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WO2025026029A1 - 靶向pd-l1蛋白的光敏剂嵌合体及其制备方法和应用 - Google Patents

靶向pd-l1蛋白的光敏剂嵌合体及其制备方法和应用 Download PDF

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WO2025026029A1
WO2025026029A1 PCT/CN2024/104970 CN2024104970W WO2025026029A1 WO 2025026029 A1 WO2025026029 A1 WO 2025026029A1 CN 2024104970 W CN2024104970 W CN 2024104970W WO 2025026029 A1 WO2025026029 A1 WO 2025026029A1
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photosensitizer
chimera
polypeptide
protein
tumor
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刘国全
刘思瑾
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Peking University
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Peking University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • 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.
  • PD-1 programmed death receptor-1
  • PD-L1 programmed death ligand-1
  • 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).
  • 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.
  • peptides Compared with antibodies, peptides generally have better physiological properties, such as better tissue and tumor permeability, higher oral bioavailability, and more tolerable half-life.
  • 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.
  • IDB immune checkpoint blockade
  • 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 >550nm; the molar absorption coefficient at the maximum absorption wavelength after 550nm is >1000M - 1cm -1 ; the quantum yield of the triplet state is >0.01;
  • the connecting arm is selected from the following structural formula:
  • 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;
  • the binding constant of the peptide to h-PD-L1 is ⁇ 20 ⁇ M.
  • the structural formula of the connecting arm is: n is an integer of 0-50; preferably n is an integer of 0-10.
  • the photosensitizer is selected from at least one of verteporfin, dihydrochlorin or pheophorbide a; preferably verteporfin.
  • polypeptide is any one of (a) to (c):
  • the chemical structural formula of the photosensitizer chimera is as shown in Formula I or 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.
  • the present invention also provides a method for preparing the photosensitizer chimera described in the above scheme, comprising the following steps:
  • 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.
  • 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.
  • the polypeptide targeting PD-L1 protein is prepared by a solid phase synthesis method based on Fmoc.
  • the non-natural D-type amino acids protected by ⁇ N-Fmoc or ⁇ N-Boc for polypeptide synthesis 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, Fmoc-D-Phe-OH.
  • 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.
  • the present invention has no particular limitation on the conditions of the first coupling.
  • the conditions of the first coupling include: a coupling temperature of 5-30° C.; and a coupling time of 8-14 h.
  • 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.
  • linker examples include, but are not limited to, Fmoc-NH-(PEG) x -CH 2 CH 2 -COOH and Fmoc-NH-(CH 2 ) y -COOH, wherein x and y are even numbers of 2-10.
  • 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.
  • DIEA is added to the solution containing the photosensitizer for the linker arm to activate the amino acid.
  • the ratio of the linker arm to the polypeptide targeting PD-L1 protein is 1-5:1.
  • the usage ratio of the first coupling agent to the photosensitizer is 1:1-4.
  • the present invention has no particular limitation on the conditions for the second coupling.
  • the conditions for the second coupling include: a coupling temperature of 5-30° C.; and a coupling time of 8-14 h.
  • 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.
  • 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.
  • 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.
  • IRB immune checkpoint blockade
  • CTLs cytotoxic T lymphocytes
  • PDT photodynamic therapy
  • Figure 1 is an LC-MS mass spectrum of the polypeptide (PPA) targeting PD-L1 protein prepared in Example 1.
  • FIG2 is a graph showing the binding ability of the polypeptide (PPA) targeting PD-L1 protein prepared in Example 1 and the protein hPD-L measured using plasma resonance technology.
  • FIG3 is the LC-MS spectrum of the photosensitizer chimera (PPA-VPF) isomers prepared in Example 1 (molecular formula: C 118 H 152 N 26 O 30 ).
  • FIG7 is a graph showing the binding ability of the photosensitizer chimera prepared in Example 1 and the protein hPD-L measured using plasma resonance technology.
  • FIG8 is a diagram 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.
  • Figure 9 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).
  • Figure 10 shows the flow cytometry results of PD-L1 protein on the surface of living cells after treatment with PBS, PPA, VPF and PPA-VPF.
  • Figure 11 shows a) the cytotoxicity of the photodegradation targeting chimera PPA-VPF after irradiation with 660nm laser at 100mW/ cm2 (5min ⁇ 2); b) the flow cytometry uptake 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 12h.
  • FIG12 is an in vivo imaging diagram of the photosensitizer VPF and the photosensitizer chimera PPA-VPF prepared in Example 1 at different time points.
  • FIG13 is an in vitro fluorescence imaging diagram of the main organs and tumor sites of the photosensitizer VPF (30 min) and the photosensitizer chimera PPA-VPF (24 h) prepared in Example 1.
  • Figure 14 shows (a) the growth curve of the tumor at the light-treated end in mice with bilateral 4T1 tumor transplantation; (b) body weight change; (c) (d) tumor photos and tumor inhibition rate (TGI) at the light-treated end.
  • TGI tumor inhibition rate
  • Figure 15 is HE staining of tumor tissue sections of mice in each group
  • Figure 16 shows (a) the 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;
  • Figure 17 is a) a representative flow cytometry graph of 4T1 tumor CD3 + CD8 + cells and a summary of their proportions; b) a representative flow cytometry graph of 4T1 tumor CD8 + IFN- ⁇ + lymphocytes and a summary of their proportions.
  • 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.
  • Non-natural d-amino acids protected with ⁇ N-Fmoc or ⁇ N-Boc for peptide synthesis 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.
  • 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 is added 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.
  • the reaction liquid is collected and blown dry with a nitrogen stream, and the obtained solid is washed with cold ether.
  • the solid is dissolved with a H 2 O/MeCN mixed solution, and a clear solution is obtained by filtering through a filter membrane for subsequent analysis and separation and purification.
  • the peptide products were analyzed and separated by HPLC, with the mobile phases being H 2 O with 0.05% TFA (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 is shown in FIG1 .
  • hPD-L1 The affinity between hPD-L1 protein and PPA series polypeptides was measured by surface plasmon resonance (SPR, Biacore 8k, GE Healthcare).
  • SPR surface plasmon resonance
  • hPD-L1 was diluted in 10mM sodium acetate buffer (pH 4.5, GE Healthcare) to obtain a protein concentration of 40 ⁇ g/mL.
  • the diluted hPD-L1 was covalently fixed to a new sensor chip (S series sensor chip CM5, GE Healthcare) through the primary amine group using a standard amine coupling kit (GE Healthcare).
  • the final fixation level of the target hPD-L1 was 9000RU by connection.
  • PBS-P buffer (10mM phosphate buffer containing 2.7mM KCl, 137mM NaCl and 0.05% surfactant P20, final pH 7.4, GE Healthcare) was used for measurement at a flow rate of 30 ⁇ L/min.
  • the PPA series peptides were dissolved in PBS-P running buffer and set up a concentration gradient, with more than 5 concentration gradients to ensure that the final affinity KD value fell within the concentration gradient range.
  • the results are shown in Table 2.
  • the PPA peptide had the strongest affinity with the PD-L1 protein, so in the end we used the PPA peptide as the parent peptide to carry out subsequent product preparation.
  • the obtained PPA had more than 5 concentration gradients to ensure that the final affinity KD value fell within the concentration gradient range, and the results are shown in Figure 2 and Table 2.
  • 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).
  • the Fmoc protecting group on the linker was further removed using a 20% piperidine solution in DMF (5 min ⁇ 2), followed by washing 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, DIEA (2.4 equiv.) was added to activate the amino acids, and the mixed solution was added to the peptide synthesis tube for preparing PPA in Preparation 1, and the reaction was carried out on a shaker overnight until the coupling was completed. The whole process was protected from light.
  • 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 is added 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.
  • the reaction liquid is collected and blown dry with a nitrogen stream, and the obtained solid is washed with cold ether.
  • the solid is dissolved with a H 2 O/MeCN mixed solution, and a clear solution is obtained by filtering through a filter membrane for subsequent analysis and separation and purification.
  • 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 phases used were H 2 O with 0.05% TFA (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.
  • hPD-L1 The affinity between hPD-L1 protein and photosensitizer chimera was measured by surface plasmon resonance (SPR, Biacore 8k, GE Healthcare).
  • SPR surface plasmon resonance
  • hPD-L1 was diluted in 10mM sodium acetate buffer (pH 4.5, GE Healthcare) to obtain a protein concentration of 40 ⁇ g/mL.
  • the diluted hPD-L1 was covalently fixed to a new sensor chip (S series sensor chip CM5, GE Healthcare) through primary amine groups using a standard amine coupling kit (GE Healthcare).
  • the final fixation level of the target hPD-L1 was 9000RU by connection.
  • PBS-P buffer (10mM phosphate buffer containing 2.7mM KCl, 137mM NaCl and 0.05% surfactant P20, final pH 7.4, GE Healthcare) was used for measurement at a flow rate of 30 ⁇ L/min.
  • 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 that the final affinity KD value fell within the concentration gradient range. The results are shown in Table 3.
  • the PPA-VPF prepared with the linker Fmoc-NH-PEG n -CH 2 CH 2 -COOH (1.2 equiv.) (n 2) was selected for subsequent testing.
  • the LC-MS characterization conditions used were: Agilent C18 analytical column, and the elution gradient was a uniform change from 30% to 90% of mobile phase B within 30 min.
  • PPA-VPF retention time is 18.68min and 20.06min.
  • ESI-MS Calculate for C 118 H 152 N 26 O 30 : 2414.67Da and 2414.67Da (average isotopes) (m/z), the highest peak of the actual molecular weight obtained is [M+3H] 3+ : 805.71Da and 805.43Da. The results are shown in Figure 3.
  • the results are shown in Figure 4.
  • the UV absorption of PPA-VPF is slightly lower than that of verteporfin.
  • 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.
  • the results are shown in FIG5 .
  • 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.
  • 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).
  • the generated ROS species were detected by X-band Bruker A200 spectrometer.
  • 4-OH-TEMP with a final concentration of 200mM was added to the PPA-VPF or verteporfin solution (20 ⁇ M) before irradiation.
  • Protein targeted photodegradation was analyzed by western blot.
  • A549 cells were seeded onto 6-well plates. After 24 h of incubation, cells were treated with the photodegradation targeting chimera PPA-VPF, verteporfin itself, pure peptide, or DMSO as a control at 37 °C for 8 h and then irradiated with a 300 W xenon arc lamp (600 nm bandpass filter, 1.5 mW/cm2).
  • 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 ⁇ _actin (ab8226, rabbit, 1:1000, Abcam).
  • PD-L1 ab125066, rabbit, 1:1000, Abcam
  • GPX1 ab108427, rabbit, 1:1000, Abcam
  • ACSL4 ab155282, rabbit, 1:1000, Abcam
  • ⁇ _actin ab8226, rabbit, 1:1000, Abcam
  • MDA-MB-231 cells were seeded on a glass-bottom 24-well plate at a density of 5 ⁇ 10 4 cells per well. After the cells were attached to the surface of a 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. After continuing to incubate for 1h, the culture medium was discarded, and the cells were fixed in 4% paraformaldehyde for 10 minutes at room temperature.
  • CEAULIGHT 300W xenon arc lamp
  • 660nm bandpass filter 660nm bandpass filter
  • 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.
  • 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).
  • the cells were illuminated (689nm, 5min ⁇ 2), and then the cells were cultured for 1 hour under light-proof conditions.
  • the culture medium was discarded, and the anti-PD-L1 protein flow antibody PEanti-mouseCD274 (biolegend, #124307) was added to treat the cells for 30 minutes, and the cells were collected for flow cytometry analysis.
  • the negative control group i.e., Blank group
  • the PBS group represents cells to which antibodies were added but no drug treatment was added.
  • the fluorescence intensity values of cells treated with PPA or VPF were almost the same as those of the PBS group, indicating that the treatment of these two drugs did not affect the PD-L1 protein content on the cell surface.
  • 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 experiment, indicating that the designed PPA-VPF can target and selectively degrade the PD-L1 protein on the surface of cancer cells.
  • 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
  • normal human embryonic kidney cells 293T were used as model cells. They were planted in a 96-well plate, and the cells were allowed to adhere to the wall in an incubator.
  • the photodegradation targeting chimera PPA-VPF was added, and after incubation for 4 hours, 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, and 10% CCK-8 solution (CCK-8 was dissolved in the culture medium) was added and incubated in an incubator for 2 hours.
  • the absorbance (480 nm) in the well plate was tested using a Biotek microplate reader and the cell survival rate was calculated. The experimental results are shown in Figure 11a.
  • the MDA-MB-231 (high expression of PD-L1 protein) and MCF-7 cells (low expression of PD-L1 protein) mentioned in 1) were inoculated in a 24-well plate at a density of 5 ⁇ 10 3 cells per well and cultured overnight. PPA-VPF or VPF was then added and incubated for 8 hours, and the cells were collected and analyzed by flow cytometry. The experimental results are shown in Figure 11b. In the flow cytometry results, the amount of PPA-VPF taken up by MDA-MB-231 cells was significantly higher than that of MCF-7 cells, and showed a certain concentration dependence, while the uptake of VPF by the two cells was not significantly different.
  • mice BALB/c mice were purchased and after one week of acclimatization, breast cancer 4T1 cell suspension was prepared for subcutaneous inoculation on the back of the mice. 5 ⁇ 10 5 cells were inoculated on the left back of each mouse.
  • mice were randomly divided into 2 groups, with 3 mice in each group.
  • 100 ⁇ L of photosensitizer VPF (with cosolvent PEG) or photodegradation targeting chimera PPA-VPF was injected through the tail vein, respectively, and 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 the mice 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 imager (IVIS SPECTRUM). The results are shown in 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 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 sites gradually decreases.
  • 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.
  • 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.
  • VPF 30min and PPA-VPF 24h were selected based on the time points where the fluorescence intensity in the tumor site is high and the fluorescence intensity in other sites is low.
  • 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 Figure 13.
  • PPA-VPF was mainly distributed in the tumor, while it was less distributed in the heart, liver, spleen, lungs and kidneys.
  • 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.
  • the optimal photodynamic therapy time was selected to avoid toxic side effects on 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.
  • the bilateral tumor model experimental method was selected to carry out in vivo anti-tumor research.
  • 4T1 cells (5 ⁇ 10 5 ) were subcutaneously injected into the right side of BALB/c mice to generate primary tumors for subsequent light treatment; 1 ⁇ 10 5 4T1 cell suspension was then subcutaneously injected into the left side of the mouse to form distal tumors.
  • the mice were randomly divided into 4 groups, 4 mice in each group, and intravenously injected with PBS, pure peptide 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 the same substance as 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 bilateral tumors were recorded daily.
  • the mice were euthanized on the 11th day for subsequent pathological and immunohistochemical evaluations. The results are shown in Figures 14a-d.
  • mice treated with PPA-VPF showed significant tumor growth inhibition after laser irradiation treatment (P ⁇ 0.001).
  • the mice in the photosensitizer VPF group also showed a certain degree of tumor growth inhibition, but it was weaker than the PPA-VPF group.
  • 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.
  • the tumor tissue of mice was pathologically examined using HE section staining.
  • the ex vivo tumor tissue was fixed with a 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.
  • a paraffin sectioning machine was used to make paraffin sections.
  • the tissue sections were stained with hematoxylin and eosin dyes, and photographed using a fully automatic digital section scanning system. The results are shown in Figure 15.
  • the tumor sections in the PBS group and the pure polypeptide PPA group did not show obvious histological changes, the tumor tissue in the VPF group was partially destroyed, and the nuclei of the tumor tissue treated with PPA-VPF were shrunken, the cells were severely damaged, and the number was significantly reduced.
  • mice 4T1 cells (5 ⁇ 10 5 ) were injected subcutaneously into the left side of BALB/c mice.
  • the mice were divided into groups, namely PBS group, PPA group, VPF group, and PPA-VPF group, and injected through the tail vein.
  • the operation was the same as above.
  • the mice in the VPF group and PPA-VPF group were irradiated with 660nm laser at an intensity of 500mW/cm 2 for 8 minutes 30min and 24h after injection. The mice were kept in the dark.
  • mice On the third day after administration, the mice were euthanized, and their spleens and tumor tissues were dissected and digested by adding collagenase (400U/mL), DNase1 (100 ⁇ g/mL) and hyaluronidase (0.04U/mL). The mixture was then passed through a 75 ⁇ m nylon cell strainer. The samples were washed three times with PBS (containing 2% FBS) and then resuspended at a density of 1 ⁇ 10 6 cells/mL for flow cytometric analysis.
  • collagenase 400U/mL
  • DNase1 100 ⁇ g/mL
  • hyaluronidase 0.04U/mL
  • Antibodies (anti-mouse CD3-FITC, anti-mouse CD4-APC, anti-mouse CD8-PE-Cy7 and anti-mouse IFN- ⁇ -PE, BioLegend) were added according to the instructions, and the membrane needed to be broken before adding the anti-mouse IFN- ⁇ -PE antibody.
  • the cells were analyzed on a flow cytometer, and the data were analyzed using FlowJo 10. The results are shown in Figure 17a. Compared with the PBS group, the proportion of CTL cells in the pure polypeptide PPA group and the photosensitizer VPF treatment group were 19.6 ⁇ 1.8% and 37.5 ⁇ 2.7%, respectively.
  • the PPA-VPF group showed the highest CTL ratio in tumor tissue, which was 54.9 ⁇ 1.45%, 6.17 times higher than that of the PBS group.
  • the expression of IFN- ⁇ evaluated further was also similar.
  • the treatment with peptide PPA alone or photodynamic therapy alone i.e., VPF group
  • VPF group was able to partially increase the percentage of CD8 + /IFN- ⁇ + T cells, which were 24.8 ⁇ 2.1% and 32.6 ⁇ 2.7%, respectively.
  • the synergistic treatment group PPA-VPF induced the highest percentage of CD8 + /IFN- ⁇ + cells, which was 54.7 ⁇ 6.0%.

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Abstract

一种靶向PD-L1蛋白的光敏剂嵌合体及其制备方法和应用。所述光敏剂嵌合体为X-R-G结构，其中X为光敏剂单元，R为连接臂，G为靶向PD-L1蛋白的多肽单元，所述光敏剂为卟啉类光敏剂，所述光敏剂的激发光波长>550nm；550nm波长以后的最大吸收波长处的摩尔吸光系数>1000M -1cm -1；三线态的量子产率>0.01；所述连接臂选自以下结构式：其中所述连接臂的氨基侧与所述光敏剂单元相连，所述连接臂的羧基侧与多肽单元相连，n为0-50的整数；所述多肽与h-PD-L1的结合常数<20μM。所述光敏嵌合体基于光降解PD-1/PD-L1中的PD-L1蛋白所诱发的免疫检查点阻断和细胞毒作用的联合治疗能够产生比单一治疗更强的免疫反应，在增强抗肿瘤治疗方面具有极大潜力。

Description

靶向PD-L1蛋白的光敏剂嵌合体及其制备方法和应用 技术领域
本发明涉及生物医药技术领域，具体涉及一种靶向PD-L1蛋白的光敏剂嵌合体及其制备方法和应用。
背景技术
肿瘤的免疫疗法已经成为继手术、化疗、放疗及靶向药物治疗后的一种火热的新兴治疗手段，并已应用于临床。近年来研究发现程序性死亡受体-1(programmed death receptor-1，PD-1)/程序性死亡配体-1(programmed death ligand-1，PD-L1)信号通路是参与肿瘤免疫逃逸的重要途径之一，该信号通路可通过抑制T细胞活化来诱导肿瘤特异性T细胞凋亡，并导致T细胞抵抗，在肿瘤、慢性炎症等病理情况下起到免疫逃逸、免疫抑制等作用。当前全球范围内共获批上市19款PD-1/PD-L1药物，其中已在中国获批上市的PD-1/PD-L1药物有13款，PD-1抑制剂9种(7种国产，2种进口)，PD-L1抑制剂4种(2种国产，2种进口)。然而，目前的治疗性抗体依旧存在缺点，包括缺乏口服生物利用度、生产困难且昂贵、与免疫相关的不良事件以及对肿瘤组织的摄取不良，还有逐渐发生的耐药性，这限制了PD-1/PD-L1抗体药物的临床应用。与抗体相比，多肽通常具有更好的生理特性，如更好的组织和肿瘤渗透性、更高的口服生物利用度和更可耐受的半衰期。此外，联合治疗的手段也是目前肿瘤免疫治疗的主要趋势，肿瘤的光动力治疗产生的细胞毒效应能够有效激活机体的免疫反应，从而更好地减少PD-1/PD-L1的耐药性问题。因此，利用光定时定位降解PD-L1蛋白诱发免疫检查点阻断(ICB)治疗并结合以光动力治疗的细胞毒作用来开发设计分子是十分有希望的PD-1/PD-L1单克隆抗体的替代品。
发明内容
有鉴于此，本发明的目的在于提供一种靶向PD-L1蛋白的光敏剂嵌合体，所述嵌合体为X-R-G结构，其中X为光敏剂单元，R为连接臂，G为靶向PD-L1蛋白的多肽单元，其中所述光敏剂为卟啉类光敏剂，所述光敏剂的激发光波长>550nm；550nm波长以后的最大吸收波长处的摩尔吸光系数>1000M-1cm-1；三线态的量子产率>0.01；
所述连接臂选自以下结构式：
其中所述连接臂的氨基侧与所述光敏剂单元相连，所述连接臂的羧基侧与多肽相连，n为0-50的整数；
所述多肽与h-PD-L1的结合常数<20μM。
优选地，所述连接臂的结构式为：n为0-50的整数；优选n为0-10的整数。
优选地，其中所述光敏剂选自维替泊芬、二氢卟或脱镁叶绿酸a中的至少一种；优选为维替泊芬。
本发明对于多肽的氨基酸序列没有特别限制，优选地，其中所述多肽为(a)-(c)中任意一项所述的多肽：
(a)具有氨基酸序列(1)-(11)的多肽；
(b)氨基酸序列(1)-(11)经过取代、缺失或增加1-3个氨基酸残基且能与PD-L1的亲和力<20μM的多肽；
(c)在(a)或(b)多肽的羧基末端连接有酯基或酰胺基的多肽；
氨基酸(序列1)-(11)如下所示：
dNdYdSdKdPdTdDdRdQdYdHdF(1)；
dNdYdSdKdPdTdDdR(2)；
dNdYdSdKdPdTdDdRdQ(3)；
dNdYdSdKdPdTdDdRdQdY(4)；
dKdPdTdDdRdQdYdHdF(5)；
dPdTdDdRdQdYdHdF(6)；
dNdYdSdKdPdTdDdRdQdYdH(7)；
dSdKdPdTdDdRdQdYdHdF(8)；
dYdSdKdPdTdDdRdQdYdHdF(9)；
dKdPdTdDdR(10)；
dSdKdPdTdDdR(11)；
优选为dNdYdSdKdPdTdDdRdQdYdHdF(1)；
dNdYdSdKdPdTdDdR(2)；
dNdYdSdKdPdTdDdRdQ(3)；
dNdYdSdKdPdTdDdRdQdY(4)；
dKdPdTdDdRdQdYdHdF(5)；
进一步优选为dNdYdSdKdPdTdDdRdQdYdHdF(1)。
在一种优选实施方式中，所述光敏剂嵌合体的化学结构式如式I或式II所示；
所述式I和式II中n独立为0-10之间的整数，优选为0-6之间的整数；G为靶向PD-L1蛋白的多肽单元，所述多肽的氨基酸序列为：dNdYdSdKdPdTdDdRdQdYdHdF(1)。所述靶向PD-L1蛋白的多肽的N端与连接臂通过酰胺键结合。
本发明还提供上述方案所述光敏剂嵌合体的制备方法，包括以下步骤：
1)将连接剂和靶向PD-L1蛋白的多肽第一混合，进行第一偶联，去除未偶联物，得到第一偶联物；
2)将所述第一偶联物和光敏剂第二混合，在避光条件下进行第二偶联，去除未偶联物，得到光敏剂嵌合体。
本发明对所述靶向PD-L1蛋白的多肽的制备方法没有特殊限制，采用本领域常规方法制备得到即可。在本发明具体实施过程中，所述靶向PD-L1蛋白的多肽采用基于Fmoc的固相合成的方法制备得到。用于多肽合成的αN-Fmoc或αN-Boc保护的非天然D型氨基酸包括：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,Fmoc-D-Phe-OH。
本发明在树脂上偶联靶向PD-L1蛋白的多肽后，脱除Fmoc保护基，洗涤，之后与溶解有连接剂的溶液第一混合；所述溶解有连接剂的溶液以DMF为溶剂，溶解有连接剂、N-羟基-7-氮杂苯并三氮唑(HATU)和HOAt；所述连接剂、HATU和HOAt的当量比优选为0.5-1.5:0.5-1.5:0.5-1.5；在第一混合前，溶解有连接剂的溶液中加入DIEA活化氨基酸。
本发明对于第一偶联的条件没有特别限制，优选第一偶联的条件包括：偶联温度为：5-30℃；偶联时间为8-14h。
得到第一偶联物后，本发明将所述第一偶联物和光敏剂第二混合，在避光条件下进行第二偶联，去除未偶联物，得到光敏剂嵌合体。
本发明对于连接剂的种类没有特别限制，连接剂的实例包括但不限于：Fmoc-NH-(PEG)x-CH2CH2-COOH、Fmoc-NH-(CH2)y-COOH。其中，x、y为2-10的偶数。
在本发明中，所述第一偶联物偶联于树脂的基础上与溶解有光敏剂的溶液进行第二混合；所述溶解有光敏剂的溶液以DMF为溶剂，溶解有光敏剂、HATU和HOAt；所述光敏剂、HATU和HOAt的当量比优选0.5-1.5:0.5-1.5:0.5-1.5。在第二混合前，溶解有连接臂光敏剂的溶液中加入DIEA活化氨基酸。
优选地，步骤1)中所述连接臂和所述靶向PD-L1蛋白的多肽用量比为：1-5：1。
在一种优选的实施方式中，步骤2)中所述第一偶联剂和光敏剂的用量比为：1：1-4。
本发明对于第二偶联的条件没有特别限制，优选第二偶联的条件包括：偶联温度为：5-30℃；偶联时间为8-14h。
本发明还提供一种抗肿瘤药物，活性成分本发明所述的光敏剂嵌合体；优选所述肿瘤为以PD-L1为靶点的肿瘤，进一步优选为乳腺癌、肺癌、结直肠癌、食管癌或胰腺癌中的任意一种；更进一步优选为乳腺癌或食管癌。
本发明提供所述的光敏剂嵌合体在制备抗肿瘤药物中的应用；优选所述肿瘤为以PD-L1为靶点的肿瘤，进一步优选为乳腺癌、肺癌、结直肠癌、食管癌或胰腺癌中的任意一种；进一步优选为乳腺癌或食管癌。
本发明将光敏剂嵌合体与肿瘤进行孵育，继而对肿瘤区域进行光照，肿瘤细胞表面上的PD-L1蛋白发生降解，产生针对于PD-1/PD-L1通路的免疫检查点阻断(ICB)治疗，进而可以调节肿瘤的免疫抑制微环境并恢复细胞毒性T淋巴细胞(CTL)的功能，再结合光动力疗法PDT实现联合治疗，最终实现在临床实践中对多种肿瘤的治疗作用。
附图说明
附图1为实施例1制备得到的靶向PD-L1蛋白的多肽(PPA)LC-MS质谱图。
附图2为应用等离子共振技术测定实施例1制备得到的靶向PD-L1蛋白的多肽(PPA)和蛋白hPD-L的结合能力图。
附图3为实施例1制备得到的光敏剂嵌合体(PPA-VPF)同分异构体的LC-MS谱图(分子式:C118H152N26O30)。
附图4为实施例1制备得到的光敏剂嵌合体(PPA-VPF)与维替泊芬(VPF)的紫外吸收谱图(a:溶剂为PBS；b:溶剂为PBS/乙腈＝1:1)。
附图5为实施例1制备得到的光敏剂嵌合体荧光光谱(a:溶剂为PBS；b:溶剂为PBS/乙腈＝1:1)。
附图6为实施例1制备得到的光敏剂嵌合体在红光照射后用捕获剂Tempo捕获后的EPR谱图(a:溶剂为PBS；b:溶剂为PBS/乙腈＝1:1)。
附图7为应用等离子共振技术测定实施例1制备得到的光敏剂嵌合体和蛋白hPD-L的结合能力图。
附图8为不同浓度下实施例1制备得到的光敏剂嵌合体对活细胞表面的PD-L1蛋白的选择性降解图。
附图9为活细胞表面的PD-L1蛋白激光共聚焦成像结果(蓝色代表细胞核；绿色代表PD-L1蛋白；红色代表VPF或PPA-VPF)。
附图10为经PBS，PPA，VPF和PPA-VPF处理后，PD-L1蛋白在活细胞表面的流式结果。
附图11为a)光降解靶向嵌合体PPA-VPF经660nm激光以100mW/cm2的光照射(5min×2)后的细胞毒性；b)不同细胞中加入实施例1制备得到的光敏剂嵌合体PPA-VPF或VPF避光孵育12h后的流式细胞术摄取荧光强度结果。
附图12为不同时间点下光敏剂VPF和实施例1制备得到的光敏剂嵌合体PPA-VPF的活体成像图。
附图13为光敏剂VPF(30min)和实施例1制备得到的光敏剂嵌合体PPA-VPF(24h)的主要脏器和肿瘤部位的离体荧光成像图。
附图14双侧移植4T1瘤的小鼠中(a)光照处理端的肿瘤的生长曲线；(b)体重变化；(c)(d)光照处理端的肿瘤照片和肿瘤抑制率(TGI)。
附图15为各组小鼠肿瘤组织切片HE染色；
附图16双侧移植4T1瘤的小鼠中(a)未光照处理端的肿瘤的生长曲线；(b)(c)未光照处理端的肿瘤照片和肿瘤抑制率(TGI)；
附图17为a)4T1瘤CD3+CD8+细胞流式代表图及比例总结；b)4T1瘤CD8+IFN-γ+淋巴细胞流式代表图及比例总结。
具体实施方式
如无特殊说明，本发明对所用原料的来源没有特殊要求，采用本领域技术人员所熟知的市售商品即可。
下面将结合本发明中的实施例，对本发明中的技术方案进行清楚、完整地描述。
制备例1
(1)靶向多肽的合成及筛选
(a)基于Fmoc的固相多肽合成
向多肽合成管中称取一定量负载有Fmoc-NH2的树脂(Fmoc loading:0.37mmol/g,总量按Fmoc计算约为5μmol)，肽段的合成以DMF作为溶剂，在多肽合成管中完成偶联。Fmoc保护基的脱除使用20％哌啶的DMF溶液处理两次，每次时间为5min；使用HATU/HOBT(1:1)为偶联试剂，通过DIEA活化羧基实现氨基酸的偶联，每次偶联时间为20min，在大位阻氨基酸(e.g.Pro、Val、Ile、Arg、Thr)之后进行的偶联一般缩合两次以保证连接效率，多肽序列见下表1。
表1靶向多肽PPA系列筛选序列

用于多肽合成的αN-Fmoc或αN-Boc保护的非天然d型氨基酸包括：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,Fmoc-d-Phe-OH。
(b)多肽的纯化及液相色谱条件
完成多肽固相合成后，将洗涤干净并除去溶剂的干燥树脂置于多肽合成管内，加入一定量的TFA/TIPS/H2O(95:2.5:2.5，-8ml/0.05mmol树脂)混合溶液并于摇床上处理2-3h实现多肽从树脂上的裂解以及多肽侧链保护基的脱除。之后收集反应液体并使用氮气流将其吹干，得到的固体用冷乙醚洗涤。最后使用H2O/MeCN混合溶液将固体溶解，经滤膜过滤得到澄清溶液，用于后续的分析和分离纯化。
使用高效液相色谱进行多肽产物的分析及分离，所用流动相为添加0.05％ TFA的H2O(流动相A)以及添加0.04％ TFA的MeCN(流动相B)。分析及分离采用梯度洗脱，所示梯度为MeCN百分比含量。其中PPA的LC-MS表征数据如附图1所示。
(c)亲和力测定及筛选
hPD-L1蛋白与PPA系列多肽之间的亲和力通过表面等离子共振(SPR，Biacore 8k，GE Healthcare)进行测量。商业购买的hPD-L1在10mM醋酸钠缓冲液(pH 4.5，GE Healthcare)中稀释，最终得到40μg/mL的蛋白质浓度。使用标准胺偶联试剂盒(GE Healthcare)，通过伯胺基团将稀释的hPD-L1共价固定到新传感器芯片(S系列传感器芯片CM5，GE Healthcare)中。通过连接将目标hPD-L1的最终固定水平为9000RU。使用PBS-P缓冲液(含2.7mM KCl、137mM NaCl和0.05％表面活性剂P20的10mM磷酸盐缓冲液，最终pH 7.4，GE Healthcare)流速30μL/min进行测量。为了获得动力学和亲和力分析的数据，将PPA系列多肽溶解在PBS-P运行缓冲液中并设置浓度梯度，5种浓度梯度以上并保证最终的亲和力KD值落在浓度梯度范围内。结果如表2所示。通过比较KD值，PPA多肽与PD-L1蛋白亲和力最强，所以最终我们使用PPA多肽为母肽，开展后续的产品制备。所得PPA的5种浓度梯度以上并保证最终的亲和力KD值落在浓度梯度范围内，结果如附图2和表2所示。
表2 PPA系列多肽与PD-L1蛋白亲和力
实施例1
(1)光敏剂嵌合体的制备
(a)交联剂和维替泊芬的偶联
Fmoc保护基使用20％哌啶的DMF溶液脱除(5min×2)，之后用DMF(×3)、DCM(×3)、DMF(×3)洗涤。将连接臂Fmoc-NH-(PEG)x-CH2CH2-COOH(1.2equiv.)(n＝2、5、10)以及Fmoc-NH-(CH2)6-COOH、HATU(1.2equiv.)和HOAt(1.2equiv.)溶于3mL DMF，加入DIEA(2.4equiv.)活化氨基酸后将该混合溶液加入到装有制备例1制备PPA多肽的合成树脂的多肽合成管中，在摇床上反应过夜至偶联完成。继续使用20％哌啶的DMF溶液脱除(5min×2)连接臂上的Fmoc保护基，之后用DMF(×3)、DCM(×3)、DMF(×3)洗涤。将维替泊芬Verteporfin(1.2equiv.)、HATU(1.2equiv.)和HOAt(1.2equiv.)溶于3mL DMF，加入DIEA(2.4equiv.)活化氨基酸后将该混合溶液加入到制备1制备PPA的多肽合成管中，在摇床上反应过夜至偶联完成，此过程全程避光操作。
(b)嵌合体制备后的处理
完成多肽固相合成后，将洗涤干净并除去溶剂的干燥树脂置于多肽合成管内，加入一定量的TFA/TIPS/H2O(95:2.5:2.5，-8ml/0.05mmol树脂)混合溶液并于摇床上处理2-3h实现多肽从树脂上的裂解以及多肽侧链保护基的脱除。之后收集反应液体并使用氮气流将其吹干，得到的固体用冷乙醚洗涤。最后使用H2O/MeCN混合溶液将固体溶解，经滤膜过滤得到澄清溶液，用于后续的分析和分离纯化。
(c)液相色谱分析及分离条件
使用高效液相色谱进行多肽产物的分析及分离，得到光敏剂嵌合体(PPA-VDF，PPA-VPF-1，PPA-VPF-2，PPA-VPF-3)。所用流动相为添加0.05％ TFA的H2O(流动相A)以及添加0.04％ TFA的MeCN(流动相B)。分析及分离采用梯度洗脱，所示梯度为MeCN百分比含量。
(d)亲和力测定及筛选
hPD-L1蛋白与光敏剂嵌合体之间的亲和力通过表面等离子共振(SPR，Biacore 8k，GE Healthcare)进行测量。商业购买的hPD-L1在10mM醋酸钠缓冲液(pH 4.5，GE Healthcare)中稀释，最终得到40μg/mL的蛋白质浓度。使用标准胺偶联试剂盒(GE Healthcare)，通过伯胺基团将稀释的hPD-L1共价固定到新传感器芯片(S系列传感器芯片CM5，GE Healthcare)中。通过连接将目标hPD-L1的最终固定水平为9000RU。使用PBS-P缓冲液(含2.7mM KCl、137mM NaCl和0.05％表面活性剂P20的10mM磷酸盐缓冲液，最终pH 7.4，GE Healthcare)流速30μL/min进行测量。为了获得动力学和亲和力分析的数据，将PPA-VDF，PPA-VPF-1，PPA-VPF-2，PPA-VPF-3溶解在PBS-P运行缓冲液中并设置浓度梯度，5种浓度梯度以上并保证最终的亲和力KD值落在浓度梯度范围内，结果如表3所示。
表3不同连接臂制备得到PPA-VPF与hPD-L1蛋白的亲和力测试结果
选取连接臂Fmoc-NH-PEGn-CH2CH2-COOH(1.2equiv.)(n＝2)制备得到的PPA-VPF进行后续测试。
(2)产品表征参数：
LC-MS表征数据：
利用LC-MS表征条件：Agilent C18分析色谱柱，洗脱梯度为30min内流动相B从30％均匀变化至90％。
PPA-VPF:保留时间为18.68min和20.06min。ESI-MS：Calculate for C118H152N26O30:2414.67Da和2414.67Da(average isotopes)(m/z),实际得到的分子量最高峰为[M+3H]3+：805.71Da和805.43Da。结果如附图3所示。
(3)紫外表征图谱
将PPA-VPF溶解在PBS或50％ACN(PBS:乙腈＝1:1)的混合溶剂中(浓度为20μM)，再分别测定其在两种溶剂中的紫外吸收谱，其中以光敏剂维替泊芬作为对照。结果如附图4所示。在PBS溶液中，PPA-VPF的紫外吸收略低于维替泊芬。而在50％ ACN混合溶剂中，PPA-VPF的紫外吸收谱图与维替泊芬本身基本无差异，原因可能是不同溶剂中的溶解性差异。
(4)荧光表征图谱
将PPA-VPF溶解在PBS或50％ACN(PBS:乙腈＝1:1)混合溶剂中(浓度为20μM)，分别测定其在两种溶剂中的荧光发射光谱，以光敏剂维替泊芬作为对照。结果如附图5所示。 PPA-VPF的最大发射波长与维替泊芬本身一致。在不同溶剂中所展示的荧光强度与上述的紫外结果较为一致，即PBS中PPA-VPF略低于VPF，而50％ACN溶液中，两者强度相当，说明的确是溶解团聚问题导致的光谱强度的变化降低，但基本光谱性质不发生改变。
(5)EPR图谱
PPA-VPF以及维替泊芬VPF在被光激发后产生的ROS被自旋捕获剂4-羟基-2,2,6,6-四甲基哌啶(4-OH-TEMP，200mM)所捕获，经X波段Bruker A200光谱仪所检测，产生的ROS种类为单线态氧。检测单线态氧时，在照射前，向PPA-VPF或维替泊芬溶液(20μM)中加入终浓度为200mM的4-OH-TEMP。经300W氙弧灯(CEAULIGHT)的660nm带通滤波器(-1.5mW/cm2)照射规定时间后，迅速将30μL等分试样吸入玻璃毛细管，并至EPR谐振器内。其中所用溶剂为PBS或50％ACN(PBS:乙腈＝1:1)。用于EPR检测的典型设置为：扫描范围，100g；扫描时间60s；微波功率19.23mw；调制幅度1g；调制频率，100kHz。结果如附图6。
(6)亲和力测定
PPA-VPF亲和力测试结果如图7所示。
(7)蛋白降解实验
蛋白质靶向光降解解采用westernblot进行分析。为了在活细胞中进行分析，将A549细胞接种到6孔板上。孵育24小时后，用光降解靶向靶向嵌合体PPA-VPF，维替泊芬本身、纯多肽或DMSO作为对照在37℃下处理细胞8小时，然后用300W氙弧灯(600nm带通滤波器，1.5mW/cm2)照射细胞。细胞在含有蛋白酶-磷酸酶抑制剂混合物(#87786，Thermo Fisher Scientific)的RIPA裂解缓冲液(P0013B，Beyotime Biotechnology)中在4℃下裂解30分钟，并通过细胞刮片收集。将收获的总蛋白质在12％聚丙烯酰胺凝胶中通过SDS-PAGE分离，然后转移到PVDF膜上(#1620177，Bio-Rad)。膜与5％脱脂牛奶一起孵育1小时，并在4℃下过夜，主要抗体如下：PD-L1(ab125066，兔子，1:1000，Abcam)、GPX1(ab108427，兔子，1:1000，Abcam)、ACSL4(ab155282，兔子，1:1000，Abcam)和β_肌动蛋白(ab8226，兔子，1:1000，Abcam)。在室温下与相应的HRP结合二级抗体(#7076，小鼠，1:2000；#7074，兔，1:2000，细胞信号)孵育2小时后，用增强化学发光试剂盒(P0018FM，Beyotime Biotechnology)检测印迹。实验结果如图8所示。实验结果表明，光降解靶向嵌合体PV-1，PV-2在细胞层面可以成功的实现PD-L1的靶向的选择性降解。
(8)细胞水平PD-L1蛋白降解验证实验
1)细胞水平的免疫荧光成像实验
将MDA-MB-231细胞接种在玻璃底24孔板上，密度为每孔5×104个细胞。将细胞附着到圆形盖玻片的表面后，将其分为四组，分别为PBS，纯多肽PPA，光降解靶向嵌合体PPA-VPF以及光敏剂维替泊芬VPF，孵育4小时后，用300W氙弧灯(CEAULIGHT)和660nm带通滤波器(-1.5mW/cm2)照射5min×2。继续孵育1h，弃去培养基，将细胞在室温下用4％多聚甲醛中固定10分钟。除去孔中的液体，用PBS缓冲液清洗细胞共三次，每次1分钟。使用山羊血清(5％)作为封闭溶液，孵育1小时，以减少背景荧光干扰。弃去封闭溶液，重复上述洗涤步骤。将连有Alexa Fluor 488的结合PD-L1抗体在PBS缓冲液中的5％山羊血清中稀释25倍，室温培养细胞1.5小时。弃去一抗，用PBS缓冲液洗涤细胞三次，然后用DAPI在室温下染核4分钟。使用激光扫描共焦显微镜拍照，实验结果如附图9所示。结果显示，纯多肽PPA组中的PD-L1蛋白荧光强度与对照组无差别，均较为完整。光敏剂VPF组经激光照射后，有少数细胞呈现一定的变圆情况，荧光强度略低于对照组，说明VPF本身造成的细胞损伤会部分无靶向的破坏细胞表面的蛋白。而在光降解靶向嵌合体PPA-VPF组中，细胞表面上的绿色荧光强度大面积降低，说明细胞表面的PD-L1蛋白被大范围降解，而细胞形态是更加完整正常，说明PPA-VPF能选择性降解细胞表面上的PD-L1蛋白。
2)流式细胞术检测细胞表面PD-L1蛋白降解情况
将MDA-MB-231细胞以每孔5×104个细胞的密度接种在24孔板中，培养过夜。设置组别，同上(即PBS组，PPA组，VPF组和PPA-VPF组)，给予药物处理4h后，进行光照(689nm，5min×2)，之后在避光条件下继续培养细胞1h，弃去培养基，加入抗PD-L1蛋白的流式抗体PEanti-mouseCD274(biolegend，#124307)处理细胞30min，收集细胞，进行流式细胞术分析。结果如附图10所示，其中阴性对照组(即Blank组)代表未向表达PD-L1蛋白的MDA-MB-231细胞中添加流式抗体，目的是为了排除细胞本身荧光。PBS组代表加入抗体但没有加药物处理的细胞。经PPA或VPF处理的细胞，荧光强度值与PBS组几乎无差异，说明这两种药物的处理并不会影响细胞表面的PD-L1蛋白含量。但令惊喜的是，经PPA-VPF处理的细胞荧光强度则显著下降，并呈现出一定的浓度依赖性，这结果和激光共聚焦实验一致，表明所设计的PPA-VPF可以靶向选择性降解癌细胞表面的PD-L1蛋白。
(9)光降解靶向嵌合体PPA-VPF抗肿瘤活性研究
1)采用CCK-8法进行检测，以人三阴性乳腺癌细胞(即MDA-MB-231细胞，高表达PD-L1蛋白)，人乳腺癌细胞(MCF-7细胞，低表达PD-L1蛋白)以及正常人胚肾细胞293T作为模型细胞，将其种植在96孔板中，于培养箱中继续待细胞贴壁，加入光降解靶向嵌合体PPA-VPF，孵育4小时后，进行光照处理(具体条件设置同上)；样品孵育24小时后，弃去原培养基，PBS洗三次，加入10％的CCK-8溶液(CCK-8溶解于培养基中)于培养箱中孵育2小时，利用Biotek酶标仪测试孔板中的吸光度(480nm)并计算细胞存活率，实验结果如附图11a所示。结果显示，在高表达PD-L1细胞MDA-MB-231细胞中PPA-VPF的细胞毒性最强，而在低表达PD-L1细胞MCF-7细胞毒性较低，在正常细胞中的毒性则可忽略，这说明，细胞毒性和细胞种受体蛋白PD-L1的含量有关。
2)用流式细胞术检测细胞摄取PPA-VPF的情况
将1)种提到了MDA-MB-231(高表达PD-L1蛋白)和MCF-7细胞(低表达PD-L1蛋白)以每孔5×103个细胞的密度接种在24孔板中，培养过夜。随后加入PPA-VPF或VPF孵育8小时，收集细胞并进行流式细胞术分析。得到实验结果如附图11b所示。流式结果中，MDA-MB-231细胞摄取PPA-VPF的量明显高于MCF-7细胞，并呈现一定的浓度依赖，而VPF的摄取两种细胞并没有显著差异，再结合附图11a结果，说明无PD-L1受体的细胞不会结合PPA-VPF，进而不会产生光敏剂的毒性，而PPA-VPF会与高表达PD-L1受体的细胞结合，光照后，降解细胞表面的PD-L1蛋白的同时，产生毒性ROS，最终导致细胞死亡。
(10)光降解靶向嵌合体PPA-VPF在体内抗肿瘤效果评价
在进行了一系列体外评价的基础上，进一步考察在BALB/c小鼠中荷瘤4T1细胞，经静脉注射PPA-VPF后肿瘤治疗的靶向性，以及光动力疗法和免疫疗法的联合治疗的应用，目的为考察这种联合疗法对小鼠原位乳腺癌的疗效及安全性。
1)4T1荷瘤小鼠的模型建立
购买BALB/c小鼠，待其适应环境一周后，制备用于小鼠背部皮下接种的乳腺癌4T1细胞悬液。每只小鼠于左侧背部接种5×105个细胞。
2)PPA-VPF体内分布实验
接种4T1细胞，每日观察小鼠肿瘤生长情况，待其肿瘤体积增长至200mm3左右，将其随机分为2组，每组重复3只，分别经尾静脉注射100μL的光敏剂VPF(加入助溶剂PEG)或光降解靶向嵌合体PPA-VPF，保证避光饲养，其中VPF组在给药后5、10、15、20、30、60和120min，PPA-VPF组则选择2、4、8、12、24、36、48h，对小鼠进行气体麻醉，随后使用小动物活体成像仪(IVIS SPECTRUM)进行拍照，结果如附图12。结果表面，光敏剂VPF在体内代谢十分迅速，给药10min内迅速分布全身，半小时左右肿瘤部位富集基本到达高峰，但富集时间较短，2h后已经基本离开肿瘤部位；而PPA-VPF注射12h后依旧在肿瘤部位有较强富集，24h后逐渐聚集到肿瘤部位，其他部位含量则逐渐降低。说明光降解靶向嵌合体PPA-VPF较光敏剂本身VPF相比，能稳定的通过EPR效应有效聚集在肿瘤部位，且可长效循环。根据上述结果，综合了肿瘤部位荧光强度较高而其余部位荧光强度较低的时间点，选定VPF 30min和PPA-VPF 24h，处死解剖小鼠，取出离体组织和肿瘤，继续考察药物的离体组织分布。结果如附图13，在所选时间点下PPA-VPF主要分布位置为肿瘤，而心脏、肝脏、脾脏、肺脏和肾脏的分布较少，而VPF组除肿瘤分布外，也有较多分布在肝脏处，说明在所选时间点下PPA-VPF能有效地富集在肿瘤，而其余组织器官较少，综合上述实验结果，选定最佳的光动力疗法时间和避免其他器官的毒副作用，决定采用静脉注射VPF 30min、PPA-VPF 24h后开始进行相应的光照小鼠处理。
(11)小鼠体内的抗肿瘤活性研究
选择双侧肿瘤模型实验方法开展体内抗肿瘤研究。将4T1细胞(5×105)皮下注射至BALB/c小鼠右侧，产生原发肿瘤用于后续的光照处理；再将1×105个4T1细胞悬液继续经皮下注射到小鼠的左侧，形成远端肿瘤。当原发肿瘤体积达到约100mm3时，将小鼠随机分为4组，每组4只，分别静脉注射PBS，纯多肽PPA，光敏剂VPF(加入助溶剂PEG)和PPA-VPF，其中PPA-VPF的剂量为2mg/kg，其他组按照与实验组等物质的量进行换算。VPF组在注射后30min，PPA-VPF组在注射后24h经660nm激光(500mW/cm2，8分钟)照射原发肿瘤。治疗频率为每2天一次。每日记录小鼠的重量变化和双侧肿瘤的体积增长情况。肿瘤体积计算公式为：V＝d×l2/2(其中：V代表肿瘤的体积，d代表肿瘤的长径，l为肿瘤的短径)。在第11天对小鼠实施安乐死，以进行后续的病理学和免疫组化评估。结果如附图14a-d所示，与对照组相比，经PPA-VPF处理的小鼠在激光照射治疗后，表现出明显的肿瘤生长抑制(P<0.001)，光敏剂VPF组的小鼠也呈现出一定的肿瘤生长抑制，但弱于PPA-VPF组，纯多肽PPA处理的小鼠无任何抑制现象。说明PPA-VPF的抑制肿瘤效果优于单独的光敏剂，说明免疫治疗联合PDT治疗可以有效抑制小鼠原位乳腺肿瘤的生长。其中小鼠体重变化稳定，无统计学差异(P>0.05)，证明这种治疗对对小鼠的生存无明显影响。
(12)肿瘤组织切片的HE染色
利用HE切片染色对小鼠的肿瘤组织进行病理学检验。使用4％多聚甲醛溶液对离体肿瘤组织进行固定，24h后使用梯度酒精去除组织块中的水份，最后将组织块放置于二甲苯中透明。将处理好的组织块经石蜡浸透和包埋后，采用石蜡切片机制成石蜡切片，使用二甲苯脱蜡后，以苏木素和伊红染料对组织切片进行染色，采用全自动数字切片扫描系统进行拍照，结果如附图15所示。经光照处理后，PBS组和纯多肽PPA组中的肿瘤切片没有显示出明显的组织学变化，VPF组肿瘤组织则部分被破坏，而经PPA-VPF处理的肿瘤组织细胞核皱缩，细胞严重损坏，数量明显减少。
(13)免疫疗法对小鼠肿瘤生长影响
为了进一步验证降解PD-L1蛋白以及联合光动力疗法对远端肿瘤的免疫治疗作用，继续监测了远端肿瘤的体积(附图16a)。与PBS组相比，PPA-VPF治疗组的远端肿瘤增殖最为缓慢。实验结束后，继续对远端肿瘤进行解剖和称重(附图16b,c)。与原发肿瘤结果类似，PPA-VPF治疗组显示出最好的肿瘤抑制效果，该组远处肿瘤的体积最小、抑瘤率最高。上述结果均证实，设计的光降解靶向嵌合体PPA-VPF在协同光动力和PD-1/PD-L1的免疫治疗中可以激发较好的抗肿瘤免疫反应。
(14)肿瘤免疫治疗的流式检测实验
1)肿瘤浸润T淋巴细胞检测
将4T1细胞(5×105)皮下注射到BALB/c小鼠的左侧。当肿瘤体积达到300mm3左右后，分组，即PBS组、PPA组、VPF组、PPA-VPF组，尾静脉注射，操作同上。VPF组和PPA-VPF组小鼠经注射后30min和24h以500mW/cm2的强度660nm激光照射8分钟。避光饲养，给药后第三天，将小鼠实施安乐死，解剖其脾脏和肿瘤组织，加入胶原酶(400U/mL)、DNase1(100μg/mL)和透明质酸酶(0.04U/mL)消化。随后将混合物通过75μm尼龙细胞过滤器。样品用PBS(含2％FBS)洗涤三次，然后以1×106个细胞/mL的密度重新悬浮进行流式细胞术分析。按照说明书添加抗体(抗小鼠CD3-FITC、抗小鼠CD4-APC、抗小鼠CD8-PE-Cy7和抗小鼠IFN-γ-PE，BioLegend)，其中在加入抗小鼠IFN-γ-PE抗体前需破膜处理。在流式细胞仪上分析细胞，并使用FlowJo 10分析数据。结果如附图17a所示，与PBS组相比，纯多肽PPA组中CTL细胞占比和光敏剂VPF处理组分别为19.6±1.8％和37.5±2.7％。而PPA-VPF组在肿瘤组织中则表现出最高的CTL比例，为54.9±1.45％，比PBS组高了6.17倍。继续评估的IFN-γ的表达情况(如附图17b)也类似，单独多肽PPA组或单独的光动力疗法的治疗(即VPF组)能够部分提高CD8+/IFN-γ+的T细胞占比，分别为24.8±2.1％和32.6±2.7％，但协同治疗组PPA-VPF则诱导最高的CD8+/IFN-γ+细胞占比为54.7±6.0％。说明基于光降解PD-1/PD-L1中的PD-L1蛋白所诱发的光动和免疫检查点阻断的联合治疗能够产生比单一治疗更强的免疫反应，在增强抗肿瘤治疗方面具有极大的潜力。

Claims (10)

  1. 一种靶向PD-L1蛋白的光敏剂嵌合体，所述嵌合体为X-R-G结构，其中X为光敏剂单元，R为连接臂，G为靶向PD-L1蛋白的多肽单元，其特征在于，
    所述光敏剂为卟啉类光敏剂，所述光敏剂的激发光波长>550nm；550nm波长以后的最大吸收波长处的摩尔吸光系数>1000M-1cm-1；三线态的量子产率>0.01；
    所述连接臂选自以下结构式：
    其中所述连接臂的氨基侧与所述光敏剂单元相连，所述连接臂的羧基侧与所述多肽单元相连，n为0-50的整数；
    所述多肽与h-PD-L1的结合常数<20μM。
  2. 根据权利要求1所述的光敏剂嵌合体，其中所述连接臂的结构式为：n为0-50的整数；优选n为0-10的整数。
  3. 根据权利要求1所述的光敏剂嵌合体，其中所述光敏剂选自维替泊芬、二氢卟吩或脱镁叶绿酸a中的至少一种；优选为维替泊芬。
  4. 根据权利要求1所述的光敏剂嵌合体，其中所述多肽为(a)-(c)中任意一项所述的多肽：
    (a)具有氨基酸序列(1)-(11)的多肽；
    (b)氨基酸序列(1)-(11)经过取代、缺失或增加1-3个氨基酸残基且能与PD-L1的亲和力<20μM的多肽；
    (c)在(a)或(b)的多肽的羧基末端连接有酯基或酰胺基的多肽；
    氨基酸序列(1)-(11)如下所示：
    dNdYdSdKdPdTdDdRdQdYdHdF(1)；
    dNdYdSdKdPdTdDdR(2)；
    dNdYdSdKdPdTdDdRdQ(3)；
    dNdYdSdKdPdTdDdRdQdY(4)；
    dKdPdTdDdRdQdYdHdF(5)；
    dPdTdDdRdQdYdHdF(6)；
    dNdYdSdKdPdTdDdRdQdYdH(7)；
    dSdKdPdTdDdRdQdYdHdF(8)；
    dYdSdKdPdTdDdRdQdYdHdF(9)；
    dKdPdTdDdR(10)；
    dSdKdPdTdDdR(11)；
    优选为dNdYdSdKdPdTdDdRdQdYdHdF(1)；
    dNdYdSdKdPdTdDdR(2)；
    dNdYdSdKdPdTdDdRdQ(3)；
    dNdYdSdKdPdTdDdRdQdY(4)；
    dKdPdTdDdRdQdYdHdF(5)；
    进一步优选为dNdYdSdKdPdTdDdRdQdYdHdF(1)。
  5. 根据权利要求1-4任意一项所述的光敏剂嵌合体，其特征在于，所述光敏剂嵌合体的化学结构式如式I或式II所示；
    所述式I和式II中n独立为1-10的整数；G为靶向PD-L1蛋白的多肽单元，所述多肽的氨基酸序列为：dNdYdSdKdPdTdDdRdQdYdHdF(1)。
  6. 权利要求1-5任意一项所述的光敏剂嵌合体的制备方法，包括以下步骤：
    1)将连接剂和靶向PD-L1蛋白的多肽第一混合，进行第一偶联，去除未偶联物，得到第一偶联物；
    2)将所述第一偶联物和光敏剂第二混合，在避光条件下进行第二偶联，去除未偶联物，得到光敏剂嵌合体。
  7. 根据权利要求6所述的制备方法，其特征在于，步骤1)中所述连接剂和所述多肽的用量比为：1-5：1。
  8. 根据权利要求6所述的衍生化的光敏剂嵌合体的制备方法，步骤2)中所述第一偶联物和所述光敏剂的用量比为：1：1-4。
  9. 一种抗肿瘤药物，活性成分包括权利要求1-5任意一项所述的光敏剂嵌合体；优选所述肿瘤为以PD-L1为靶点的肿瘤；进一步优选为乳腺癌、肺癌、结直肠癌、食管癌或胰腺癌中的任意一种；更进一步优选为乳腺癌或食管癌癌。
  10. 权利要求1-5任意一项所述的光敏剂嵌合体在制备抗肿瘤药物中的应用；优选所述肿瘤为以PD-L1为靶点的肿瘤；进一步优选为乳腺癌、肺癌、结直肠癌、食管癌或胰腺癌的任意一种；更进一步优选为乳腺癌或食管癌。
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