot new treatment option, following surgery, chemotherapy, radiotherapy, and targeted drug therapy, and has been applied clinically. Recent studies have revealed that the programmed death receptor-1 (PD-1) / programmed death ligand-1 (PD-L1) signaling pathway is a key pathway involved in tumor immune escape. This signaling pathway can induce tumor-specific T cell apoptosis by inhibiting T cell activation, leading to T cell resistance and contributing to immune escape and immunosuppression in pathological conditions such as tumors and chronic inflammation. Currently, 19 PD-1 / PD-L1 drugs have been approved for marketing worldwide, of which 13 have been approved for marketing in China. These include nine PD-1 inhibitors (seven domestically produced and two imported) and four PD-L1 inhibitors (two domestically produced and two imported). However, current therapeutic antibodies still have shortcomings, including lack of oral bioavailability, difficult and expensive production, immune-related adverse events, poor uptake into tumor tissues, and gradual development of drug resistance, which limits the clinical application of PD-1 / PD-L1 antibody drugs. Compared with antibodies, peptides generally have better physiological properties, such as better tissue and tumor penetration, higher oral bioavailability, and more tolerable half-life. In addition, combination therapy is also the main trend in current tumor immunotherapy. The cytotoxic effect produced by photodynamic therapy of tumors can effectively activate the body's immune response, thereby better reducing the problem of PD-1 / PD-L1 resistance. Therefore, the use of light-timed targeted degradation of PD-L1 protein to induce immune checkpoint blockade (ICB) therapy and combined with the cytotoxic effect of photodynamic therapy to develop and design molecules is a very promising alternative to PD-1 / PD-L1 monoclonal antibodies.

[0003] Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a photosensitizer chimera targeting PD-L1 protein, wherein the chimera has an XRG structure, wherein X is a photosensitizer unit, R is a linker arm, and G is a polypeptide unit targeting PD-L1 protein, wherein the photosensitizer is a porphyrin photosensitizer, and the excitation wavelength of the photosensitizer is greater than 550nm; the molar absorptivity at the maximum absorption wavelength after 550nm is greater than 1000M-1 cm -1 ;The quantum yield of triplet state is >0.01;

[0005] The connecting arm is selected from the following structural formula:

[0006] wherein the amino side of the connecting arm is connected to the photosensitizer unit, the carboxyl side of the connecting arm is connected to the polypeptide, and n is an integer of 0-50;

[0007] The binding constant of the peptide to h-PD-L1 is <20 μM.

[0008] Preferably, the structural formula of the connecting arm is: n is an integer of 0-50; preferably n is an integer of 0-10.

[0009] Preferably, the photosensitizer is selected from at least one of verteporfin, dihydrochlorin or pheophorbide a; preferably verteporfin.

[0010] The present invention has no particular limitation on the amino acid sequence of the polypeptide. Preferably, the polypeptide is any one of (a) to (c):

[0011] (a) a polypeptide having the amino acid sequence (1)-(11);

[0012] (b) a polypeptide having amino acid sequences (1)-(11) with substitution, deletion or addition of 1-3 amino acid residues and capable of binding to PD-L1 with an affinity of <20 μM;

[0013] (c) a polypeptide having an ester group or an amide group linked to the carboxyl terminus of the polypeptide of (a) or (b);

[0014] Amino acids (sequence 1)-(11) are shown below:

[0015] dNdYdSdKdPdTdDdRdQdYdHdF(1);

[0016] dNdYdSdKdPdTdDdR(2);

[0017] dNdYdSdKdPdTdDdRdQ(3);

[0018] dNdYdSdKdPdTdDdRdQdY(4);

[0019] dKdPdTdDdRdQdYdHdF(5);

[0020] dPdTdDdRdQdYdHdF(6);

[0021] dNdYdSdKdPdTdDdRdQdYdH(7);

[0022] dSdKdPdTdDdRdQdYdHdF(8);

[0023] dYdSdKdPdTdDdRdQdYdHdF(9);

[0024] dKdPdTdDdR(10);

[0025] dSdKdPdTdDdR(11);

[0026] Preferably, dNdYdSdKdPdTdDdRdQdYdHdF(1);

[0027] dNdYdSdKdPdTdDdR(2);

[0028] dNdYdSdKdPdTdDdRdQ(3);

[0029] dNdYdSdKdPdTdDdRdQdY(4);

[0030] dKdPdTdDdRdQdYdHdF(5);

[0031] More preferably, it is dNdYdSdKdPdTdDdRdQdYdHdF(1).

[0032] In a preferred embodiment, the chemical structural formula of the photosensitizer chimera is as shown in Formula I or Formula II;

[0033] In Formula I and Formula II, n is independently an integer between 0 and 10, preferably an integer between 0 and 6; G is a polypeptide unit targeting the PD-L1 protein, and the amino acid sequence of the polypeptide is: dNdYdSdKdPdTdDdRdQdYdHdF (1). The N-terminus of the polypeptide targeting the PD-L1 protein is bound to the linker arm via an amide bond.

[0034] The present invention also provides a method for preparing the photosensitizer chimera described in the above scheme, comprising the following steps:

[0035] 1) first mixing the linker and the polypeptide targeting the PD-L1 protein, performing a first coupling, and removing uncoupled substances to obtain a first conjugate;

[0036] 2) mixing the first conjugate and the photosensitizer for a second time, performing a second coupling under light-shielding conditions, removing unconjugated substances, and obtaining a photosensitizer chimera.

[0037] The present invention has no particular limitation on the preparation method of the polypeptide targeting PD-L1 protein, and it can be prepared by conventional methods in the art. In the specific implementation of the present invention, the polypeptide targeting PD-L1 protein is prepared by a solid phase synthesis method based on Fmoc. α N-Fmoc or α N-Boc protected non-natural D-amino acids include: Fmoc-D-Asn(Trt)-OH, Fmoc-D-Tyr(OtBu)-OH, Fmoc-D-Ser(tBu)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Pro-OH, Fmoc-D-Thr(tBu)-OH, Fmoc-D-Asp(OtBu)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-His(Trt)-OH, and Fmoc-D-Phe-OH.

[0038] In the present invention, after coupling a polypeptide targeting the PD-L1 protein to the resin, the Fmoc protecting group is removed, washed, and then mixed with a solution containing a linker for the first time; the solution containing the linker uses DMF as a solvent and contains a linker, N-hydroxy-7-azabenzotriazole (HATU) and HOAt; the equivalent ratio of the linker, HATU and HOAt is preferably 0.5-1.5:0.5-1.5:0.5-1.5; before the first mixing, DIEA is added to the solution containing the linker to activate the amino acid.

[0039] The present invention has no particular limitation on the conditions for the first coupling. Preferably, the conditions for the first coupling include: a coupling temperature of 5-30° C.; and a coupling time of 8-14 h.

[0040] After obtaining the first conjugate, the present invention mixes the first conjugate and the photosensitizer for a second time, performs a second coupling under light-proof conditions, removes the unconjugated substance, and obtains a photosensitizer chimera.

[0041] The present invention has no particular limitation on the type of linker, and examples of linkers include but are not limited to: Fmoc-NH-(PEG) x -CH2CH2-COOH, Fmoc-NH-(CH2) y -COOH, wherein x and y are even numbers ranging from 2 to 10.

[0042] In the present invention, the first conjugate is coupled to the resin and then secondarily mixed with a solution containing a photosensitizer. The photosensitizer solution is prepared using DMF as a solvent and contains the photosensitizer, HATU, and HOAt. The equivalent ratio of the photosensitizer, HATU, and HOAt is preferably 0.5-1.5:0.5-1.5:0.5-1.5. Prior to the second mixing, DIEA is added to the solution containing the linker photosensitizer to activate the amino acid.

[0043] Preferably, in step 1), the ratio of the linker arm to the polypeptide targeting the PD-L1 protein is 1-5:1.

[0044] In a preferred embodiment, in step 2), the usage ratio of the first coupling agent to the photosensitizer is 1:1-4.

[0045] The present invention has no particular limitation on the conditions for the second coupling. Preferably, the conditions for the second coupling include: a coupling temperature of 5-30° C.; and a coupling time of 8-14 h.

[0046] The present invention also provides an anti-tumor drug, the active ingredient of which is the photosensitizer chimera described in the present invention; preferably, the tumor is a tumor targeting PD-L1, further preferably any one of breast cancer, lung cancer, colorectal cancer, esophageal cancer or pancreatic cancer; further preferably, breast cancer or esophageal cancer.

[0047] The present invention provides the use of the photosensitizer chimera in the preparation of an anti-tumor drug; preferably, the tumor is a tumor targeting PD-L1, more preferably any one of breast cancer, lung cancer, colorectal cancer, esophageal cancer or pancreatic cancer; more preferably, breast cancer or esophageal cancer.

[0048] The present invention incubates the photosensitizer chimera with the tumor, and then irradiates the tumor area with light, causing the PD-L1 protein on the surface of the tumor cells to degrade, resulting in immune checkpoint blockade (ICB) therapy targeting the PD-1 / PD-L1 pathway, thereby regulating the tumor's immunosuppressive microenvironment and restoring the function of cytotoxic T lymphocytes (CTLs). Combined treatment is then achieved with photodynamic therapy (PDT), ultimately achieving a therapeutic effect on multiple tumors in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is an LC-MS mass spectrum of the polypeptide (PPA) targeting PD-L1 protein prepared in Example 1.

[0050] FIG2 is a graph showing the binding ability of the PD-L1 protein-targeting polypeptide (PPA) prepared in Example 1 and the hPD-L protein measured using plasmon resonance technology.

[0051] Figure 3 is the LC-MS spectrum of the photosensitizer chimera (PPA-VPF) isomers prepared in Example 1 (molecular formula: C 118 H 152 N 26 O 30 ).

[0052] FIG4 is a UV absorption spectrum of the photosensitizer chimera (PPA-VPF) and verteporfin (VPF) prepared in Example 1 (a: solvent is PBS; b: solvent is PBS / acetonitrile = 1:1).

[0053] FIG5 is the fluorescence spectrum of the photosensitizer chimera prepared in Example 1 (a: solvent is PBS; b: solvent is PBS / acetonitrile = 1:1).

[0054] FIG6 is an EPR spectrum of the photosensitizer chimera prepared in Example 1 after being irradiated with red light and captured by the capture agent Tempo (a: solvent is PBS; b: solvent is PBS / acetonitrile = 1:1).

[0055] FIG7 is a graph showing the binding ability of the photosensitizer chimera prepared in Example 1 and the protein hPD-L measured using plasmon resonance technology.

[0056] FIG8 is a graph showing the selective degradation of PD-L1 protein on the surface of living cells by the photosensitizer chimera prepared in Example 1 at different concentrations.

[0057] Figure 9 is the laser confocal imaging result of PD-L1 protein on the surface of living cells (blue represents the cell nucleus; green represents PD-L1 protein; red represents VPF or PPA-VPF).

[0058] Figure 10 shows the flow cytometry results of PD-L1 protein on the surface of living cells after treatment with PBS, PPA, VPF and PPA-VPF.

[0059] Figure 11 is a) photodegradation targeting chimera PPA-VPF by 660nm laser at 100mW / cm 2 b) Flow cytometry fluorescence intensity results of different cells after adding the photosensitizer chimera PPA-VPF or VPF prepared in Example 1 and incubating in the dark for 12 hours.

[0060] FIG12 is an in vivo imaging diagram of the photosensitizer VPF and the photosensitizer chimera PPA-VPF prepared in Example 1 at different time points.

[0061] FIG13 is an in vitro fluorescence imaging diagram of the main organs and tumor sites of the photosensitizer VPF (30 min) and the photosensitizer chimera PPA-VPF (24 h) prepared in Example 1.

[0062] Figure 14 shows (a) the growth curve of the tumor at the light-treated end in mice with bilateral 4T1 tumor transplantation; (b) the weight change; (c) (d) the tumor photos and tumor inhibition rate (TGI) at the light-treated end.

[0063] Figure 15 shows HE staining of tumor tissue sections of mice in each group;

[0064] Figure 16 shows (a) tumor growth curve of the untreated side of the mouse with bilateral 4T1 tumor transplantation; (b) (c) tumor photos and tumor inhibition rate (TGI) of the untreated side;

[0065] Figure 17 a) 4T1 tumor CD3 + CD8 + Representative flow cytometry images and ratio summary; b) 4T1 tumor CD8 + IFN-γ + Representative lymphocyte flow cytometry plots and ratio summary. DETAILED DESCRIPTION

[0066] Unless otherwise specified, the present invention has no special requirements on the sources of the raw materials used, and commercially available products known to those skilled in the art can be used.

[0067] The technical solutions of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention.

[0068] Preparation Example 1

[0069] (1) Synthesis and screening of targeting peptides

[0070] (a) Fmoc-based solid-phase peptide synthesis

[0071] A certain amount of resin loaded with Fmoc-NH2 (Fmoc loading: 0.37 mmol / g, total amount calculated as Fmoc is approximately 5 μmol) was weighed into a peptide synthesis tube. Peptide synthesis was performed using DMF as the solvent, and coupling was completed in the peptide synthesis tube. The Fmoc protecting group was removed using a 20% piperidine solution in DMF twice, each time for 5 minutes. HATU / HOBT (1:1) was used as a coupling reagent, and the carboxyl group was activated by DIEA to achieve amino acid coupling. Each coupling time was 20 minutes. Coupling after sterically hindered amino acids (eg, Pro, Val, Ile, Arg, Thr) was generally condensed twice to ensure connection efficiency. The peptide sequences are shown in Table 1 below.

[0072] Table 1 Targeted peptide PPA series screening sequences

[0073] For peptide synthesis α N-Fmoc or α N-Boc protected non-natural d-amino acids include: Fmoc-d-Asn(Trt)-OH, Fmoc-d-Tyr(OtBu)-OH, Fmoc-d-Ser(tBu)-OH, Fmoc-d-Lys(Boc)-OH, Fmoc-d-Pro-OH, Fmoc-d-Thr(tBu)-OH, Fmoc-d-Asp(OtBu)-OH, Fmoc-d-Arg(Pbf)-OH, Fmoc-d-Gln(Trt)-OH, Fmoc-d-His(Trt)-OH, and Fmoc-d-Phe-OH.

[0074] (b) Peptide purification and liquid chromatography conditions

[0075] After completing solid-phase peptide synthesis, the washed and solvent-free dried resin is placed in a peptide synthesis tube. A certain amount of TFA / TIPS / H2O (95:2.5:2.5, 8 ml / 0.05 mmol resin) mixture is added and shaken for 2-3 hours to achieve cleavage of the peptide from the resin and removal of the peptide side chain protecting groups. The reaction liquid is then collected and blown dry with a stream of nitrogen. The resulting solid is washed with cold ether. Finally, the solid is dissolved in a H2O / MeCN mixture and filtered through a filter membrane to obtain a clear solution for subsequent analysis and separation and purification.

[0076] The peptide products were analyzed and separated using high-performance liquid chromatography (HPLC) using mobile phases consisting of H₂O supplemented with 0.05% TFA (mobile phase A) and MeCN supplemented with 0.04% TFA (mobile phase B). Gradient elution was used for analysis and separation, with the gradient shown as percentage of MeCN. The LC-MS characterization data for PPA are shown in Figure 1.

[0077] (c) Affinity determination and screening

[0078] The affinity between hPD-L1 protein and PPA series polypeptides was measured by surface plasmon resonance (SPR, Biacore 8k, GE Healthcare). Commercially purchased hPD-L1 was diluted in 10mM sodium acetate buffer (pH 4.5, GE Healthcare) to obtain a final protein concentration of 40μg / mL. Using a standard amine coupling kit (GE Healthcare), the diluted hPD-L1 was covalently fixed to a new sensor chip (S series sensor chip CM5, GE Healthcare) via primary amine groups. The final fixation level of the target hPD-L1 was 9000RU by connection. PBS-P buffer (10mM phosphate buffer containing 2.7mM KCl, 137mM NaCl and 0.05% surfactant P20, final pH 7.4, GE Healthcare) was used for measurement at a flow rate of 30μL / min. In order to obtain data for kinetic and affinity analysis, the PPA series peptides were dissolved in PBS-P running buffer and a concentration gradient was set. More than 5 concentration gradients were used to ensure the final affinity K D The values ​​fall within the concentration gradient range. The results are shown in Table 2. By comparing K D The PPA peptide had the strongest affinity with the PD-L1 protein, so we finally used the PPA peptide as the mother peptide to carry out subsequent product preparation. The obtained PPA had more than 5 concentration gradients and ensured the final affinity K D The values ​​fall within the concentration gradient range, and the results are shown in Figure 2 and Table 2.

[0079] Table 2 Affinity of PPA series peptides to PD-L1 protein

[0080] Example 1

[0081] (1) Preparation of photosensitizer chimeras

[0082] (a) Coupling of crosslinker and verteporfin

[0083] The Fmoc protecting group was removed using a 20% piperidine solution in DMF (5 min × 2), followed by washing with DMF (× 3), DCM (× 3), and DMF (× 3). )x-CH2CH2-COOH (1.2 equiv.) (n=2, 5, 10), Fmoc-NH-(CH2)6-COOH, HATU (1.2 equiv.), and HOAt (1.2 equiv.) were dissolved in 3 mL of DMF. DIEA (2.4 equiv.) was added to activate the amino acids, and the resulting mixture was added to a peptide synthesis tube containing the synthetic resin used to prepare the PPA peptide in Preparation Example 1. The reaction was allowed to proceed on a shaker overnight until the coupling was complete. The Fmoc protecting group on the linker arm was subsequently removed using a 20% piperidine solution in DMF (5 min x 2), followed by washing with DMF (x 3), DCM (x 3), and DMF (x 3). Verteporfin (1.2 equiv.), HATU (1.2 equiv.), and HOAt (1.2 equiv.) were dissolved in 3 mL of DMF. DIEA (2.4 equiv.) was added to activate the amino acids. The mixed solution was added to the peptide synthesis tube prepared in Preparation 1 for PPA. The reaction was allowed to proceed on a shaker overnight until the coupling was complete. The whole process was protected from light.

[0084] (b) Post-production processing of chimeras

[0085] After completing solid-phase peptide synthesis, the washed and solvent-free dried resin is placed in a peptide synthesis tube. A certain amount of TFA / TIPS / H2O (95:2.5:2.5, 8 ml / 0.05 mmol resin) mixture is added and shaken for 2-3 hours to achieve cleavage of the peptide from the resin and removal of the peptide side chain protecting groups. The reaction liquid is then collected and blown dry with a stream of nitrogen. The resulting solid is washed with cold ether. Finally, the solid is dissolved in a H2O / MeCN mixture and filtered through a filter membrane to obtain a clear solution for subsequent analysis and separation and purification.

[0086] (c) Liquid chromatography analysis and separation conditions

[0087] High-performance liquid chromatography (HPLC) was used to analyze and separate the peptide products, yielding photosensitizer chimeras (PPA-VDF, PPA-VPF-1, PPA-VPF-2, and PPA-VPF-3). The mobile phases used were H₂O supplemented with 0.05% TFA (mobile phase A) and MeCN supplemented with 0.04% TFA (mobile phase B). Gradient elution was used for analysis and separation, with the gradient shown as the percentage of MeCN.

[0088] (d) Affinity determination and screening

[0089] The affinity between hPD-L1 protein and photosensitizer chimera was measured by surface plasmon resonance (SPR, Biacore 8k, GE Healthcare). Commercially purchased hPD-L1 was diluted in 10mM sodium acetate buffer (pH 4.5, GE Healthcare) to a final protein concentration of 40μg / mL. The diluted hPD-L1 was covalently fixed to a new sensor chip (S series sensor chip CM5, GE Healthcare) via primary amine groups using a standard amine coupling kit (GE Healthcare). The final fixation level of the target hPD-L1 was 9000RU by connection. PBS-P buffer (10mM phosphate buffer containing 2.7mM KCl, 137mM NaCl and 0.05% surfactant P20, final pH 7.4, GE Healthcare) was used for measurement at a flow rate of 30μL / min. In order to obtain the data for kinetic and affinity analysis, PPA-VDF, PPA-VPF-1, PPA-VPF-2, and PPA-VPF-3 were dissolved in PBS-P running buffer and a concentration gradient was set. More than 5 concentration gradients were used to ensure the final affinity K D The values ​​fall within the concentration gradient range, and the results are shown in Table 3.

[0090] Table 3 Affinity test results of PPA-VPF prepared with different linkers and hPD-L1 protein

[0091] Select the linker Fmoc-NH-PEG n -CH2CH2-COOH (1.2 equiv.) (n = 2) to prepare the obtained PPA-VPF for subsequent testing.

[0092] (2) Product characterization parameters:

[0093] LC-MS characterization data:

[0094] LC-MS characterization conditions: Agilent C18 analytical column, elution gradient: mobile phase B changed uniformly from 30% to 90% within 30 min.

[0095] PPA-VPF: retention time is 18.68min and 20.06min. ESI-MS: Calculate for C 118 H 152 N 26 O 30 :2414.67Da and 2414.67Da (average isotopes) (m / z), the highest peak of the actual molecular weight obtained is [M+3H]3+ : 805.71Da and 805.43Da. The results are shown in Figure 3.

[0096] (3) UV characterization spectrum

[0097] PPA-VPF was dissolved in either PBS or a 50% ACN (PBS:acetonitrile = 1:1) mixture (at a concentration of 20 μM), and its UV absorption spectra were measured in both solvents, using the photosensitizer verteporfin as a control. The results are shown in Figure 4. In the PBS solution, PPA-VPF's UV absorption was slightly lower than that of verteporfin. In the 50% ACN mixture, however, the UV absorption spectrum of PPA-VPF was essentially identical to that of verteporfin itself, likely due to differences in solubility in the different solvents.

[0098] (4) Fluorescence characterization spectrum

[0099] PPA-VPF was dissolved in PBS or a 50% ACN (PBS:acetonitrile = 1:1) mixture (at a concentration of 20 μM), and its fluorescence emission spectra were measured in both solvents, using the photosensitizer verteporfin as a control. The results are shown in Figure 5. The maximum emission wavelength of PPA-VPF is consistent with that of verteporfin itself. The fluorescence intensities exhibited in different solvents are consistent with the UV results described above. In PBS, PPA-VPF is slightly lower than VPF, while in 50% ACN, the intensities are comparable, indicating that the decrease in spectral intensity is indeed due to dissolution aggregation, but the fundamental spectral properties remain unchanged.

[0100] (5) EPR spectrum

[0101] ROS generated by PPA-VPF and verteporfin VPF upon photoexcitation were trapped by the spin-trapping agent 4-hydroxy-2,2,6,6-tetramethylpiperidine (4-OH-TEMP, 200 mM). Singlet oxygen was detected using an X-ray spectrometer on a Bruker A200. For singlet oxygen detection, 4-OH-TEMP was added to a 200 mM final concentration of 4-OH-TEMP to the PPA-VPF or verteporfin solution (20 μM) before irradiation. After irradiation for the specified time using a 300 W xenon arc lamp (CEAULIGHT) with a 660 nm bandpass filter (-1.5 mW / cm²), a 30 μL aliquot was quickly drawn into a glass capillary and transferred to the EPR resonator. The solvents used were PBS or 50% ACN (PBS:acetonitrile = 1:1). Typical settings for EPR detection were: scan range, 100 g; scan time, 60 s; microwave power, 19.23 mW; modulation amplitude, 1 g; and modulation frequency, 100 kHz. The results are shown in FIG6 .

[0102] (6) Affinity determination

[0103] The results of the PPA-VPF affinity test are shown in FIG7 .

[0104] (7) Protein degradation experiment

[0105] Protein targeted photodegradation was analyzed by western blot. For analysis in living cells, A549 cells were seeded onto 6-well plates. After 24 hours of incubation, cells were treated with the photodegradation-targeted chimera PPA-VPF, verteporfin itself, pure peptide, or DMSO as a control at 37°C for 8 hours and then irradiated with a 300W xenon arc lamp (600nm bandpass filter, 1.5mW / cm2). Cells were lysed in RIPA lysis buffer (P0013B, Beyotime Biotechnology) containing a protease-phosphatase inhibitor cocktail (#87786, Thermo Fisher Scientific) at 4°C for 30 minutes and collected by cell scraping. The harvested total protein was separated by SDS-PAGE in a 12% polyacrylamide gel and then transferred to a PVDF membrane (#1620177, Bio-Rad). The membrane was incubated with 5% skim milk for 1 hour and overnight at 4°C with the following primary antibodies: PD-L1 (ab125066, rabbit, 1:1000, Abcam), GPX1 (ab108427, rabbit, 1:1000, Abcam), ACSL4 (ab155282, rabbit, 1:1000, Abcam), and β-actin (ab8226, rabbit, 1:1000, Abcam). After incubation with corresponding HRP-conjugated secondary antibodies (#7076, mouse, 1:2000; #7074, rabbit, 1:2000, Cell Signaling) for 2 hours at room temperature, the blot was detected using an enhanced chemiluminescence kit (P0018FM, Beyotime Biotechnology). The results are shown in Figure 8. These results demonstrate that the photodegradation-targeting chimeras PV-1 and PV-2 can successfully achieve targeted and selective degradation of PD-L1 at the cellular level.

[0106] (8) PD-L1 protein degradation verification experiment at the cellular level

[0107] 1) Immunofluorescence imaging experiments at the cellular level

[0108] MDA-MB-231 cells were seeded in 24-well glass-bottom plates at a density of 5 × 10 cells per well. 4After attaching the cells to the surface of a circular coverslip, they were divided into four groups: PBS, pure peptide PPA, photodegradation-targeted chimera PPA-VPF, and photosensitizer verteporfin VPF. After incubation for 4 hours, the cells were irradiated with a 300W xenon arc lamp (CEAULIGHT) and a 660nm bandpass filter (-1.5mW / cm2) for 5min x 2. After incubation for another 1h, the culture medium was discarded, and the cells were fixed in 4% paraformaldehyde for 10 minutes at room temperature. The liquid in the wells was removed and the cells were washed three times with PBS buffer for 1 minute each. Goat serum (5%) was used as a blocking solution and incubated for 1 hour to reduce background fluorescence interference. The blocking solution was discarded and the above washing steps were repeated. The PD-L1 antibody conjugated with Alexa Fluor 488 was diluted 25-fold in 5% goat serum in PBS buffer, and the cells were incubated at room temperature for 1.5 hours. The primary antibody was discarded, the cells were washed three times with PBS buffer, and then the nuclei were stained with DAPI for 4 minutes at room temperature. Photographs were taken using a laser scanning confocal microscope, and the experimental results are shown in Figure 9. The results showed that the fluorescence intensity of the PD-L1 protein in the pure polypeptide PPA group was no different from that in the control group, and both were relatively intact. After laser irradiation, a small number of cells in the photosensitizer VPF group showed a certain degree of rounding, and the fluorescence intensity was slightly lower than that of the control group, indicating that the cell damage caused by VPF itself would partially and non-targetedly destroy the protein on the cell surface. In the photodegradation targeted chimera PPA-VPF group, the green fluorescence intensity on the cell surface was greatly reduced, indicating that the PD-L1 protein on the cell surface was degraded on a large scale, while the cell morphology was more complete and normal, indicating that PPA-VPF can selectively degrade the PD-L1 protein on the cell surface.

[0109] 2) Flow cytometry detection of cell surface PD-L1 protein degradation

[0110] MDA-MB-231 cells were seeded in 24-well plates at a density of 5×104 cells per well and cultured overnight. The groups were set up as above (i.e., PBS group, PPA group, VPF group, and PPA-VPF group). After 4 hours of drug treatment, the cells were illuminated (689nm, 5min×2). After that, the cells were cultured for another hour in the dark. The culture medium was discarded, and the anti-PD-L1 protein flow cytometry antibody PEanti-mouseCD274 (biolegend, #124307) was added to treat the cells for 30 minutes. The cells were collected and analyzed by flow cytometry. The results are shown in Figure 10, where the negative control group (i.e., Blank group) represents the MDA-MB-231 cells expressing PD-L1 protein without the addition of flow cytometry antibodies in order to exclude the fluorescence of the cells themselves. The PBS group represents cells with antibodies but no drug treatment. The fluorescence intensity values ​​of cells treated with PPA or VPF were almost the same as those of the PBS group, indicating that the treatment of these two drugs did not affect the PD-L1 protein content on the cell surface. But surprisingly, the fluorescence intensity of cells treated with PPA-VPF decreased significantly and showed a certain concentration dependence. This result was consistent with the laser confocal microscopy experiment, indicating that the designed PPA-VPF can target and selectively degrade the PD-L1 protein on the surface of cancer cells.

[0111] (9) Study on the anti-tumor activity of the photodegradation-targeted chimera PPA-VPF

[0112] 1) The CCK-8 assay was used for detection. Human triple-negative breast cancer cells (i.e., MDA-MB-231 cells, high expression of PD-L1 protein), human breast cancer cells (MCF-7 cells, low expression of PD-L1 protein), and normal human embryonic kidney cells 293T were used as model cells and seeded in 96-well plates. The cells were allowed to adhere to the wall in an incubator, and the photodegradation-targeted chimera PPA-VPF was added. After incubation for 4 hours, the cells were irradiated with light (the specific conditions were the same as above). After incubation for 24 hours, the original culture medium was discarded, the cells were washed three times with PBS, and 10% CCK-8 solution (CCK-8 was dissolved in the culture medium) was added and incubated in an incubator for 2 hours. The absorbance (480 nm) in the well plate was measured using a Biotek microplate reader and the cell viability was calculated. The experimental results are shown in Figure 11a. The results showed that PPA-VPF had the strongest cytotoxicity in MDA-MB-231 cells that highly expressed PD-L1, while it had lower cytotoxicity in MCF-7 cells that lowly expressed PD-L1, and its cytotoxicity in normal cells was negligible, indicating that cytotoxicity was related to the content of the cell receptor protein PD-L1.

[0113] 2) Detection of cellular uptake of PPA-VPF by flow cytometry

[0114] 1) MDA-MB-231 (high expression of PD-L1 protein) and MCF-7 (low expression of PD-L1 protein) cells were cultured at 5×10 3 The cells were seeded in a 24-well plate at a density of 10 cells and cultured overnight. PPA-VPF or VPF was then added and incubated for 8 hours. The cells were collected and analyzed by flow cytometry. The experimental results are shown in Figure 11b. In the flow cytometry results, the amount of PPA-VPF taken up by MDA-MB-231 cells was significantly higher than that of MCF-7 cells, and showed a certain concentration dependence, while the uptake of VPF by the two cells was not significantly different. Combined with the results of Figure 11a, it can be seen that cells without PD-L1 receptors will not bind to PPA-VPF, and thus will not produce the toxicity of the photosensitizer, while PPA-VPF will bind to cells with high expression of PD-L1 receptors. After illumination, while degrading the PD-L1 protein on the cell surface, toxic ROS are produced, eventually leading to cell death.

[0115] (10) Evaluation of the anti-tumor effect of the photodegradation-targeted chimera PPA-VPF in vivo

[0116] Based on a series of in vitro evaluations, we further investigated the targeting of tumor treatment after intravenous injection of PPA-VPF in BALB / c mice bearing 4T1 cells, as well as the application of combined photodynamic therapy and immunotherapy, in order to investigate the efficacy and safety of this combined therapy for in situ breast cancer in mice.

[0117] 1) Establishment of 4T1 tumor-bearing mouse model

[0118] BALB / c mice were purchased and allowed to acclimate to the environment for one week before preparing a breast cancer 4T1 cell suspension for subcutaneous inoculation on the back of the mice. Each mouse was inoculated with 5×10 5 cells.

[0119] 2) PPA-VPF in vivo distribution experiment

[0120] Inoculate 4T1 cells and observe the tumor growth of mice every day until the tumor volume grows to 200mm 3The mice were randomly divided into two groups with three mice in each group and injected with 100 μL of photosensitizer VPF (with cosolvent PEG) or photodegradation targeting chimera PPA-VPF through the tail vein respectively. The mice were kept in the dark. The VPF group was anesthetized at 5, 10, 15, 20, 30, 60 and 120 minutes after administration, while the PPA-VPF group was anesthetized at 2, 4, 8, 12, 24, 36 and 48 hours. The mice were then photographed using a small animal in vivo imaging device (IVIS SPECTRUM). The results are shown in Figure 12. The results show that the photosensitizer VPF is metabolized very rapidly in the body. It is quickly distributed throughout the body within 10 minutes of administration. The enrichment in the tumor site basically reaches its peak in about half an hour, but the enrichment time is short. It has basically left the tumor site after 2 hours. PPA-VPF is still strongly enriched in the tumor site 12 hours after injection. After 24 hours, it gradually accumulates in the tumor site, and the content in other sites gradually decreases. This suggests that the photodegradation-targeting chimera PPA-VPF can more stably accumulate in tumors via the EPR effect and maintain long-term circulation compared to the photosensitizer VPF itself. Based on these results, considering time points with higher fluorescence intensity in the tumor and lower fluorescence intensity in other areas, we selected VPF 30 minutes and PPA-VPF 24 hours. Mice were sacrificed and dissected, and ex vivo tissues and tumors were removed to further investigate the drug's in vitro tissue distribution. The results, as shown in Figure 13, show that at the selected time points, PPA-VPF was primarily distributed in the tumor, with less accumulation in the heart, liver, spleen, lungs, and kidneys. In addition to tumor accumulation, the VPF group also showed significant accumulation in the liver, indicating that PPA-VPF was effectively enriched in the tumor at the selected time points, with less accumulation in other tissues and organs. Based on these experimental results, we determined the optimal photodynamic therapy time to avoid toxic side effects in other organs. We decided to initiate the corresponding light-irradiated mouse treatment 30 minutes after intravenous injection of VPF and 24 hours after intravenous injection of PPA-VPF.

[0121] (11) Anti-tumor activity study in mice

[0122] The bilateral tumor model was selected to conduct in vivo anti-tumor studies. 4T1 cells (5×10 5 ) were injected subcutaneously into the right side of BALB / c mice to generate primary tumors for subsequent light treatment; 1×10 5 The 4T1 cell suspension was then subcutaneously injected into the left side of the mouse to form distal tumors. When the primary tumor volume reached approximately 100 mm 3Mice were randomly divided into four groups, each with four mice, and intravenously injected with PBS, pure peptide PPA, photosensitizer VPF (with cosolvent PEG), and PPA-VPF. The dose of PPA-VPF was 2 mg / kg, and the other groups were converted according to the amount of the same substance as the experimental group. The primary tumors were irradiated with 660 nm laser (500 mW / cm2, 8 minutes) 30 minutes after injection in the VPF group and 24 hours after injection in the PPA-VPF group. The treatment frequency was once every two days. The weight changes of the mice and the volume growth of the bilateral tumors were recorded daily. The tumor volume was calculated as: V = d × l2 / 2 (where: V represents the volume of the tumor, d represents the long diameter of the tumor, and l represents the short diameter of the tumor). Mice were euthanized on the 11th day for subsequent pathological and immunohistochemical evaluation. The results, shown in Figures 14a-d, show that compared to the control group, mice treated with PPA-VPF exhibited significant tumor growth inhibition after laser irradiation (P < 0.001). Mice in the photosensitizer VPF group also showed some tumor growth inhibition, but this was less pronounced than in the PPA-VPF group. Mice treated with pure PPA showed no inhibition. This suggests that PPA-VPF is more effective in suppressing tumors than photosensitizer alone, demonstrating that immunotherapy combined with PDT can effectively inhibit the growth of orthotopic breast tumors in mice. The weight changes of the mice were stable, with no statistically significant difference (P > 0.05), demonstrating that this treatment had no significant effect on the survival of the mice.

[0123] (12) HE staining of tumor tissue sections

[0124] The tumor tissues of mice were pathologically examined using HE section staining. The ex vivo tumor tissues were fixed with 4% paraformaldehyde solution, and after 24 hours, gradient alcohol was used to remove moisture from the tissue blocks. Finally, the tissue blocks were placed in xylene for transparency. The treated tissue blocks were paraffin-infiltrated and embedded, and paraffin sections were made using a paraffin sectioning machine. After dewaxing with xylene, the tissue sections were stained with hematoxylin and eosin dyes, and photographed using a fully automatic digital section scanning system. The results are shown in Figure 15. After light treatment, the tumor sections in the PBS group and the pure polypeptide PPA group did not show obvious histological changes, while the tumor tissue in the VPF group was partially destroyed. The nuclei of the tumor tissues treated with PPA-VPF shrank, the cells were severely damaged, and the number was significantly reduced.

[0125] (13) Effects of immunotherapy on tumor growth in mice

[0126] In order to further verify the immunotherapeutic effect of degrading PD-L1 protein and combining it with photodynamic therapy on distal tumors, the volume of the distal tumor was monitored (Figure 16a). Compared with the PBS group, the distal tumors in the PPA-VPF treatment group proliferated the slowest. After the experiment, the distal tumors were dissected and weighed (Figures 16b, c). Similar to the results of the primary tumor, the PPA-VPF treatment group showed the best tumor inhibition effect. The volume of the distant tumor in this group was the smallest and the tumor inhibition rate was the highest. The above results all confirm that the designed photodegradation targeting chimera PPA-VPF can stimulate a better anti-tumor immune response in synergistic photodynamic and PD-1 / PD-L1 immunotherapy.

[0127] (14) Flow cytometry experiments for tumor immunotherapy

[0128] 1) Detection of tumor-infiltrating T lymphocytes

[0129] 4T1 cells (5×10 5 ) was injected subcutaneously into the left flank of BALB / c mice. When the tumor volume reached 300 mm 3 After about 24 hours, the mice were divided into PBS group, PPA group, VPF group and PPA-VPF group, and the tail vein injection was carried out as above. The mice in VPF group and PPA-VPF group were injected with 500 mW / cm 2 The mice were irradiated with a 660 nm laser of 800 nm intensity for 8 minutes. The mice were kept in the dark. On the third day after administration, the mice were euthanized, and their spleens and tumor tissues were dissected and digested with collagenase (400 U / mL), DNase1 (100 μg / mL), and hyaluronidase (0.04 U / mL). The mixture was then passed through a 75 μm nylon cell strainer. The samples were washed three times with PBS (containing 2% FBS) and then filtered with 1×10 6 The cells were resuspended at a density of 10 cells / mL for flow cytometry analysis. Antibodies (anti-mouse CD3-FITC, anti-mouse CD4-APC, anti-mouse CD8-PE-Cy7 and anti-mouse IFN-γ-PE, BioLegend) were added according to the instructions, and the membrane needed to be broken before adding the anti-mouse IFN-γ-PE antibody. The cells were analyzed on a flow cytometer, and the data were analyzed using FlowJo 10. The results are shown in Figure 17a. Compared with the PBS group, the proportion of CTL cells in the pure polypeptide PPA group and the photosensitizer VPF treatment group were 19.6±1.8% and 37.5±2.7%, respectively. The PPA-VPF group showed the highest CTL ratio in tumor tissue, which was 54.9±1.45%, which was 6.17 times higher than that of the PBS group. The expression of IFN-γ that was further evaluated (as shown in Figure 17b) was also similar. The treatment of the polypeptide PPA group alone or the photodynamic therapy group alone (ie, the VPF group) was able to partially increase the CD8 + / IFN-γ + The proportion of T cells in the synergistic treatment group was 24.8±2.1% and 32.6±2.7%, respectively. However, the PPA-VPF group induced the highest CD8 + / IFN-γ + The cell proportion was 54.7±6.0%. This indicates that the combination therapy of photoactivation induced by photodegradation of PD-L1 protein in PD-1 / PD-L1 and immune checkpoint blockade can produce a stronger immune response than single therapy and has great potential in enhancing anti-tumor therapy.

Claims

1. A photosensitizer chimera targeting PD-L1 protein, wherein the chimera is an XRG structure, wherein X is a photosensitizer unit, R is a connecting arm, and G is a polypeptide unit targeting PD-L1 protein, characterized in that: The photosensitizer is a porphyrin photosensitizer, the excitation light wavelength of the photosensitizer is greater than 550nm; the molar absorption coefficient at the maximum absorption wavelength after 550nm is greater than 1000M -1 cm -1 ;The quantum yield of triplet state>0.01; The connecting arm is selected from the following structural formula: wherein the amino side of the connecting arm is connected to the photosensitizer unit, the carboxyl side of the connecting arm is connected to the polypeptide unit, and n is an integer of 0-50; The binding constant of the peptide to h-PD-L1 is <20 μM.

2. The photosensitizer chimera according to claim 1, wherein the structural formula of the connecting arm is: n is an integer of 0-50; preferably n is an integer of 0-10.

3. The photosensitizer chimera according to claim 1, wherein the photosensitizer is at least one selected from verteporfin, dihydrochlorin or pheophorbide a; preferably verteporfin.

4. The photosensitizer chimera according to claim 1, wherein the polypeptide is the polypeptide described in any one of (a) to (c): (a) a polypeptide having the amino acid sequence (1) to (11); (b) a polypeptide having an amino acid sequence (1)-(11) with a substitution, deletion or addition of 1-3 amino acid residues and an affinity for PD-L1 of less than 20 μM; (c) a polypeptide having an ester group or an amide group linked to the carboxyl terminus of the polypeptide of (a) or (b); The amino acid sequences (1)-(11) are as follows: dNdYdSdKdPdTdDdRdQdYdHdF(1); dNdYdSdKdPdTdDdR(2); dNdYdSdKdPdTdDdRdQ(3); dNdYdSdKdPdTdDdRdQdY(4); dKdPdTdDdRdQdYdHdF(5); dPdTdDdRdQdYdHdF(6); dNdYdSdKdPdTdDdRdQdYdH(7); dSdKdPdTdDdRdQdYdHdF(8); dYdSdKdPdTdDdRdQdYdHdF(9); dKdPdTdDdR(10); dSdKdPdTdDdR(11); Preferably, dNdYdSdKdPdTdDdRdQdYdHdF (1); dNdYdSdKdPdTdDdR(2); dNdYdSdKdPdTdDdRdQ(3); dNdYdSdKdPdTdDdRdQdY(4); dKdPdTdDdRdQdYdHdF(5); More preferably, it is dNdYdSdKdPdTdDdRdQdYdHdF(1).

5. The photosensitizer chimera according to any one of claims 1 to 4, characterized in that: The chemical structural formula of the photosensitizer chimera is shown in Formula I or Formula II; In Formula I and Formula II, n is independently an integer of 1-10; G is a polypeptide unit targeting PD-L1 protein, and the amino acid sequence of the polypeptide is: dNdYdSdKdPdTdDdRdQdYdHdF(1).

6. The method for preparing the photosensitizer chimera according to any one of claims 1 to 5, comprising the following steps: 1) first mixing the linker and the polypeptide targeting the PD-L1 protein, performing a first coupling, removing uncoupled substances, and obtaining a first conjugate; 2) The first conjugate and the photosensitizer are mixed for a second time, and a second coupling is performed under light-proof conditions to remove unconjugated substances to obtain a photosensitizer chimera.

7. The preparation method according to claim 6, characterized in that: In step 1), the ratio of the linker to the polypeptide is 1-5:

1.

8. The method for preparing a derivatized photosensitizer chimera according to claim 6, wherein in step 2), the dosage ratio of the first conjugate to the photosensitizer is 1:1-4.

9. An anti-tumor drug, the active ingredient of which includes the photosensitizer chimera according to any one of claims 1 to 5; preferably, the tumor is a tumor targeting PD-L1; further preferably, it is any one of breast cancer, lung cancer, colorectal cancer, esophageal cancer or pancreatic cancer; further preferably, it is breast cancer or esophageal cancer.

10. Use of the photosensitizer chimera according to any one of claims 1 to 5 in the preparation of anti-tumor drugs; preferably, the tumor is a tumor targeting PD-L1; further preferably, it is any one of breast cancer, lung cancer, colorectal cancer, esophageal cancer or pancreatic cancer; further preferably, it is breast cancer or esophageal cancer.

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