Photosensitizer chimera targeting pd-l1 protein, and preparation method and application thereof

Abstract

The present application relates to a photosensitizer chimera targeting PD-L1 protein and a preparation method and application thereof. The photosensitizer chimera of the present application has an X-R-G structure, wherein X is a photosensitizer unit, R is a connecting arm, and G is a polypeptide unit targeting PD-L1 protein. The photosensitizer is a porphyrin photosensitizer, the excitation light wavelength of the photosensitizer is >550 nm, the molar absorption coefficient at the maximum absorption wavelength after 550 nm is >1000 M ‑1 cm ‑1 ; the quantum yield of the triplet state is >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; and the binding constant of the polypeptide to h-PD-L1 is <20 μM. The photosensitizer chimera of the present application can produce a stronger immune response than single treatment based on the combined treatment of the immune checkpoint blockade and cytotoxicity induced by the photodegradation of PD-L1 protein in PD-1 / PD-L1, and has great potential in 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 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, 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. 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. 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 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;

[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)-(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]

[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 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.

[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, and removing uncoupled substances to obtain a first conjugate;

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[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 a 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 -CH2CH2-COOH, Fmoc-NH-(CH2) y -COOH, wherein x and y are even numbers ranging from 2 to 10.

[0044] 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.

[0045] Preferably, in step 1), the ratio of the linker arm to the polypeptide targeting the 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, further preferably any one of breast cancer, lung cancer, colorectal cancer, esophageal cancer or pancreatic cancer; further preferably, breast cancer or esophageal cancer.

[0049] 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.

[0050] 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

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

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

[0053] Attachment 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] Attachment Figure 4 The UV absorption spectra of the photosensitizer chimera (PPA-VPF) and verteporfin (VPF) prepared in Example 1 (a: solvent is PBS; b: solvent is PBS / acetonitrile = 1:1).

[0055] Attachment Figure 5 Fluorescence spectra of the photosensitizer chimera prepared in Example 1 (a: solvent is PBS; b: solvent is PBS / acetonitrile = 1:1).

[0056] Attachment Figure 6 These are the EPR spectra 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).

[0057] Attachment 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 plasmon resonance technology.

[0058] Attachment Figure 8 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.

[0059] Attachment Figure 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] Attachment Figure 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] Attachment Figure 11 a) Photodegradation targeting chimera PPA-VPF was irradiated 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.

[0062] Attachment Figure 12These are in vivo imaging images of the photosensitizer VPF and the photosensitizer chimera PPA-VPF prepared in Example 1 at different time points.

[0063] Attachment Figure 13 These are in vitro fluorescence imaging images of major organs and tumor sites of the photosensitizer VPF (30 min) and the photosensitizer chimera PPA-VPF (24 h) prepared in Example 1.

[0064] Attachment Figure 14 (a) Tumor growth curve of the light-treated side in mice with bilateral 4T1 tumor transplantation; (b) body weight change; (c) (d) Tumor photos and tumor inhibition rate (TGI) of the light-treated side.

[0065] Attachment Figure 15 HE staining of tumor tissue sections of mice in each group;

[0066] Attachment Figure 16 (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;

[0067] Attachment 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

[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 can be used.

[0069] The technical solutions of the present invention will be described clearly and completely 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] 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.

[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 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.

[0080] The peptide products were analyzed and separated using high performance liquid chromatography (HPLC) with 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. The LC-MS characterization data for PPA are shown in the attached figure. 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 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 the attached Figure 2 and as shown in Table 2.

[0083] Table 2 Affinity of PPA series peptides to 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 -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.

[0089] (b) Post-production processing of chimeras

[0090] 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.

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

[0092] 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.

[0093] (d) Affinity determination and screening

[0094] The affinity between the hPD-L1 protein and the 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 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 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.

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

[0096]

[0097] Select the linker Fmoc-NH-PEG n -CH2CH2-COOH (1.2 equiv.) (n = 2) to prepare the obtained PPA-VPF for subsequent testing.

[0098] (2) Product characterization parameters:

[0099] LC-MS characterization data:

[0100] LC-MS characterization conditions: Agilent C18 analytical column, elution gradient: mobile phase B changed uniformly from 30% to 90% within 30 min.

[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 PBS or 50% ACN (PBS: acetonitrile = 1:1) mixed solvent (concentration was 20 μM), and its UV absorption spectrum in the two solvents was measured respectively, with the photosensitizer verteporfin as a control. The results are shown in the attached figure. Figure 4 As shown in Figure 2, in PBS solution, the UV absorption of PPA-VPF is slightly lower than that of verteporfin. However, in a 50% ACN mixed solvent, the UV absorption spectrum of PPA-VPF is essentially the same as that of verteporfin itself, which may be due to differences 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 the 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 UV results above. In PBS, PPA-VPF is slightly lower than VPF, while in 50% ACN solution, the intensities are comparable. This indicates that the decrease in spectral intensity is indeed caused by dissolution and aggregation, but the basic spectral properties remain unchanged.

[0106] (5) EPR spectrum

[0107] 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). The typical settings for EPR detection are: scan range, 100g; scan time, 60s; microwave power, 19.23mW; modulation amplitude, 1g; modulation frequency, 100kHz. The results are shown in the attached figure. Figure 6 .

[0108] (6) Affinity determination

[0109] The results of the PPA-VPF affinity test 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 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, ThermoFisher 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 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 using 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 and selective degradation of PD-L1 at the cellular level.

[0112] (8) PD-L1 protein degradation verification experiment at the cellular 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 time. 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 stain the nuclei with DAPI for 4 minutes at room temperature. Take pictures using a laser scanning confocal microscope, and the experimental results are shown in the attached figure. Figure 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 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 decreased over a large area, 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 detection of 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 4 hours of drug treatment, the cells were illuminated (689 nm, 5 min × 2). After that, the cells were cultured for 1 hour under dark conditions. The culture medium was discarded and the anti-PD-L1 protein flow cytometry antibody PE anti-mouse CD274 (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. Figure 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 that have been treated with antibodies but not drugs. The fluorescence intensity values ​​of cells treated with PPA or VPF are almost the same as those of the PBS group, indicating that the treatment of these two drugs does not affect the cells.

[0117] 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 selectively degrade the PD-L1 protein on the surface of cancer cells.

[0118] (9) Study on the anti-tumor activity of the 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, 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. 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 and incubated for 4 hours. The light treatment was performed (the specific conditions were the same as above); after the sample was incubated 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 in the well plate was tested using a Biotek microplate reader (480 nm) and the cell viability was calculated. The experimental results are shown in the attached figure. Figure 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 cytotoxicity in normal cells was negligible, indicating that cytotoxicity is related to the content of the cell receptor protein PD-L1.

[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 100 cells / well 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. Figure 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 the uptake of VPF by the two cells did not show significant difference. Figure 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 exposure to light, 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 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 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.

[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. They were 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 with gas at 5, 10, 15, 20, 30, 60 and 120 min after administration, while the PPA-VPF group was anesthetized at 2, 4, 8, 12, 24, 36 and 48 h. The mice were then photographed using a small animal in vivo imaging system (IVIS SPECTRUM). The results are shown in the attached figure. Figure 12. The results show that the photosensitizer VPF is metabolized very quickly in the body and is quickly distributed throughout the body within 10 minutes of administration. The accumulation in the tumor site basically reaches its peak in about half an hour, but the accumulation 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 sites gradually decreases. This shows that the photodegradation targeted chimera PPA-VPF can stably accumulate in the tumor site through the EPR effect compared to the photosensitizer itself VPF, and can circulate for a long time. Based on the above results, considering the time points where the fluorescence intensity in the tumor site is higher and the fluorescence intensity in other sites is lower, VPF 30min and PPA-VPF 24h were selected, the mice were killed and dissected, the in vitro tissues and tumors were removed, and the in vitro tissue distribution of the drug was further investigated. The results are shown in the attached figure. Figure 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 distribution, the VPF group also had a large distribution in the liver, indicating that at the selected time point, PPA-VPF could be effectively enriched in the tumor, while other tissues and organs were less abundant. Based on the above experimental results, the optimal photodynamic therapy time was selected to avoid toxic side effects in other organs. It was decided to start the corresponding light irradiation treatment of mice 30 minutes after intravenous injection of VPF and 24 hours after PPA-VPF.

[0128] (11) Anti-tumor 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 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 tumors were 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 the mice and the volume growth of the bilateral tumors were recorded daily. The formula for calculating the 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 Figure 14As shown in Figures ad, compared with the control group, mice treated with PPA-VPF showed significant tumor growth inhibition after laser irradiation (P<0.001). Mice in the photosensitizer VPF group also showed some tumor growth inhibition, but weaker than the PPA-VPF group. Mice treated with pure peptide PPA showed no inhibition. This indicates that PPA-VPF has a better tumor inhibition effect than the photosensitizer alone, indicating that immunotherapy combined with PDT can effectively inhibit the growth of in situ breast tumors in mice. The weight changes of the mice were stable, with no statistical difference (P>0.05), proving that this treatment had no significant effect on the survival of the mice.

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

[0131] The tumor tissue of mice was pathologically examined using HE staining. 4% paraformaldehyde solution was used to fix the isolated tumor tissue. After 24 hours, gradient alcohol was used to remove the water in the tissue block. Finally, the tissue block was placed in xylene for transparency. The processed tissue block was paraffin-soaked and embedded. 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 slice scanning system. The results are shown in the attached figure. Figure 15 After light treatment, the tumor sections in the PBS group and the pure peptide PPA group showed no obvious histological changes, while the tumor tissue in the VPF group was partially destroyed. In the tumor tissue treated with PPA-VPF, the nuclei 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 Figure 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 Figure 16 b, c). Similar to the primary tumor results, the PPA-VPF treatment group demonstrated the best tumor suppression, with the smallest distant tumor size and the highest tumor inhibition rate. These results confirm that the designed photodegradable targeted chimera PPA-VPF can stimulate a robust anti-tumor immune response when used in synergistic photodynamic therapy 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 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 manufacturer's instructions. 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. Figure 17 As shown in a, compared with the PBS group, the CTL cell ratio 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 attached). Figure 17 b) Similarly, the treatment of the peptide PPA group or the photodynamic therapy group (ie, VPF group) can 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 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, characterized in that: The photosensitizer is verteporfin, and the connecting arm is , 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, n is an integer of 0-50, and n is not 0, The amino acid sequence of the polypeptide unit is: dNdYdSdKdPdTdDdRdQdYdHdF.

2. The photosensitizer chimera according to claim 1, characterized in that n is an integer from 0 to 10.

3. The photosensitizer chimera according to claim 1 or 2, 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 the polypeptide unit targeting the PD-L1 protein, and the amino acid sequence of the polypeptide unit is: dNdYdSdKdPdTdDdRdQdYdHdF.

4. The method for preparing the photosensitizer chimera according to any one of claims 1 to 3, comprising the following steps: 1) first mixing the linker of claim 1 and the polypeptide unit targeting the PD-L1 protein of claim 1, performing a first coupling, and removing uncoupled substances to obtain a first conjugate; 2) Mixing the first conjugate obtained in step 1) with the photosensitizer of claim 1, performing a second coupling under light-shielding conditions, removing unconjugated substances, and obtaining a photosensitizer chimera.

5. The preparation method according to claim 4, characterized in that In step 1), the ratio of the linker to the polypeptide unit is 1-5:

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

7. An anti-tumor drug, characterized in that: The active ingredient comprises the photosensitizer chimera according to any one of claims 1 to 3.

8. Use of the photosensitizer chimera according to any one of claims 1 to 3 in the preparation of an anti-tumor drug, wherein the tumor is any one of breast cancer, lung cancer, colorectal cancer, esophageal cancer or pancreatic cancer.

9. The use according to claim 8, wherein the tumor is breast cancer or esophageal cancer.

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