Photosensitizer chimera targeting PD-L1 protein as well as preparation method and application of photosensitizer chimera

Abstract

The invention relates to a PD-L1 protein targeted photosensitizer chimera as well as a preparation method and application thereof. The photosensitizer chimera is of an X-R-G structure, X is a photosensitizer unit, R is a connecting arm, G is a polypeptide unit targeting PD-L1 protein, a photosensitizer is a porphyrin photosensitizer, and the excitation light wavelength of the photosensitizer is gt; the wavelength is 550nm; the molar absorption coefficient gt at the maximum absorption wavelength after the wavelength of 550 nm; 1000 M <-1 > cm <-1 >; the quantum yield of the triplet state is gt; 0.01, 0.01; the connecting arm is selected from the following structural formula: # imgabs0 #, the amino side of the connecting arm is connected with the photosensitizer unit, the carboxyl side of the connecting arm is connected with the polypeptide unit, and n is an integer of 0-50; the binding constant of the polypeptide and h-PD-L1 is lt; and 20 [mu] M. According to the photosensitive chimera disclosed by the invention, combined treatment based on immune checkpoint blocking and cytotoxic effects induced by photodegradation of PD-L1 protein in PD-1 / PD-L1 can generate immune response stronger than single treatment, and the photosensitive chimera has great potential in the aspect of enhancing anti-tumor treatment.

Description

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 method after surgery, chemotherapy, radiotherapy and targeted drug therapy, and has been applied in clinical practice. In recent years, studies have found that the programmed death receptor-1 (PD-1) / programmed death ligand-1 (PD-L1) signaling pathway is one of the important pathways involved in tumor immune escape. This signaling pathway can induce tumor-specific T cell apoptosis by inhibiting T cell activation and lead to T cell resistance, playing a role in immune escape and immunosuppression in pathological conditions such as tumors and chronic inflammation. Currently, a total of 19 PD-1 / PD-L1 drugs have been approved for marketing worldwide, of which 13 have been approved for marketing in China, 9 PD-1 inhibitors (7 domestic and 2 imported), and 4 PD-L1 inhibitors (2 domestic and 2 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 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 permeability, higher oral bioavailability, and more tolerable half-life. In addition, combined therapy is also the main trend in tumor immunotherapy. The cytotoxic effect produced by photodynamic therapy of tumors can effectively activate the body's immune response, thereby better reducing the drug resistance problem of PD-1 / PD-L1. Therefore, the use of light-timed localized 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. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a photosensitizer chimera targeting PD-L1 protein, wherein the chimera is 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 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;

[0004] The connecting arm is selected from the following structural formula:

[0005]

[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) to (11);

[0012] (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;

[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] The 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]

[0034]

[0035] 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 PD-L1 protein, and the amino acid sequence of the polypeptide is: dNdYdSdKdPdTdDdRdQdYdHdF (1). The N-terminus of the polypeptide targeting PD-L1 protein is bonded to the connecting arm via an amide bond.

[0036] The present invention also provides a method for preparing the photosensitizer chimera described in the above scheme, comprising the following steps:

[0037] 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;

[0038] 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.

[0039] The present invention has no special restrictions 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.

[0040] After coupling the polypeptide targeting PD-L1 protein on 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.

[0041] The present invention has no particular limitation on the conditions of the first coupling. Preferably, the conditions of the first coupling include: a coupling temperature of 5-30° C.; and a coupling time of 8-14 h.

[0042] 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 the photosensitizer chimera.

[0043] 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 -CH 2 CH 2 -COOH, Fmoc-NH-(CH 2 ) y -COOH. wherein x and y are even numbers of 2-10.

[0044] In the present invention, the first conjugate is coupled to the resin and then mixed with a solution containing a photosensitizer for a second time; the solution containing the photosensitizer uses DMF as a solvent and contains 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. Before the second mixing, DIEA is added to the solution containing the photosensitizer for the linker arm to activate the amino acid.

[0045] Preferably, in step 1), the ratio of the linker arm to the polypeptide targeting PD-L1 protein is 1-5:1.

[0046] In a preferred embodiment, in step 2), the usage ratio of the first coupling agent to the photosensitizer is 1:1-4.

[0047] 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.

[0048] 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, and more preferably, it is any one of breast cancer, lung cancer, colorectal cancer, esophageal cancer or pancreatic cancer; and even more preferably, it is breast cancer or esophageal cancer.

[0049] The present invention provides the use of the photosensitizer chimera in the preparation of anti-tumor drugs; preferably, the tumor is a tumor targeting PD-L1, and more preferably, it is any one of breast cancer, lung cancer, colorectal cancer, esophageal cancer or pancreatic cancer; more preferably, it is breast cancer or esophageal cancer.

[0050] The present invention incubates the photosensitizer chimera with the tumor, and then irradiates the tumor area, degrading the PD-L1 protein on the surface of the tumor cells, producing 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), and then combining with photodynamic therapy (PDT) to achieve combined treatment, ultimately achieving a therapeutic effect on a variety of tumors in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Attached Figure 1 This is the LC-MS mass spectrum of the polypeptide (PPA) targeting PD-L1 protein prepared in Example 1.

[0052] Attached Figure 2 This is a graph showing the binding ability of the PD-L1 protein-targeting polypeptide (PPA) prepared in Example 1 and the protein hPD-L using plasma resonance technology.

[0053] Attached Figure 3 LC-MS spectrum of the photosensitizer chimera (PPA-VPF) isomer prepared in Example 1 (molecular formula: C 118 H 152 N 26 O 30 ).

[0054] Attached Figure 4 The UV absorption spectra of the photosensitizer chimera (PPA-VPF) and verteporfin (VPF) prepared in Example 1 (a: the solvent is PBS; b: the solvent is PBS / acetonitrile = 1:1).

[0055] Attached Figure 5 The fluorescence spectrum of the photosensitizer chimera prepared in Example 1 (a: the solvent is PBS; b: the solvent is PBS / acetonitrile = 1:1).

[0056] Attached Figure 6 This is the EPR spectrum of the photosensitizer chimera prepared in Example 1 after being captured by the capture agent Tempo after red light irradiation (a: the solvent is PBS; b: the solvent is PBS / acetonitrile = 1:1).

[0057] Attached Figure 7 This is a graph showing the binding ability of the photosensitizer chimera prepared in Example 1 and the protein hPD-L determined using plasma resonance technology.

[0058] Attached Figure 8 The diagram shows the selective degradation of PD-L1 protein on the surface of living cells by the photosensitizer chimera prepared in Example 1 at different concentrations.

[0059] Attached Fig. 9 This is the result of laser confocal imaging 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).

[0060] Attached Fig.10 The flow cytometry results of PD-L1 protein on the surface of living cells after treatment with PBS, PPA, VPF and PPA-VPF.

[0061] Attached Fig.11 a) The photodegradation targeting chimera PPA-VPF was irradiated by 660 nm laser at 100 mW / cm 2 b) cytotoxicity after light irradiation (5min×2); b) flow cytometry fluorescence intensity results after adding the photosensitizer chimera PPA-VPF or VPF prepared in Example 1 to different cells and incubating in the dark for 12h.

[0062] Attached Fig.12The in vivo imaging images of the photosensitizer VPF and the photosensitizer chimera PPA-VPF prepared in Example 1 at different time points.

[0063] Attached Fig.13 These are in vitro fluorescence imaging images 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.

[0064] Attached Fig.14 (a) Growth curve of the tumor at the light-treated end in mice with bilaterally transplanted 4T1 tumors; (b) Body weight changes; (c) (d) Tumor photos and tumor inhibition rate (TGI) at the light-treated end.

[0065] Attached Fig.15 HE staining of tumor tissue sections of mice in each group;

[0066] Attached Fig.16 (a) Growth curve of the tumor at the non-light-treated end in mice with bilateral 4T1 tumor transplantation; (b) (c) Tumor photos and tumor inhibition rate (TGI) at the non-light-treated end;

[0067] Attached Fig.17 a) 4T1 tumor CD3 + CD8 + Representative flow cytometry images and ratio summary; b) 4T1 tumor CD8 + IFN-γ + Representative graphs and ratio summary of lymphocyte flow cytometry. DETAILED DESCRIPTION

[0068] 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 may be used.

[0069] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0070] Preparation Example 1

[0071] (1) Synthesis and screening of targeting peptides

[0072] (a) Fmoc-based solid-phase peptide synthesis

[0073] Weigh a certain amount of Fmoc-NH 2The resin (Fmoc loading: 0.37mmol / g, the total amount calculated by Fmoc is about 5μmol), the synthesis of peptides uses DMF as solvent, and the coupling is completed in the peptide synthesis tube. The removal of the Fmoc protecting group is treated twice with a 20% piperidine DMF solution, each time for 5 minutes; HATU / HOBT (1:1) is used as a coupling reagent, and the amino acid coupling is achieved by activating the carboxyl group with DIEA, and each coupling time is 20 minutes. The coupling after the large sterically hindered amino acids (egPro, Val, Ile, Arg, Thr) is generally condensed twice to ensure the connection efficiency. The peptide sequence is shown in Table 1 below.

[0074] Table 1 Targeted peptide PPA series screening sequences

[0075]

[0076]

[0077] 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.

[0078] (b) Peptide purification and liquid chromatography conditions

[0079] After completing the solid phase synthesis of the peptide, the washed and solvent-free dry resin is placed in a peptide synthesis tube, and a certain amount of TFA / TIPS / H 2 O (95:2.5:2.5, -8ml / 0.05mmol resin) mixed solution and treated on a shaker for 2-3h to achieve the cleavage of the peptide from the resin and the removal of the peptide side chain protecting group. Then the reaction liquid was collected and dried with a nitrogen stream, and the obtained solid was washed with cold ether. Finally, H 2 The solid was dissolved in the O / MeCN mixed solution and filtered through a filter membrane to obtain a clear solution for subsequent analysis and separation and purification.

[0080] The peptide products were analyzed and separated using high performance liquid chromatography (HPLC) with 0.05% TFA added to the mobile phase. 2O (mobile phase A) and MeCN with 0.04% TFA (mobile phase B). Gradient elution was used for analysis and separation, and the gradient shown is the percentage content of MeCN. The LC-MS characterization data of PPA are shown in the attached Figure 1 shown.

[0081] (c) Affinity determination and screening

[0082] 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 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) through a primary amine group. 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 that 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 value falls within the concentration gradient range, and the results are shown in the attached Figure 2 And as shown in Table 2.

[0083] Table 2 Affinity of PPA series peptides and PD-L1 protein

[0084]

[0085] Example 1

[0086] (1) Preparation of photosensitizer chimeras

[0087] (a) Coupling of crosslinker and verteporfin

[0088] 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 -CH 2 CH 2 -COOH (1.2 equiv.) (n = 2, 5, 10) and Fmoc-NH-(CH 2 ) 6 -COOH, HATU (1.2 equiv.) and HOAt (1.2 equiv.) were dissolved in 3 mL DMF, and DIEA (2.4 equiv.) was added to activate the amino acid, and the mixed solution was added to the polypeptide synthesis tube containing the synthetic resin for preparing the PPA polypeptide in Preparation Example 1, and the reaction was carried out on a shaker overnight until the coupling was completed. The Fmoc protecting group on the linker was removed (5 min × 2) using a 20% piperidine DMF solution, and then washed with DMF (× 3), DCM (× 3), and DMF (× 3). Verteporfin (1.2 equiv.), HATU (1.2 equiv.) and HOAt (1.2 equiv.) were dissolved in 3 mL DMF, and DIEA (2.4 equiv.) was added to activate the amino acid, and the mixed solution was added to the polypeptide synthesis tube for preparing PPA in Preparation Example 1, and the reaction was carried out on a shaker overnight until the coupling was completed. The whole process was protected from light.

[0089] (b) Treatment after chimera preparation

[0090] After completing the solid phase synthesis of the peptide, the washed and solvent-free dry resin is placed in a peptide synthesis tube, and a certain amount of TFA / TIPS / H 2 O (95:2.5:2.5, -8ml / 0.05mmol resin) mixed solution and treated on a shaker for 2-3h to achieve the cleavage of the peptide from the resin and the removal of the peptide side chain protecting group. Then the reaction liquid was collected and dried with a nitrogen stream, and the obtained solid was washed with cold ether. Finally, H 2 The solid was dissolved in the O / MeCN mixed solution and filtered through a filter membrane to obtain a clear solution for subsequent analysis and separation and purification.

[0091] (c) Liquid chromatography analysis and separation conditions

[0092] The peptide products were analyzed and separated by high performance liquid chromatography to obtain photosensitizer chimeras (PPA-VDF, PPA-VPF-1, PPA-VPF-2, PPA-VPF-3). The mobile phase used was H2O with 0.05% TFA added. 2O (mobile phase A) and MeCN supplemented with 0.04% TFA (mobile phase B). Gradient elution was used for analysis and separation, and the gradient shown is the percentage of MeCN.

[0093] (d) Affinity determination and screening

[0094] The affinity between hPD-L1 protein and photosensitizer chimera was measured by surface plasmon resonance (SPR, Biacore8k, GE Healthcare). Commercially purchased hPD-L1 was diluted in 10mM sodium acetate buffer (pH 4.5, GE Healthcare) to obtain a protein concentration of 40 μg / 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) through a primary amine group. 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. To obtain 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 set up a concentration gradient. More than 5 concentration gradients were used to ensure that the final affinity K D The values ​​fall within the concentration gradient range, and the results are shown in Table 3.

[0095] Table 3 Affinity test results of PPA-VPF prepared with different linker arms and hPD-L1 protein

[0096]

[0097] Select the linker Fmoc-NH-PEG n -CH 2 CH 2 -COOH (1.2 equiv.) (n = 2) to prepare the PPA-VPF for subsequent testing.

[0098] (2) Product characterization parameters:

[0099] LC-MS characterization data:

[0100] The LC-MS characterization conditions were: Agilent C18 analytical column, and the elution gradient was that the mobile phase B changed evenly from 30% to 90% within 30 minutes.

[0101] 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 the attached Figure 3 shown.

[0102] (3) UV characterization spectrum

[0103] PPA-VPF was dissolved in a mixed solvent of PBS or 50% ACN (PBS: acetonitrile = 1:1) (concentration of 20 μM), and its UV absorption spectra in the two solvents were measured respectively, with the photosensitizer verteporfin as a control. The results are shown in the attached figure. Figure 4 As shown in the figure, in PBS solution, the UV absorption of PPA-VPF is slightly lower than that of verteporfin. In 50% ACN mixed solvent, the UV absorption spectrum of PPA-VPF is basically the same as that of verteporfin itself, which may be due to the difference in solubility in different solvents.

[0104] (4) Fluorescence characterization spectrum

[0105] PPA-VPF was dissolved in PBS or 50% ACN (PBS: acetonitrile = 1:1) mixed solvent (concentration of 20 μM), and its fluorescence emission spectra in the two solvents were measured respectively, with photosensitizer verteporfin as a control. The results are shown in the attached figure. Figure 5 As shown. The maximum emission wavelength of PPA-VPF is consistent with that of verteporfin itself. The fluorescence intensity displayed in different solvents is consistent with the above UV results, that is, PPA-VPF in PBS is slightly lower than VPF, while in 50% ACN solution, the intensity of the two is comparable, indicating that the change in spectral intensity is indeed reduced due to the dissolution and agglomeration problem, but the basic spectral properties do not change.

[0106] (5) EPR spectrum

[0107] The ROS generated by PPA-VPF and Verteporfin VPF after photoexcitation were captured by the spin trap 4-hydroxy-2,2,6,6-tetramethylpiperidine (4-OH-TEMP, 200mM), and the type of ROS generated was singlet oxygen detected by X-band Bruker A200 spectrometer. When detecting singlet oxygen, 4-OH-TEMP with a final concentration of 200mM was added to the PPA-VPF or Verteporfin solution (20μM) before irradiation. After irradiation for a specified time with a 660nm bandpass filter (-1.5mW / cm2) of a 300W xenon arc lamp (CEAULIGHT), a 30μL aliquot was quickly aspirated into a glass capillary and into the EPR resonator. The solvent used was PBS or 50% ACN (PBS: acetonitrile = 1:1). The typical settings for EPR detection are: scanning range, 100g; scanning time, 60s; microwave power, 19.23mw; modulation amplitude, 1g; modulation frequency, 100kHz. The results are shown in the attached Figure 6 .

[0108] (6) Affinity determination

[0109] PPA-VPF affinity test results are as follows Figure 7 shown.

[0110] (7) Protein degradation experiment

[0111] 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 photodegradation targeting 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 at 4°C for 30 minutes in RIPA lysis buffer (P0013B, Beyotime Biotechnology) containing a protease-phosphatase inhibitor cocktail (#87786, ThermoFisher Scientific) 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 the 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 with an enhanced chemiluminescence kit (P0018FM, Beyotime Biotechnology). The experimental results are shown in Figure 2. Figure 8 The experimental results show that the photodegradation targeting chimeras PV-1 and PV-2 can successfully achieve targeted selective degradation of PD-L1 at the cellular level.

[0112] (8) PD-L1 protein degradation verification experiment at the cell level

[0113] 1) Immunofluorescence imaging experiments at the cellular level

[0114] MDA-MB-231 cells were seeded in 24-well glass-bottom plates at a density of 5 × 10 cells per well. 4cells. After the cells were attached to the surface of the circular coverslip, they were divided into four groups, namely PBS, pure peptide PPA, photodegradation targeting chimera PPA-VPF and photosensitizer verteporfin VPF. After incubation for 4 hours, they were irradiated with a 300W xenon arc lamp (CEAULIGHT) and a 660nm bandpass filter (-1.5mW / cm2) for 5min×2. Continue incubation for 1h, discard the culture medium, and fix the cells in 4% paraformaldehyde at room temperature for 10 minutes. Remove the liquid in the well and wash the cells three times with PBS buffer for 1 minute each. Use goat serum (5%) as a blocking solution and incubate for 1 hour to reduce background fluorescence interference. Discard the blocking solution and repeat the above washing steps. The PD-L1 antibody linked to Alexa Fluor 488 was diluted 25 times in 5% goat serum in PBS buffer, and the cells were incubated at room temperature for 1.5 hours. Discard the primary antibody, wash the cells three times with PBS buffer, and then use DAPI to stain the nucleus for 4 minutes at room temperature. Take pictures using a laser scanning confocal microscope, and the experimental results are shown in the attached Fig. 9 As shown. 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 complete. After laser irradiation, a few cells in the photosensitizer VPF group showed a certain degree of rounding, and the fluorescence intensity was slightly lower than that in the control group, indicating that the cell damage caused by VPF itself would partially and non-targetedly destroy the proteins 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.

[0115] 2) Flow cytometry to detect cell surface PD-L1 protein degradation

[0116] MDA-MB-231 cells were seeded in a 24-well plate 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 drug treatment for 4 hours, the cells were illuminated (689nm, 5min×2), and then the cells were cultured for 1 hour in a dark environment. 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 the attached figure. Fig.10As shown in the figure, the negative control group (i.e., Blank group) represents the MDA-MB-231 cells expressing PD-L1 protein without adding 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 cells.

[0117] Surprisingly, the fluorescence intensity of cells treated with PPA-VPF decreased significantly and showed a certain concentration dependence, which was consistent with the laser confocal microscopy experiment, indicating that the designed PPA-VPF can selectively degrade the PD-L1 protein on the surface of cancer cells.

[0118] (9) Study on the anti-tumor activity of photodegradation targeted chimera PPA-VPF

[0119] 1) The CCK-8 method was used for detection. Human triple-negative breast cancer cells (i.e., MDA-MB-231 cells, highly expressing PD-L1 protein), human breast cancer cells (MCF-7 cells, low expressing PD-L1 protein) and normal human embryonic kidney cells 293T were used as model cells. They were planted in 96-well plates and continued to be attached to the wall in an incubator. The photodegradation targeting chimera PPA-VPF was added. After incubation for 4 hours, the light treatment was performed (the specific conditions were set as above); after the sample was incubated for 24 hours, the original culture medium was discarded, PBS was washed three times, 10% CCK-8 solution (CCK-8 was dissolved in the culture medium) was added and incubated in an incubator for 2 hours, and the absorbance in the well plate was tested using a Biotek microplate reader (480nm) and the cell survival rate was calculated. The experimental results are shown in the attached figure. Fig.11 The results showed that PPA-VPF had the strongest cytotoxicity in MDA-MB-231 cells with high PD-L1 expression, while it had lower cytotoxicity in MCF-7 cells with low PD-L1 expression, and its toxicity in normal cells was negligible, indicating that cytotoxicity was related to the content of PD-L1 receptor protein in cell species.

[0120] 2) Detection of cellular uptake of PPA-VPF by flow cytometry

[0121] 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 / mL 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 the attached figure. Fig.11As shown in b. 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 there was no significant difference in the uptake of VPF between the two cells. Fig.11 The results indicate that cells without PD-L1 receptors will not bind to PPA-VPF, and thus will not produce the toxicity of the photosensitizer. However, PPA-VPF will bind to cells that highly express PD-L1 receptors. After illumination, it will degrade the PD-L1 protein on the cell surface and produce toxic ROS, ultimately leading to cell death.

[0122] (10) Evaluation of the anti-tumor effect of the photodegradation targeted chimera PPA-VPF in vivo

[0123] 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 treatment with photodynamic therapy and immunotherapy, in order to investigate the efficacy and safety of this combined therapy for in situ breast cancer in mice.

[0124] 1) Establishment of 4T1 tumor-bearing mouse model

[0125] BALB / c mice were purchased and allowed to adapt to the environment for one week before preparing breast cancer 4T1 cell suspension for subcutaneous inoculation on the back of mice. Each mouse was inoculated with 5×10 5 cells.

[0126] 2) PPA-VPF in vivo distribution experiment

[0127] Inoculate 4T1 cells and observe the tumor growth of mice every day until the tumor volume grows to 200mm 3 The 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 mice in the VPF group were anesthetized with gas at 5, 10, 15, 20, 30, 60 and 120 min after administration, and those in the PPA-VPF group were anesthetized at 2, 4, 8, 12, 24, 36 and 48 h, and then photographed using a small animal in vivo imaging device (IVIS SPECTRUM). The results are shown in the attached figure. Fig.12. The results show that the photosensitizer VPF is metabolized very quickly in the body. It is quickly distributed throughout the body within 10 minutes of administration. The enrichment in the tumor site basically reaches a peak in about half an hour, but the enrichment time is short, and it has basically left the tumor site after 2 hours; while PPA-VPF is still strongly enriched in the tumor site 12 hours after injection, and gradually accumulates in the tumor site after 24 hours, and the content in other parts gradually decreases. This shows that the photodegradation targeted chimera PPA-VPF can stably accumulate in the tumor site through the EPR effect compared with the photosensitizer itself VPF, and can circulate for a long time. Based on the above results, VPF 30min and PPA-VPF 24h were selected based on the time points when the fluorescence intensity in the tumor site was higher and the fluorescence intensity in other parts was lower. The mice were killed and dissected, and the ex vivo tissues and tumors were removed to continue to investigate the in vitro tissue distribution of the drug. The results are shown in the attached figure. Fig.13 At the selected time point, PPA-VPF was mainly distributed in the tumor, while it was less distributed in the heart, liver, spleen, lungs and kidneys. In addition to the tumor, the VPF group also had a large distribution in the liver, indicating that PPA-VPF could be effectively enriched in the tumor at the selected time point, while other tissues and organs were less. Based on the above experimental results, the optimal photodynamic therapy time was selected to avoid toxic side effects on other organs. It was decided to start the corresponding light irradiation mouse treatment 30 minutes after intravenous injection of VPF and 24 hours after PPA-VPF.

[0128] (11) Antitumor activity study in mice

[0129] 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 3 At the same time, the mice were randomly divided into 4 groups, 4 mice in each group, and intravenously injected with PBS, pure polypeptide PPA, photosensitizer VPF (with cosolvent PEG) and PPA-VPF, respectively. The dose of PPA-VPF was 2 mg / kg, and the other groups were converted according to the amount of substances in the experimental group. The primary tumor was irradiated with 660nm laser (500mW / 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 2 days. The weight changes of mice and the volume growth of bilateral tumors were recorded daily. The formula for calculating tumor volume is: V=d×l2 / 2 (where: V represents the volume of the tumor, d represents the long diameter of the tumor, and l is the short diameter of the tumor). The mice were euthanized on the 11th day for subsequent pathological and immunohistochemical evaluations. The results are shown in the attached Fig.14As shown in Figures ad, compared with the control group, mice treated with PPA-VPF showed significant tumor growth inhibition after laser irradiation treatment (P<0.001). Mice in the photosensitizer VPF group also showed a certain tumor growth inhibition, but weaker than the PPA-VPF group. Mice treated with pure peptide PPA showed no inhibition. This indicates that the tumor inhibition effect of PPA-VPF is better than that of photosensitizer alone, indicating that immunotherapy combined with PDT treatment can effectively inhibit the growth of in situ breast tumors in mice. Among them, the weight changes of mice were stable and there was no statistical difference (P>0.05), proving that this treatment had no significant effect on the survival of mice.

[0130] (12) HE staining of tumor tissue sections

[0131] HE section staining was used to perform pathological examination on the tumor tissue of mice. The ex vivo tumor tissue was fixed with 4% paraformaldehyde solution, and after 24 hours, gradient alcohol was used to remove the water in the tissue block, and finally the tissue block was placed in xylene for transparency. After the processed tissue block was infiltrated and embedded in paraffin, a paraffin section machine was used to make paraffin sections. After dewaxing with xylene, the tissue sections were stained with hematoxylin and eosin dyes, and photographed with a fully automatic digital section scanning system. The results are shown in the attached figure. Fig.15 After light treatment, the tumor sections in the PBS group and the pure polypeptide PPA group showed no obvious histological changes, while the tumor tissue in the VPF group was partially destroyed, and the nuclei of the tumor tissue treated with PPA-VPF shrank, the cells were severely damaged, and the number was significantly reduced.

[0132] (13) Effects of immunotherapy on tumor growth in mice

[0133] To further verify the immunotherapy effect of PD-L1 protein degradation and combined with photodynamic therapy on distal tumors, the volume of distal tumors was monitored (see Appendix Fig.16 a). 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 (see Appendix Fig.16 b, c). Similar to the results of primary tumors, the PPA-VPF treatment group showed the best tumor inhibition effect, with the smallest volume of distant tumors and the highest tumor inhibition rate. The above results all confirm that the designed photodegradable targeted chimera PPA-VPF can stimulate a good anti-tumor immune response in synergistic photodynamic and PD-1 / PD-L1 immunotherapy.

[0134] (14) Flow cytometry experiments for tumor immunotherapy

[0135] 1) Detection of tumor-infiltrating T lymphocytes

[0136] 4T1 cells (5×105 ) 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 performed in the same way as above. The mice in the VPF group and PPA-VPF group were exposed to 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 by adding 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 was permeabilized 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 the attached figure. Fig.17 As shown in Figure a, compared with the PBS group, the CTL cell proportions in the pure peptide 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%, 6.17 times higher than that of the PBS group. The expression of IFN-γ was further evaluated (see Appendix Fig.17 b) Similarly, the treatment with peptide PPA alone or photodynamic therapy alone (i.e., 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 combined 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 selected from at least one of 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 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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