WO2025218256A1 - 一种肿瘤特异性溶酶体靶向嵌合体及其在肿瘤治疗中的应用 - Google Patents

一种肿瘤特异性溶酶体靶向嵌合体及其在肿瘤治疗中的应用

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Publication number
WO2025218256A1
WO2025218256A1 PCT/CN2024/143027 CN2024143027W WO2025218256A1 WO 2025218256 A1 WO2025218256 A1 WO 2025218256A1 CN 2024143027 W CN2024143027 W CN 2024143027W WO 2025218256 A1 WO2025218256 A1 WO 2025218256A1
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tumor
lysosome
sequence
switch element
response region
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French (fr)
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刘庄
李雨晴
杨宇
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Suzhou University
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Suzhou University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention belongs to the technical field of immunotherapy, and in particular relates to a tumor-specific lysosome-targeted chimera and its application in tumor treatment.
  • Immune checkpoint blockade therapy is the main means of tumor immunotherapy.
  • a variety of antibody drugs have been used clinically, but the actual clinical response rate of existing immune checkpoint blockade therapies is low, and there is a need to develop efficient and safe immune checkpoint blockade drugs.
  • PD-L1 downregulation can also be achieved by silencing PD-L1-related genes, such as using siRNA or the gene-editing tool CRISPR.
  • siRNA siRNA
  • CRISPR gene-editing tool
  • these gene-editing strategies still face challenges in delivery efficiency, hindering their widespread application.
  • existing technologies primarily focus on the development of novel molecular tools and their application at the cellular level, but have yet to be implemented and promoted at the biological level or in practical applications. The existing technologies are difficult to translate due to their high process complexity, poor tissue selectivity, significant safety concerns related to on-target off-tumor toxicity in vivo, and high production costs. This is why the present invention is proposed.
  • the present invention provides a tumor-specific lysosome-targeting chimera (LYTAC), which contains a specific response switch.
  • the switch is combined with a lysosomal shuttle receptor nucleic acid aptamer and can be well responsive to the characteristic physical and chemical properties of the tumor microenvironment, such as low pH, high potassium ion concentration, and high ATP.
  • the switch element undergoes a configurational change and dissociates from the lysosomal shuttle receptor aptamer, thereby achieving the degradation of tumor cell membrane proteins at the tumor site, ensuring that membrane proteins on the surface of normal cells of the body are not degraded in the physiological environment.
  • the first objective of the present invention is to provide a responsive switch element, which is essentially a customized DNA sequence with multiple interconnected DNA functional regions.
  • the responsive switch element binds to a lysosomal shuttle receptor nucleic acid aptamer when unresponsive and dissociates from the lysosomal shuttle receptor nucleic acid aptamer when responsive.
  • the responsive switch element is capable of responding to at least pH, ions (such as potassium ions), and biomolecules (such as ATP and membrane proteins).
  • the responsive switch element has at least the following structure:
  • the responsive switch element When used to respond to pH, the responsive switch element is provided with a response region, a connecting sequence, a response region, a base complementary region, a response region, a connecting sequence, and a response region that are interconnected;
  • the response region contains a number of cytosines
  • the responsive switch element When used to respond to ions, the responsive switch element is provided with a first response region, a connecting sequence, a base complementary region, a connecting sequence, and a second response region connected to each other;
  • the first response region and the second response region are obtained by splitting a sequence containing the core sequence 5'-GGGTAGGGCGGGTTGGG-3';
  • the responsive switch element When used to respond to biomolecules, the responsive switch element is provided with a third response region, a base complementary region, and a fourth response region connected to each other;
  • the K + (potassium ion) responsive switch element comprises a sequence shown in any one of SEQ ID NOs. 16-29.
  • the switch element is an ATP-responsive switch element.
  • the third and fourth response regions further contain an extension sequence for stabilizing the first and/or second sequences.
  • the extension sequence is 1-3 bases, wherein each base is independently selected from G, C, A, and T.
  • the extension sequence is located at the 3' end of the first sequence and the 5' end of the second sequence, and the two extension sequence bases are complementary.
  • the ATP nucleic acid aptamer contains at least the following sequence: ACCTGGGGGAGTATGCGGAGGAAGGT.
  • the nucleic acid aptamer is split into: ACCTGGGGGAGTA (core sequence of the third response region) and TGCGGAGGAAGGT (core sequence of the fourth response region).
  • the ATP-responsive switch element has at least five consecutive bases participating in complementary pairing starting from the 5' end, or the ATP-responsive switch element has at least five consecutive bases participating in complementary pairing starting from the 3' end, and the target nucleic acid aptamer has at least five consecutive bases participating in complementary pairing.
  • the ATP-responsive switch element has a maximum of 40 bases involved in complementary pairing, preferably 5 to 20.
  • the ATP-responsive switch element comprises a sequence shown in any one of SEQ ID NO.30-52.
  • the switch element is a switch element responsive to other biomolecules, such as a membrane protein responsive element.
  • the switch element can also realize biomarker response, wherein the biomarker used is a membrane protein, and the membrane protein includes but is not limited to any one of HER2 (human epidermal growth factor receptor-2), EpCAM (epithelial cell adhesion molecule), Nucleolin (nucleolin), PTK7 (protein tyrosine pseudokinase 7), VEGF (vascular endothelial growth factor), and EGFR (epidermal growth factor receptor) membrane proteins.
  • HER2 human epidermal growth factor receptor-2
  • EpCAM epidermal growth factor receptor 2
  • Nucleolin nucleolin
  • PTK7 protein tyrosine pseudokinase 7
  • VEGF vascular endothelial growth factor
  • EGFR epidermal growth factor receptor
  • the ATP-responsive switch element splits the ATP nucleic acid aptamer into two oligonucleotide chains, so that they are distributed on both sides of the base complementary pairing region, one end contains the third response region ACCTGGGGGAGTA sequence, and the other end contains the fourth response region TGCGGAGGAAGGT sequence.
  • the third response and the fourth response sequence interact with ATP to form a hairpin structure, so that the ATP nucleic acid aptamer is detached from the switch element.
  • the cation-independent mannose 6-phosphate receptor (CI-M6PR) nucleic acid aptamer When applied to the LYTAC structure, the cation-independent mannose 6-phosphate receptor (CI-M6PR) nucleic acid aptamer is released for binding to CI-M6PR and restoring the protein function of LYTAC, thereby achieving tumor-specific targeted protein degradation.
  • CI-M6PR mannose 6-phosphate receptor
  • the second object of the present invention is to provide a combination of a specific response target, comprising the responsive switch element and a nucleic acid aptamer targeting a lysosomal shuttle receptor, thereby constructing a tumor-specific lysosomal targeting chimera. Therefore:
  • the third object of the present invention is to provide a tumor-specific lysosome-targeting chimera, comprising at least one lysosomal shuttle receptor recognition structure (which may contain at least one nucleic acid aptamer targeting a lysosomal shuttle receptor) and at least one responsive switch element, wherein the lysosomal shuttle receptor recognition structure can bind to a base complementary region on the responsive switch element.
  • the tumor-specific lysosome-targeting chimera also includes at least one target protein recognition structure, which is interconnected with a lysosomal shuttle receptor recognition structure.
  • the tumor-specific lysosomal-targeting chimera is a lysosomal-targeting chimera that is responsive to the tumor microenvironment.
  • the switch element binds to the lysosomal shuttle receptor aptamer and dissociates after a specific response in the tumor microenvironment, thereby exposing the lysosomal shuttle receptor aptamer and providing a binding site for the LYTAC and lysosomal shuttle receptor, causing degradation of the target protein and further inducing tumor cell apoptosis.
  • target protein recognition structure and the lysosomal shuttle receptor recognition structure are connected via a connecting molecule.
  • the target protein recognition structure is capable of binding to the target protein.
  • the target protein recognition structure is selected from one or more of antibodies, nanobodies, nucleic acid aptamers, and polypeptides.
  • the tumor-specific lysosome targeting chimera includes the following structure: a target protein recognition structure and a lysosomal shuttle receptor recognition structure connected by a connecting molecule, and the molar ratio of the lysosomal shuttle receptor recognition structure to the target protein recognition structure is n ⁇ 1; when n>1, it has a multivalent effect.
  • the linker molecule is a molecule that is non-degradable in the tumor microenvironment.
  • the lysosomal shuttle receptor recognition structure or the target protein recognition structure is connected to the connecting molecule via a flexible segment or directly connected to the connecting molecule.
  • the flexible segment is selected from one or more of polyethylene glycol, carbon chain, and base chain.
  • the linker molecule contains at least one of the following structures: an amide bond, a triazole ring, a thioether bond, and a disulfide bond.
  • linker molecule contains at least one of the following structures:
  • the target protein recognition structure is selected from commercially available or homemade monoclonal or polyclonal antibodies.
  • the target protein recognition structure includes PD-L1 antibody, HER2 antibody, platelet-derived growth factor antibody, protein tyrosine kinase 7 antibody, mesenchymal epithelial transition factor antibody, G protein-coupled receptor 65 antibody, CD47 antibody, CD71 antibody, EGFR antibody, and ABCG2 antibody.
  • the lysosomal shuttle receptor recognition structure includes one or more of CI-M6PR nucleic acid aptamer, ASGPR nucleic acid aptamer, transferrin receptor nucleic acid aptamer, integrin nucleic acid aptamer, cytokine receptor nucleic acid aptamer, and poly-oligonucleotide.
  • the CI-M6PR nucleic acid aptamer is selected from one or more of the following sequences:
  • the ASGPR nucleic acid aptamer is the following sequence:
  • the base complementary region in Table 1 is replaced with a sequence complementary to a sequence at the 5' end of the above nucleic acid aptamer.
  • the length of the base complementary region is preferably 5-20 nt, such as CGCCTCCCC (corresponding to SEQ ID NO. 54).
  • the structure of the multivalent lysosomal targeting chimera includes but is not limited to any one of the following structures:
  • X is the target protein recognition structure
  • R1 and R2 are flexible chain segments
  • Y is the lysosomal shuttle receptor recognition structure.
  • R1 and R2 are independently selected from PEG, a carbon chain or a base chain.
  • the present invention also provides the use of the above-mentioned responsive switch element or tumor-specific lysosome-targeting chimera in the preparation of a protein degrader or an immunotherapy drug.
  • the immunotherapy drug can be prepared into immune checkpoint blocking drugs, etc. for anti-tumor use.
  • the present invention also provides a multivalent lysosome-targeting chimera and its application in protein degradation and immunotherapy.
  • the specific scheme is as follows:
  • Immune checkpoint blockade therapy is the main means of tumor immunotherapy.
  • a variety of antibody drugs have been used clinically, but the actual clinical response rate of existing immune checkpoint blockade therapies is low, and there is a need to develop efficient and safe immune checkpoint blockade drugs.
  • PDL1 downregulation can also be achieved by silencing PDL1-related genes, such as using siRNA or the gene editing tool CRISPR to silence the PDL1 gene.
  • siRNA or the gene editing tool CRISPR to silence the PDL1 gene.
  • these gene editing strategies still face challenges such as low delivery efficiency, which has prevented their widespread application.
  • the protein degradation targeting chimera (PROTAC) structure has received widespread attention.
  • the two ends of the PROTAC molecule recognize the target protein and the E3 ubiquitin ligase respectively, achieving the connection between the target protein and the E3 ubiquitin ligase.
  • the target protein is labeled with a ubiquitin tag, which is then recognized by the proteasome in the cell and further degraded by the proteasome. Because the ubiquitination process mainly occurs inside the cell, this strategy has limited effect on the degradation of membrane proteins.
  • LYTAC lysosome-targeting chimera
  • One end of the chimera targets the target protein through an antibody, while the other end targets the cation-independent mannose 6-phosphate receptor (CI-M6PR) through an oligosaccharide peptide group.
  • CI-M6PR cation-independent mannose 6-phosphate receptor
  • This lysosome-targeting chimera can form a ternary complex with the target membrane protein and the lysosomal shuttling receptor. Through the lysosomal shuttling activity of CI-M6PR, it further mediates the target membrane protein to the lysosome for complete degradation, thereby downregulating the target protein level on the cell membrane.
  • the present invention further provides a multivalent lysosome targeting chimera, comprising any one of the following structures: a target protein recognition structure and a lysosomal shuttle receptor recognition structure connected by a connecting molecule, and the molar ratio n of the lysosomal shuttle receptor recognition structure to the target protein recognition structure is greater than 1.
  • the lysosomal shuttle receptor recognition structure or the target protein recognition structure is connected to the connecting molecule via a flexible segment or directly.
  • the linker molecule is a molecule that is non-degradable in the tumor microenvironment.
  • the flexible segment is selected from one or more of polyethylene glycol, carbon chain, and base chain.
  • the linker molecule contains at least one of the following structures: an amide bond, a triazole ring, a thioether bond, and a disulfide bond.
  • linker molecule contains at least one of the following structures:
  • the target protein recognition structure includes PDL1 antibody, HER2 antibody, platelet-derived growth factor antibody, protein tyrosine kinase 7 antibody, mesenchymal epithelial transition factor antibody, G protein-coupled receptor 65 antibody, CD47 antibody, CD71 antibody, EGFR antibody, ABCG2 antibody, etc.
  • the target protein recognition structure is selected from commercially available or homemade monoclonal or polyclonal antibodies.
  • the lysosomal shuttle receptor recognition structure includes one or more of CI-M6PR nucleic acid aptamer, ASGPR nucleic acid aptamer, transferrin receptor (CD71) nucleic acid aptamer, and poly-oligonucleotide.
  • sequence of the CI-M6PR nucleic acid aptamer is selected from any one of SEQ ID NO.53-56; the sequence of the ASGPR nucleic acid aptamer is SEQ ID NO.57.
  • the structure of the multivalent lysosomal targeting chimera includes but is not limited to any one of the following structures:
  • X is the target protein recognition structure
  • R1 and R2 are flexible chain segments
  • Y is the lysosomal shuttle receptor recognition structure.
  • R1 and R2 are independently selected from PEG, a carbon chain or a base chain.
  • the present invention also provides the use of the multivalent lysosome-targeting chimera in the preparation of protein degradation agents or immunotherapy drugs.
  • the immunotherapy drug can be prepared into immune checkpoint blocking drugs, etc. for anti-tumor use.
  • the specific lysosome-targeting chimera of the present invention can bind to the target membrane protein on the surface of tumor cells, utilize the lysosomal degradation pathway to degrade the target protein, further leading to tumor apoptosis.
  • the released tumor-specific antigens further induce an anti-tumor immune response, thereby achieving efficient anti-tumor immunotherapy.
  • the specific lysosome-targeting chimera described in the present invention has the characteristics of responding to the tumor microenvironment or specific markers, and can only degrade membrane proteins in tumor tissue, overcoming the problem of current LYTAC lacking tumor selectivity. Therefore, through intravenous administration, in the normal blood circulation, the tumor-specific lysosome-targeting chimera will not cause degradation of normal cell surface proteins. This reduces the impact on the patient's normal cells and avoids on-target off-tumor toxicity, thus having better safety. It also helps to improve the efficiency of immune cells in killing tumors and enhance the effectiveness of anti-tumor immunotherapy.
  • the specific lysosome-targeting chimera described in the present invention can combine multiple lysosomal shuttle receptor aptamers with antibodies, has a multivalent effect, and increases the efficiency of the lysosome-targeting chimera in simultaneously binding to the target protein and the lysosomal shuttle receptor, thereby increasing the probability of degrading the target protein and increasing the efficiency, further increasing the probability of causing tumor cell apoptosis, improving the efficacy of LYTAC technology in tumor treatment, and increasing the application potential of LYTAC technology in tumor treatment.
  • the specific lysosome-targeting chimera of the present invention has the advantages of controllable structure, simple preparation, and high yield, which is convenient for mass production and clinical application.
  • the lysosomal targeting chimera of the present invention has a multivalent effect, which makes the target protein recognition structure connected to multiple lysosomal shuttle receptor recognition structures, and the efficiency of binding to lysosomal shuttle receptors is higher, thereby improving the degradation efficiency of the target protein. It can also act continuously at the target site, achieving the beneficial effect of low dose and high efficacy.
  • the efficient induction of cell surface PDL1 degradation will lead to tumor cell apoptosis, release tumor antigens, stimulate immune cells to enhance anti-tumor immune response, and integrate the continuous reaction of immunogenic death-antigen presentation and immune cell activation-immune checkpoint blockade effect, thereby achieving a coherent anti-tumor immune response and amplifying the therapeutic effect.
  • FIG1 is a schematic diagram showing the response of a switching element according to the present invention.
  • FIG2 is a statistical graph showing the changes in fluorescence signals of different tumor-specific lysosome-targeting chimeras as pH values change.
  • FIG3 is a statistical graph of FAM fluorescence signal intensity on the cell surface in different groups.
  • FIG4 is a Western blotting diagram of PD-L1 protein in cells of different groups.
  • FIG5 shows the PD-L1 content of cells in different groups in Example 5.
  • FIG6 is a statistical diagram of the apoptosis ratio of DC cells in different groups in Example 6.
  • Figure 7 is a statistical graph of the PD-L1 content in CD45+ and CD11+ cells in the peripheral blood of mice in Example 7.
  • FIG8 is a statistical graph showing weight changes of tumor-bearing mice in Example 8.
  • FIG9 is a statistical diagram of the number of CD45+ and CD11+ cells in the peripheral blood of mice in Example 8.
  • FIG10 is a protein immunoblot image of PDL1 protein on the surface of tumor cells.
  • FIG11 shows the changes in tumor volume in mice in Example 9.
  • FIG12 shows the weight changes of mice in Example 9.
  • FIG13 is the results of immunoblotting of PD-L1 protein in PC-3 prostate cancer cell lines after different treatments in Example 10. The relative contents of PD-L1 in other groups were calculated based on the control.
  • FIG14 shows the results of immunoblotting of PD-L1 protein in Hela cervical cancer cell lines after different treatments in Example 10.
  • the relative contents of PD-L1 in other groups were calculated based on the control.
  • FIG15 is a tumor growth curve of the mouse 4T1 subcutaneous tumor model in Example 11.
  • FIG16 is a statistical graph showing changes in body weight of mice in the mouse 4T1 subcutaneous tumor model in Example 11.
  • Figure 17 is a statistical graph of the fluorescence signal intensity of the tumor-specific lysosome-targeting chimeras whose switching elements are sequences SEQ ID NO. 42 to 45 at different ATP concentrations in Example 12.
  • Figure 18 is a statistical graph of the fluorescence signal intensity of the tumor-specific lysosome-targeting chimeras whose switching elements are sequences SEQ ID NO. 49 to 52 at different ATP concentrations in Example 12.
  • FIG19 is a Western blot of PDL1 detected on B19F10 tumor cells in different samples as described in Example B1;
  • FIG20 is a Western blot of PDL1 in tumor cells from different groups in Example B2;
  • FIG21 is a statistical graph of PDL1 content in cells after 24 hours of incubation at different sample concentrations in Example B3;
  • FIG22 is a statistical graph showing the relative content of cell membrane PDL1 protein at different time points during the co-incubation of samples and cells in Example B4;
  • FIG23 is a statistical graph showing the percentage of apoptosis in different groups in Example B5;
  • FIG24 is a statistical diagram of the relative activity of cells in different groups in Example B6;
  • FIG25 is a fluorescence signal image of cell surface calreticulin captured by laser confocal microscopy in Example B7;
  • FIG26 is a statistical graph showing ATP release in different groups of cells in Example B7;
  • FIG27 is a statistical graph showing the maturation ratios of dendritic cells in different groups in Example B8;
  • FIG28 is a graph showing the tumor growth curves of mice in different groups in Example C1.
  • A represents adenine
  • T represents thymine
  • C represents cytosine
  • G represents guanine
  • switch element refers to a structure that typically shields lysosomal shuttling receptor aptamers, but can release this shielding function under specific circumstances. Specific circumstances include a specific pH range, the presence of potassium ions, ATP (adenosine triphosphate), or other biomarkers.
  • the tumor-specific lysosome-targeting chimera used in the examples is based on the structure of the above-mentioned non-tumor-specific lysosome-targeting chimera, with Y combined with a switch element, and the switch element is Seq.1.
  • a tumor-specific lysosome targeting chimera comprises a target protein recognition structure, a lysosome shuttle receptor recognition structure and a specific response switch structure switch element.
  • FIG. 1 is a schematic diagram of a tumor-specific lysosomal targeting chimera, using the CI-M6PR nucleic acid aptamer as an example.
  • the switch element has a complementary pairing region with the M6PR.
  • the pH-responsive switch element is represented by the regular font portion, the italicized bold portion is the linker sequence, and the underlined portion is the base-pairing region complementary to the aptamer.
  • the pH-responsive tumor-specific lysosome-targeting chimera can open the switch structure switch element under specific pH conditions (slightly acidic conditions of the tumor microenvironment), thereby removing the shielding effect on the lysosomal shuttle receptor aptamer sequence. That is, under specific pH conditions, the lysosomal shuttle receptor aptamer can bind to the lysosomal shuttle receptor, allowing LYTAC to cause lysosomal targeted degradation of the target protein.
  • the 5' end of the M6PR aptamer is labeled with the fluorescent group tetramethylrhodamine (TAMRA), and the switch element is labeled with the quencher group BHQ-2.
  • TAMRA fluorescent group tetramethylrhodamine
  • BHQ-2 quencher group
  • the pH T value By analyzing the pH T value, the midpoint of the structural transition of the switch element, the pH T value can be used to assess the suitability of the designed switch element for this system, ensuring that the switch element undergoes sensitive conformational transitions between the physiological pH of 7.4 and the tumor microenvironment pH of 6.5.
  • the optimal pH T value should be between pH 6.5 and 7.4, sufficient to inhibit or relieve the inhibition of the CI-M6PR aptamer.
  • Figure 2 shows a statistical plot of fluorescence signal changes for different samples as pH values change.
  • the fluorescence intensity is approximately 90% suppressed, indicating that the switch element essentially completely inhibits aptamer activity.
  • the fluorescence intensity gradually increases.
  • the fluorescence signal intensity reaches equilibrium, releasing approximately 80% of the signal. This indicates that Seq. 1 fully opens the switch element in a slightly acidic environment with a pH of no greater than 6.6, releasing the barrier effect on the lysosomal shuttling receptor aptamer.
  • Example 2.1 is the best sequence for constructing the switching element.
  • a tumor-specific lysosome-targeting chimera with a switch structure was further designed to degrade specific target proteins in tumor tissues.
  • the tumor-specific lysosome-targeting chimera described in this application can open the switch element and expose the lysosomal shuttle receptor aptamer as the pH conditions change, further demonstrating that the tumor-specific lysosome-targeting chimera described in this application can perform the function of carrying target proteins to lysosomes under the slightly acidic microenvironment conditions of the tumor site, and cannot affect other cells expressing the same protein in a normal physiological environment.
  • the pH conditions were changed to detect the binding ability between the nucleic acid aptamer end of the tumor-specific lysosome-targeting chimera and the lysosomal shuttle protein on the surface of tumor cells.
  • the lysosomal shuttling receptor aptamer (a nucleic acid aptamer that recognizes and binds to CI-M6PR on the cell surface) of a tumor-specific lysosomal targeting chimera was labeled with a FAM fluorophore at the 5' end.
  • LYTAC was then co-incubated with B16F10 cells, and the pH of the incubation system was adjusted. Note that in vitro, without additional IFN- ⁇ stimulation, PD-L1 expression on the surface of B16F10 cells is low, so the antibody end of the LYTAC is essentially incapable of cell binding.
  • a FAM signal is detected on the cell surface, it indicates that only the nucleic acid aptamer end has bound to CI-M6PR. Therefore, quantitative measurement of the fluorescence signal intensity on the cell surface by flow cytometry can determine whether the switch structure on the LYTAC inhibits/disinhibits CI-M6PR binding activity under different pH conditions, thereby preventing/enabling B16F10 cell binding. Furthermore, after binding to different samples, B16F10 cells were washed three times with PBS before analysis. FAM fluorescence was largely unaffected by pH.
  • FIG. 3 is a statistical graph of the FAM fluorescence signal intensity on the cell surface in different groups.
  • the results show that in Comparative Example 3.1, there are only tumor cells, and almost no fluorescence signal can be detected, indicating that the background fluorescence signal content in the system is extremely low.
  • the FAM fluorescence signal in Example 3.2 is weak, close to that in Comparative Example 3.1, indicating that under the condition of pH 7.4, the lysosome targeting chimera does not bind to the tumor cells, that is, under the condition of pH 7.4, the switch element of the tumor-specific lysosome targeting chimera has a shielding effect on the targeting group.
  • Example 3.3 the pH is around 6.5, simulating the pH conditions in the tumor microenvironment of the solid tumor site.
  • the tumor-specific lysosome targeting chimera successfully binds to the tumor cells, indicating that the tumor-specific lysosome targeting chimera is in a slightly acidic environment, the switch element is opened, the binding site is exposed, and it can bind to the tumor cells, and the binding efficiency is relatively high.
  • the free CI-M6PR nucleic acid aptamer was able to successfully bind to tumor cells at a pH of 7.4, serving as a positive control group.
  • Example 3.3 This demonstrates that the switch element in Example 3.3 can fully relieve the inhibitory effect in the slightly acidic environment of the tumor, restoring the interaction between the tumor-specific lysosome-targeting chimera and CI-M6PR, thereby further promoting the degradation of the target protein.
  • the combined experimental data from several groups in this example demonstrate that the tumor-specific lysosome-targeting chimera described herein can bind to tumor microenvironments at specific pH values, but not at the neutral pH of general body fluids, thereby avoiding binding to other cells carrying the same receptor during circulation and improving utilization efficiency.
  • Tumor cells were co-incubated with different samples, the pH was adjusted, and the PD-L1 protein content on the tumor cell surface was measured. Lower protein levels indicate degradation of PD-L1 protein on the tumor cell surface, indicating that the lysosome-targeting chimera is effective.
  • the tumor cells used were the B16F10 cell line.
  • the experimental groups are as follows:
  • Example 4.3 under pH 7.4, the non-tumor-specific lysosome-targeted chimera (compared with the tumor-specific lysosome-targeted chimera, the only difference is that it does not contain a switch element, the same below) degraded about 57% of PD-L1, indicating that the lysosome-targeted chimera can function to degrade PD-L1.
  • the switch element can basically completely inhibit/restore the activity of PD-L1 degradation.
  • Tumor-specific lysosome-targeting chimeras do not cause damage to immune cells under normal physiological conditions.
  • DC cells dendritic cells
  • the samples were processed and detected by Western blotting.
  • the grouping and pH conditions were as follows:
  • Example 5 shows the PD-L1 levels of cells in different groups in Example 5.
  • the sample lacks tumor specificity, resulting in partial degradation of PD-L1 protein on the surface of DC cells at a pH of 7.4.
  • the specific lysosome-targeting chimera of the present invention did not affect PD-L1 protein levels on the surface of DC cells.
  • the side effects of degrading PD-L1 on the surface of DC cells will be further investigated.
  • FIG. 6 is a statistical graph of the proportion of DC cell apoptosis in different groups in Example 6.
  • the results show that in Comparative Examples 6.1 and 6.2, the number of apoptotic cells is relatively small, basically maintained at around 10%.
  • Example 6.3 DC cells produced significant apoptosis (over 60%), indicating that the non-tumor-specific lysosome-targeted chimera will affect the activity of DC cells and may bring side effects.
  • Example 6.4 avoided the side effects on DC cells very well, and there was no significant difference compared to Comparative Example 6.1, indicating that the tumor-specific targeted chimera of the present invention can reverse the negative effects on DC cells, thereby being more beneficial to the overall anti-tumor treatment.
  • the extent to which the lysosome-targeted chimera affects DC cells in actual use needs further exploration and verification.
  • mice were intravenously infused with sample solutions at 20 or 40 ⁇ g/mouse (in 200 ⁇ L of normal saline). The sample doses are shown in the table below.
  • peripheral blood was collected from the mice.
  • the PD-L1 content on the surface of CD45+ and CD11+ cells in the peripheral blood of different groups was detected by flow cytometry, and statistical graphs were prepared.
  • the tumor-specific lysosome-targeting chimera of the present invention does not reduce the level of PD-L1 on the surface of DCs in the peripheral blood of mice, that is, the tumor-specific lysosome-targeting chimera does not cause degradation of PD-L1 on the surface of DCs in peripheral blood, thereby avoiding the risk of potential side effects on DCs in peripheral blood and having better safety.
  • Example 8 Changes in the number of CD45+CD11+ cells in mouse peripheral blood
  • Tumor-bearing mice were intravenously infused with sample solutions at a dose of 20 or 40 ⁇ g/mouse (in 200 ⁇ L of normal saline) for one injection.
  • the sample dosages are shown in the table below.
  • CD45+CD11c+ cells i.e. DC cells in peripheral blood
  • CD45+ cells i.e. total immune cells
  • the non-tumor-specific lysosome-targeted chimera had a fluctuating effect on peripheral blood DC cells.
  • DC basically recovered to the same level as in Example 8.1 after 7 days of administration.
  • Example 8.3 after administration of a higher dose, DC in the peripheral blood of mice did not fully recover within a week and decreased significantly by about 50%.
  • the tumor-specific lysosome-targeted chimera did not significantly affect peripheral blood DC, proving that the activity of the tumor-specific lysosome-targeted chimera in degrading peripheral blood DC-PD-L1 under physiological conditions can be basically completely inhibited. It can be inferred that the tumor-specific lysosome-targeted chimera has higher utilization and safety.
  • FIG. 10 shows the protein immunoblot of PD-L1 protein on the surface of tumor cells, and its content relative to the blank control group was statistically analyzed. The results showed that lysosome-targeted chimeras, regardless of whether they are tumor-specific or not, can achieve the effect of degrading the PD-L1 content on the surface of tumor cells. Moreover, tumor-specific lysosome-targeted chimeras have a superior degradation effect compared to non-specific ones.
  • Equal doses of tumor-specific lysosome-targeted chimeras degrade more PD-L1 on the cell membrane surface of the tumor site than lysosome-targeted chimeras, resulting in better utilization, higher efficiency, and higher safety.
  • the therapeutic effect of the tumor-specific lysosome-targeting chimera described in this application on solid tumors was verified in a mouse subcutaneous tumor model.
  • the groups are as follows:
  • mice were inoculated on the backs of mice to establish a subcutaneous melanoma model in mice.
  • the first intravenous infusion of the sample was designated as day 0.
  • the average tumor volume of the mice was approximately 50 cubic millimeters.
  • the above-mentioned doses of sample were injected through the tail vein on days 0, 3, 6, and 9, respectively.
  • the tumor volume was recorded every two days, and the statistical results are shown in Figure 11.
  • the mouse weight was recorded every two days, and the statistical results are shown in Figure 12.
  • the tumor-specific lysosome-targeted chimera has a tumor therapeutic effect.
  • increasing the dose of the sample at the tumor site helps to increase the therapeutic effect.
  • the tumor-specific lysosome-targeted chimera reduces binding at non-tumor sites, thereby increasing the effective amount of molecules in the entire system that exert tumor-killing effects, thereby further increasing the therapeutic effect and being more conducive to clinical application.
  • mice did not decrease significantly during the entire treatment period, indicating that the sample is relatively safe.
  • the weight gain of mice in Example 9.3 was relatively slow, which may be due to the nonspecific adsorption of lysosome-targeted chimeras to immune cells, which had a slight impact on the health of mice.
  • Specific lysosome-targeted chimeras have better safety.
  • the experimental method is the same as that of Example 4, except that the cells used are human Hela cervical cancer cell line and PC-3 prostate cancer cell line.
  • Figure 13 shows the relative content of PD-L1 in cells of the PC-3 prostate cancer cell line after different treatments.
  • Figure 14 shows the relative content of PD-L1 in cells of the Hela cervical cancer cell line after different treatments.
  • the results show that the tumor-specific lysosome-targeted chimera described in the present application can degrade PD-L1 on the surface of tumor cells in an environment with a pH value of approximately 6.5, and does not have the function of degrading PD-L1 in an environment with a pH of approximately 7.4.
  • the tumor-specific lysosome-targeted chimera described in the present application has the characteristic of pH response, and the pH environment in general solid tumors is weakly acidic, indicating that the present invention has tissue selectivity and will not degrade the same antigen on the surface of other cells in other environments in the body, thereby having better safety.
  • the experimental method was the same as that of Example 9, except that the tumor model was different.
  • a mouse 4T1 subcutaneous tumor model was established in the early stage, and the mice were randomly divided into groups and then given medication.
  • Figure 15 shows the tumor growth curve of mice in Example 11
  • Figure 16 shows the weight change curve of mice in Example 11.
  • the results show that the tumor volume growth rate of mice treated with a 40 ⁇ g dose of the tumor-specific lysosome-targeting chimera was significantly reduced compared to other groups (** represents p ⁇ 0.01), indicating that the tumor-specific lysosome-targeting chimera described in this application has a better therapeutic effect. It is also more effective than the same dose of non-specific lysosome-targeting chimera.
  • ATP-responsive, tumor-specific lysosomal targeting chimeras can activate the switch element in the switch structure under the action of ATP, removing the shielding effect on the lysosomal shuttle receptor aptamer sequence.
  • the lysosomal shuttle receptor aptamer can bind to the lysosomal shuttle receptor, allowing the LYTAC to trigger lysosomal targeted degradation of the target protein. Sequences were constructed and tested for ATP responsiveness.
  • the 5' end of the M6PR aptamer is labeled with the fluorescent group tetramethylrhodamine (TAMRA), and the switch element is labeled with the quencher group BHQ-2.
  • TAMRA fluorescent group tetramethylrhodamine
  • BHQ-2 quencher group
  • ATP levels in peripheral blood and healthy tissue interstitium are extremely low, ranging from 10 to 100 nM, while ATP levels in the tumor microenvironment are approximately 1 to 5 mM.
  • the ATP-responsive switch element can open within this concentration range and does not respond at the nM level, it indicates that the switch element meets the requirements for specific response.
  • Figure 17 is a statistical graph of the fluorescence signal intensity of tumor-specific lysosome-targeted chimeras whose switch elements are sequences SEQ ID NO. 42 to 45 at different ATP concentrations.
  • the results show that all four sequences can respond in an ATP environment at the mM concentration level, open the switch element, expose the nucleic acid aptamer and trigger fluorescence, and almost no switch element is triggered at lower concentrations, which means that these sequences meet the requirements of ATP-specific response switch elements and can achieve the function of lysosomal targeted degradation in an ATP concentration environment that is different from that in other normal tissues.
  • Figure 18 shows a statistical plot of the fluorescence signal intensity of tumor-specific lysosome-targeting chimeras whose switch elements are SEQ ID NOs. 49 to 52, respectively, at different ATP concentrations.
  • the results show that all four sequences are able to respond to ATP concentrations at the mM level, opening the switch element, exposing the aptamer, and triggering fluorescence. At lower concentrations, the switch element is almost completely untriggered.
  • SEQ ID NO. 49 as the switch element, exhibits the highest response efficiency. This demonstrates that these sequences meet the requirements for ATP-specific responsive switch elements and can achieve lysosomal-targeted degradation in tumor-specific ATP environments.
  • the present invention also provides a multivalent lysosomal targeting chimera (LYTAC), which contains a target protein recognition structure and a ligand structure targeting a lysosomal shuttle receptor, and one target protein recognition structure is connected to more than one ligand structure.
  • LYTAC multivalent lysosomal targeting chimera
  • Example A1 A multivalent lysosomal targeting chimera, comprising a target protein recognition structure and a lysosomal shuttle receptor recognition structure.
  • the specific structural composition is shown in the following table:
  • Example A2 Preparation of a multivalent lysosome-targeting chimera:
  • the amino group of the PDL1 antibody was replaced with azide to obtain an azide-modified PDL1 antibody;
  • the CI-M6PR nucleic acid aptamer was modified with DBCO to obtain a DBCO-modified CI-M6PR nucleic acid aptamer;
  • the azide-modified PDL1 antibody and the DBCO-modified CI-M6PR nucleic acid aptamer were linked via click chemistry.
  • the product is filtered using a 50 kD ultrafiltration centrifuge tube and washed with a neutral PBS buffer to fully remove excess bifunctional cross-linking agent NHS-PEG 4 -N 3 .
  • a multivalent lysosomal-targeting chimera By adjusting the feed ratio of the azide-modified PDL1 antibody and the DBCO-modified CI-M6PR nucleic acid aptamer, a multivalent lysosomal-targeting chimera can be obtained.
  • the feed amounts are shown in the following table:
  • the multivalent lysosome-targeting chimera is an antibody connected to multiple nucleic acid aptamers, which can increase the affinity (efficiency) of the antibody binding to the lysosomal shuttle protein after recognition and coordination, thereby improving the lysosomal targeting effect.
  • Example B1 Experimental study on the PDL1 degradation efficiency of lysosome-targeted chimeras.
  • 10 5 B19F10 tumor cells were incubated with 10 nM of the lysosome-targeting chimeras from Examples A1.1 to A1.4, and the PDL1 content on the tumor cells was detected using western blot. Additionally, increasing the dosage of the lysosome-targeting chimeras was used, and 10 5 tumor cells were incubated with 50 nM of the lysosome-targeting chimeras from Examples A1.1 to A1.4, and the PDL1 content in the tumor cells was also detected using western blot.
  • Figure 19 shows a Western blot of PDL1 in B19F10 tumor cells from different samples.
  • the depth of the bands represents the PDL1 content, with darker bands indicating higher PDL1 content and lighter bands indicating lower PDL1 content.
  • the lysosome-targeting chimeras of Examples A1.1 to A1.4 were all able to reduce PDL1 content in tumor cells, demonstrating that the lysosome-targeting chimeras of the present invention have the ability to downregulate PDL1 levels.
  • the efficiency of PDL1 degradation increases. Among them, the efficiency of PDL1 degradation in Example A1.1, Example A1.2 and Example A1.3 is more correlated with the dosage.
  • the lysosome-targeting chimera of Example A1.4 whose dosage increases, does not improve the degradation efficiency to a corresponding extent. It is speculated that the reason may be that too many nucleic acid aptamers are coupled to the antibody, which affects the recognition efficiency between the antibody and PDL1, thereby interfering with the binding of the lysosome-targeting chimera and the target protein, and thus affecting the efficiency of lysosome-targeted dissolution.
  • the lysosome-targeting chimera described in the present invention can cause a significant multivalent effect by increasing the number of nucleic acid aptamers linked to the antibody. Compared with linking a single nucleic acid aptamer, it significantly improves the efficiency of the lysosome-targeting chimera in degrading the target protein, can achieve downregulation of the target protein content at a lower dose, and enhance the effect of anti-tumor immunotherapy.
  • Example B2 By adding different inhibitors, it was verified that the pathway for degrading PDL1 by the lysosome-targeting chimera of the present invention is the lysosomal degradation pathway.
  • Example B2.2 B16F10 cells were co-incubated with the lysosome-targeting chimera of Example A1.2 and the MG132 proteasome inhibitor;
  • Example B2.3 B16F10 cells were co-incubated with the lysosome-targeting chimera of Example A1.2 and the lysosomal inhibitor Chloroquine.
  • Figure 20 is a Western blot of PDL1 in tumor cells from different groups in Example B2.
  • the results show that the addition of a proteasome inhibitor in Example B2.2 had no effect on the efficiency of the lysosome-targeted chimera in Example A1.2 in degrading PDL1 protein.
  • the addition of a lysosome inhibitor in Example B2.3 significantly interfered with the degradation of PDL1 protein by the lysosome-targeted chimera in Example A1.2, virtually preventing PDL1 degradation. This indicates that the lysosome-targeted chimera of the present invention degrades PDL1 via the lysosomal pathway rather than the proteasomal pathway.
  • Example B1 The method of Example B1 was followed, except that the multivalent lysosome-targeting chimera was from Example A1.2, with concentrations of 2 nM, 5 nM, 10 nM, 50 nM, and 100 nM, respectively. Each group of samples was co-incubated with 10 5 B16F10 cells for 24 hours.
  • Figure 21 is a statistical graph of PDL1 content after 24 hours of cell incubation at different sample concentrations in Example B3.
  • the abscissa represents the concentration of the multivalent lysosome-targeting chimera, and the ordinate represents the relative content of cell surface PDL1.
  • the results show that as the concentration of the multivalent lysosome-targeting chimera increases, more cell surface PDL1 protein is degraded within the same timeframe, indicating that a high concentration of the multivalent lysosome-targeting chimera is more efficient at degrading cell membrane surface PDL1 protein. This also demonstrates that the good uniformity of the multivalent lysosome-targeting chimera of the present invention ensures consistency in degradation efficiency and concentration.
  • Example B4 Experimental verification of the sustained effect of the lysosome-targeted chimera of the present invention in degrading PDL1.
  • Figure 22 is a statistical graph of the relative content of PDL1 protein on the cell membrane at different time points during the incubation process. The results show that within 12 hours, the content of PDL1 antibody was significantly downregulated. After about 12 hours, the PDL1 antibody was almost completely degraded.
  • the rate of downregulation of the PDL1 protein content on the membrane surface of the sample slowed down, and the relative content reached a very low level, indicating that the lysosome-targeting chimera of the present invention can achieve sustained downregulation of PDL1 antibody expression on the cell membrane surface and maintain the PDL1 content at a low level. That is, the lysosome-targeting chimera of the present invention can downregulate PDL1 antibody on the cell membrane surface of tumors in vivo for a long time, thereby achieving the effect of anti-tumor immunotherapy.
  • Example B5 The lysosome-targeting chimera of the present invention downregulates the level of PDL1 protein on the surface of tumor cells, which can cause cell apoptosis.
  • Example B5.4 Lysosomal targeting chimera of Example A1.2
  • Figure 23 is a statistical graph of the apoptosis ratio of different groups of cells in Example B5.
  • the results show that the lysosome-targeted chimera of the present invention can cause apoptosis of most tumor cells. That is, when the PDL1 protein on the cell membrane is degraded, it directly leads to apoptosis of tumor cells. This phenomenon has never been reported in the prior art. It is unexpected that the lysosome-targeted chimera of the present invention has the effect of inducing apoptosis of tumor cells.
  • the lysosome-targeted chimera with a multivalent effect of the present invention has a stronger targeted degradation efficiency than the lysosome-targeted chimera in the prior art. Therefore, in addition to having an immune checkpoint blocking effect, it can also cause spontaneous apoptosis of tumor cells.
  • the lysosome-targeted chimera of the present invention has an unexpected technical effect.
  • This example is used to explore the effective concentration of the multivalent lysosome-targeting chimera.
  • Figure 24 is a statistical graph of the relative activity of cells in different groups in Example B6.
  • the experimental results show that the relative survival rate of cells in the blank control group was 100%.
  • concentration of the multivalent targeting chimera sample increased, the killing effect of the multivalent lysosome-targeting chimera on tumor cells gradually increased over the same period of time.
  • the half-inhibitory concentration (IC50) of the lysosome-targeting chimera of the present invention was approximately 4.2nM. A smaller IC50 indicates a stronger ability to kill tumor cells, indicating that the lysosome-targeting chimera of the present invention has the effect of inducing tumor cell death.
  • the lysosome-targeting chimera described in the present invention has a multivalent effect, significantly improving the degradation efficiency of the PDL1 protein.
  • the degradation of large amounts of PDL1 leads to the death of tumor cells. Therefore, the lysosome-targeting chimera described in the present invention can not only completely remove PDL1, causing immune checkpoint blockade and enhancing the ability of immune cells to recognize and kill tumor cells, but also directly induce tumor cell apoptosis, expose tumor-associated antigens, and stimulate immune cells to enhance anti-tumor immune responses. This has multiple effects and significantly enhances the efficacy of tumor treatment.
  • Example B7 To demonstrate that the apoptosis of tumor cells induced by the lysosome-targeting chimera of the present invention can enhance the immunogenicity of tumor cells, this example was designed to demonstrate that after the action of the lysosome-targeting chimera, calreticulin on the surface of tumor cells increases.
  • Example B7.4 Co-incubation of the lysosome-targeting chimera of Example A1.2 with tumor cells
  • the treated cells in the different groups were labeled with calreticulin using a calreticulin detection kit according to the manufacturer's instructions.
  • the cells were then fixed with 4% paraformaldehyde and stained with Hoechst to label the nuclei.
  • the stained cells were then examined using a laser confocal microscope.
  • Figure 25 is a fluorescence signal image of cell surface calreticulin captured by laser confocal microscopy in Example B7.
  • FIG25 shows that the expression of calreticulin in the cells of Example B7.4 is increased, indicating that the lysosome-targeting chimera causes an increase in calreticulin on the surface of tumor cells, thereby helping immune cells to recognize tumor cells and enhancing the efficacy of anti-tumor immunotherapy.
  • ATP release is another indicator of the immunogenicity of tumor cells; the higher the ATP release, the stronger the immunogenicity.
  • Figure 26 is a statistical graph of ATP release from cells in different groups in Example B7.
  • the results show that the lysosome-targeted chimera of Example A1.2 can cause a significant increase in the amount of ATP released by tumor cells after acting on tumor cells.
  • the above-released ATP and exposed tectum proteins are both damage-associated molecular patterns (DAMPs), which are positively correlated with the immunogenicity of tumor cells, indicating that the lysosome-targeted chimera of the present invention can enhance the immunogenicity of tumor cells.
  • the enhanced immunogenicity of tumor cells will cause a strong anti-tumor immune response. Therefore, the lysosome-targeted chimera of the present invention can induce a strong anti-tumor immune response, thereby achieving the purpose of anti-tumor immunotherapy.
  • Example B8 The apoptotic cell products in Example 9 were used to stimulate the maturation of bone marrow-derived dendritic cells (BMDCs).
  • BMDCs bone marrow-derived dendritic cells
  • Comparative Example B8.1 The apoptotic products of Comparative Example B7.1 were co-incubated with bone marrow-derived dendritic cells
  • Comparative Example B8.2 The apoptotic products of Comparative Example B7.2 were co-incubated with bone marrow-derived dendritic cells
  • Example B8.3 Co-incubation of the apoptotic products of Example B7.4 with bone marrow-derived dendritic cells
  • Bone marrow cells were isolated from the femur and tibia of male C57BL/6 mice and cultured in a medium containing granulocyte macrophage colony-stimulating factor (GM-CSF, concentration 20 ng/mL) and interleukin-4 (IL-4, concentration 10 ng/mL). After 5 days of culture, a small amount of cells were collected and stained with CD11c antibody flow cytometry to test their maturity, confirming that the myeloid cells had differentiated into DCs. Simultaneously, B16F10 tumor cells were co-incubated with different samples (50 nM) for 48 hours. Tumor cells treated with different samples were then co-incubated with DCs in a 1:1 ratio for 3 days. Finally, DCs were collected and flow cytometry was used to measure the expression of CD80 and CD86 on DCs. CD80 and CD86 are key markers of DC maturation, and cells that are double-positive for both CD80 and CD86 are mature myeloid DCs.
  • GM-CSF
  • Bone marrow-derived dendritic cells are the most powerful antigen-presenting cells discovered to date, and they are the only antigen-presenting cells capable of significantly stimulating the proliferation of naive T cells. Therefore, dendritic cells are a key step in initiating adaptive T cell immune responses and play a crucial role in tumor immunotherapy. The maturity of dendritic cells influences the extent of subsequent anti-tumor immune responses; higher levels of maturity are expected to elicit stronger anti-tumor immune responses.
  • Figure 27 is a statistical graph of the maturation ratios of dendritic cells in different groups in Example B8. The results show that the apoptotic products of Example B7.4 can induce the maturation of bone marrow-derived dendritic cells, indicating that the lysosome-targeted chimera of the present invention can induce an anti-tumor immune response and achieve in vivo anti-tumor immunotherapy.
  • Example C1 Animal experiments demonstrate that the lysosome-targeting chimera of the present invention can induce anti-tumor immune responses and inhibit tumor growth and metastasis.
  • Tumor cells (about 5 ⁇ 10 5 B16F10 cells) were inoculated on the back of mice. When the tumors grew to 25 mm 3 , the mice were randomly divided into groups as follows:
  • Example C1.3 Tumor-bearing mice were treated with a mixture of PDL1 antibody and M6PR nucleic acid aptamer, with a dosage ratio of 1:6
  • Example C1.4 Treatment of tumor-bearing mice with the lysosome-targeted chimera of Example A1.2
  • Example C1.2 In Comparative Example C1.2, Example C1.3, and Example C1.4, 20 ⁇ g of PDL1 antibody or LYTAC of equal concentration (quantified by PDL1) was administered intravenously once every 3 days for a total of 4 times. The changes in tumor volume were recorded, and a tumor growth curve was prepared. The results are shown in Figure 28.
  • Figure 28 is a graph showing the tumor growth curves of mice in different groups in Example C1. The results show that the traditional immune checkpoint blockade therapy used in Comparative Example C1.2 does not inhibit tumor growth well.
  • Example C1.3 only the nucleic acid aptamer of the lysosome-targeting receptor is mixed with the PDL1 antibody, and its effect is no different from that of the immune checkpoint blockade therapy alone.
  • Example C1.4 the lysosome-targeting chimera described in the present invention is used for treatment, which has a significant effect of inhibiting tumor growth. This shows that the lysosome-targeting chimera described in the present invention has an application prospect of inducing anti-tumor treatment in living bodies, and has a good therapeutic effect.
  • the lysosome-targeting chimera described in the present invention can be administered intravenously, which is convenient and efficient.
  • Example D Study on multivalent lysosome-targeting chimeras linked to other molecules and their protein degradation efficiency.
  • Example A2 According to the preparation method of Example A2, a variety of multivalent lysosome-targeting chimeras were prepared, all of which had the effect of degrading target proteins, and the degradation efficiency was better than that of monovalent lysosome-targeting chimeras.
  • the detection equipment and methods used in the above examples include: BD FACSCalibur flow cytometer; Leica TCS SP8 confocal microscope; and Western Blot.
  • the above multivalent lysosomal targeting chimeras connect the target protein recognition structure to multiple lysosomal shuttle receptor recognition structures, resulting in higher efficiency in binding to lysosomal shuttle receptors, thereby improving the degradation efficiency of the target protein.
  • the multivalent lysosomal targeting chimeras of the present invention are prepared by covalent coupling reactions, with a simple synthesis method, high reaction efficiency, convenient purification operations, and low overall preparation costs.
  • the multivalent lysosome-targeting chimera of the present invention has good stability in the body, does not cause reactions that are detrimental to physiological activities, and due to its high recognition efficiency, can be better enriched at the tumor site, thereby improving safety in use.
  • the multivalent lysosomal targeting chimera of the present invention can act continuously at the target site, achieving the beneficial effect of low dose and high efficacy.
  • immune checkpoint blockade In addition to achieving the results of immune checkpoint blockade, it can also effectively induce the degradation of cell surface PDL1, leading to tumor cell apoptosis, releasing tumor antigens, and stimulating immune cells to enhance anti-tumor immune responses.
  • This combines the continuous reaction of immunogenic death-antigen presentation and immune cell activation-immune checkpoint blockade effects, thereby achieving a coherent anti-tumor immune response and amplifying the therapeutic effect.
  • numbers are used to describe the quantities of components and attributes. It should be understood that such numbers used in the description of the embodiments are modified in some examples by the modifiers "about,” “approximately,” or “substantially.” Unless otherwise specified, “about,” “approximately,” or “substantially” indicate that the numbers described are subject to ⁇ variations. Accordingly, in some embodiments, the numerical parameters used in the description and claims are approximate values, which may vary depending on the desired characteristics of individual embodiments.

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Abstract

提供能够特异性响应的开关元件及含有该响应开关的溶酶体靶向嵌合体，经验证仅在肿瘤微环境中能够特异性降解肿瘤细胞中含有的目标蛋白，而不影响机体正常细胞的功能，且制备步骤简便，可用于制备免疫检查点阻断药物，提高了肿瘤治疗效果，解决了现有技术的转化难题和在靶脱肿瘤毒性问题，有利于实现高效且安全的抗肿瘤免疫治疗。

Description

一种肿瘤特异性溶酶体靶向嵌合体及其在肿瘤治疗中的应用 技术领域
本发明属于免疫治疗技术领域，尤其涉及一种肿瘤特异性溶酶体靶向嵌合体及其在肿瘤治疗中的应用。
背景技术
免疫检查点阻断治疗是肿瘤免疫治疗的主要手段，已有多种抗体药物在临床上应用，但是现有的免疫检查点阻断治疗的实际临床响应率较低，需要开发高效且安全的免疫检查点阻断药物。
近年研究表明，程序性死亡受体配体1(PD-L1)除了具有免疫检查点的功能外，其跨膜结构的胞内部分还会广泛参与到肿瘤细胞的生长、增殖以及转移等相关信号通路中，促进肿瘤细胞的发生发展，因此单单通过抗体等手段阻断PD-L1的免疫检查点功能，仅能在一定程度上实现肿瘤免疫治疗的目标。目前有几种下调肿瘤细胞表面PD-L1表达水平的策略，例如基于PD-L1表达的信号通路，通过引入一些小分子抑制剂来减少PD-L1的表达，但这种策略仅能干扰其中一条表达通路，肿瘤细胞可能通过其他代偿性通路来增加PD-L1的表达，导致难以实现预期的PD-L1下调效果。除了干扰PD-L1表达的策略，还可以通过沉默PD-L1相关基因来实现PD-L1的下调，比如使用siRNA或者基因编辑工具CRISPR等等来实现PD-L1基因的沉默，但是这种涉及基因编辑的策略在实操上，仍然面临递送效率低等问题，导致其不能被广泛应用。另外，现有技术主要聚焦于新型分子工具的开发和在细胞层面的应用，在生物体层面和实际应用中尚未实践和推广，现有技术的难点在于工艺难度大、组织选择性差、生物体内具有在靶脱肿瘤毒性这一重要安全性隐患、生产成本高等方面，转化较困难。因此提出本发明。
发明内容
为解决上述问题，本发明提供了一种肿瘤特异性溶酶体靶向嵌合体(LYTAC)，其中含有特异性响应开关，所述开关与溶酶体穿梭受体核酸适体结合，能对肿瘤微环境的特征理化特性如低pH、高钾离子浓度和高ATP具有良好响应性，在肿瘤微环境中，开关元件发生构型变化，与溶酶体穿梭受体适体解离，从而实现肿瘤部位的肿瘤细胞膜蛋白降解，保证在生理环境中，机体正常细胞表面的膜蛋白不被降解。
本发明的第一个目的是提供一种响应型开关元件，本质上是一条定制的DNA序列，所述响应型开关元件上设有相互连接的多个DNA功能区域，在未响应时与溶酶体穿梭受体核酸适体结合，响应时与溶酶体穿梭受体核酸适体分离，至少能够响应pH、离子(如钾离子)、生物分子(如ATP、膜蛋白等)等。其至少具有以下结构：
Ⅰ.用于响应pH时，所述响应型开关元件上设有相互连接的响应区、连接序列、响应区、碱基互补区域、响应区、连接序列、响应区；
所述响应区含有若干胞嘧啶；
Ⅱ.用于响应离子时，所述响应型开关元件上设有相互连接的第一响应区、连接序列、碱基互补区域、连接序列、第二响应区；
所述第一响应区和第二响应区由含有核心序列5’-GGGTAGGGCGGGTTGGG-3’的序列拆分得到；
Ⅲ.用于响应生物分子时，所述响应型开关元件上设有相互连接的第三响应区、碱基互补区域、第四响应区；
将生物分子核酸适体拆分为第一序列和第二序列，所述第三响应区包括第一序列，第四响应区包括第二序列，且所述第三响应区和第四响应区可与生物分子形成发卡结构；
所述碱基互补区域用于与靶标核酸适体互补。
本发明的一个实施例中，所述开关元件为pH响应型开关元件。
进一步地，pH响应型开关元件的组成单元从5’端到3’端依次包括响应区、连接序列、响应区域、碱基互补配对区域、响应区域、连接序列、响应区域。
进一步地，所述响应区至少包含4-8个胞嘧啶，所述碱基互补区域与靶标核酸适体至少有任意5个连续的碱基参与互补配对。
进一步地，pH响应型开关元件从5’端开始至少有任意5个连续的碱基参与互补配对，或pH响应型开关元件从3’端开始至少有任意5个连续的碱基参与互补配对，所述靶标核酸适体至少有任意5个连续的碱基参与互补配对。
进一步地，pH响应型开关元件最多有40个碱基参与互补配对，优选为5～20。
进一步地，所述连接序列的核苷酸选自A和T中的任意一种或两种，所述连接序列的核苷酸的数量为1-7个。其中，T和A的数量对开关元件响应条件有细微影响，例如当连接序列为3个T即可恰好满足pH约为6.5的环境中打开开关元件时，进一步将T替换成A会降低发生构象转变的pH值，导致A的数量越多，则引起发生构象转变的pH值越低，所述开关原件中连接序列可以按照需要进行调整。
最优选地，pH响应型开关元件包含SEQ ID NO.1-15任一所示的序列。
本发明的一个实施例中，所述开关元件为钾离子响应型开关元件。
进一步地，K+(钾离子)响应型开关元件的组成单元从5’端到3’端依次包括第一响应区、连接序列、碱基互补配对区域、连接序列和第二响应区。
进一步地，所述第一响应区和第二响应区通过含有核心序列5’-GGGTAGGGCGGGTTGGG-3’的序列拆分得到，且拆分位点位于所述核心序列上，拆分后第一响应区和第二响应区上核心序列的G碱基数目比为1：1或者3：1。具体地：
第一响应区G数目：第二响应区G数目＝1：1或者3：1，
1：1拆分时，第一响应区序列为5’-GGGTTGGG-3’，第二响应区序列为5’-GGGTAGGG-3’，均包含6个G(即为1：1)；
3：1拆分时，第一响应区序列为5’-GGGTAGGGCGGG-3’，第二响应区序列为5’-GGG-3’，分别包含9个G和3个G(即为3：1)。
进一步地，所述连接序列的核苷酸选自A和T中的任意一种或两种，所述连接序列的核苷酸的数量为2-8个。优选地，所述连接序列为3～6个T。
进一步地，所述响应区至少包含4-8个胞嘧啶，所述碱基互补区域与靶标核酸适体至少有任意5个连续的碱基参与互补配对。
进一步地，K+(钾离子)响应型开关元件从5’端开始至少有任意5个连续的碱基参与互补配对，或K+(钾离子)响应型开关元件从3’端开始至少有任意5个连续的碱基参与互补配对，所述靶标核酸适体至少有任意5个连续的碱基参与互补配对。
进一步地，K+(钾离子)响应型开关元件最多有40个碱基参与互补配对，优选为5～20。
最优选地，K+(钾离子)响应型开关元件包含SEQ ID NO.16-29任一所示的序列。
本发明的一个实施例中，所述开关元件为ATP响应型开关元件。
进一步地，ATP响应型开关元件的组成单元从5’端到3’端依次包括第三响应区、碱基互补配对区域、第四响应区。
进一步地，所述第三响应区和第四响应区还含有用于稳定第一序列和/或第二序列的延长序列，所述延长序列为1-3个碱基，其中每个碱基独立地选自G、C、A、T，所述延长序列位于第一序列的3’端和第二序列的5’端，且两个延长序列碱基是互补配对的。。
进一步地，ATP核酸适体至少含有如下所示的序列：ACCTGGGGGAGTATGCGGAGGAAGGT，优选地，将该核酸适体拆分为：ACCTGGGGGAGTA(第三响应区的核心序列)，TGCGGAGGAAGGT(第四响应区的核心序列)。
进一步地，ATP响应型开关元件从5’端开始至少有任意5个连续的碱基参与互补配对，或ATP响应型开关元件从3’端开始至少有任意5个连续的碱基参与互补配对，所述靶标核酸适体至少有任意5个连续的碱基参与互补配对。
进一步地，ATP响应型开关元件最多有40个碱基参与互补配对，优选为5～20。
最优选地，ATP响应型开关元件包含SEQ ID NO.30-52任一所示的序列。
本发明的一个实施例中，所述开关元件为其他生物分子响应型开关元件，如膜蛋白响应元件。
进一步地，所述开关元件还可实现生物标志物响应，其中使用的生物标志物为膜蛋白，所述膜蛋白包括但不限于HER2(人表皮生长因子受体-2)、EpCAM(上皮细胞粘附分子)、Nucleolin(核仁素)、PTK7(蛋白酪氨酸假性激酶7)、VEGF(血管内皮生长因子)、EGFR(表皮生长因子受体)膜蛋白中的任意一种。由于核酸适体识别靶标分子的存在共性，可通过形成一些独特结构如茎(Stem)、环(Loop)、发卡(Hairpin)等区域实现对靶标分子的识别，因此基于本发明中对ATP响应型开关元件的设计，理论上可拓展至针对于蛋白类的其他生物标志物响应。根据靶标分子的不同，响应区域的核酸适体序列将有所不同。
具体来说，本发明中ATP响应型开关元件将ATP核酸适体分裂成两条寡核苷酸链，使其分布在碱基互补配对区域两侧，一端包含第三响应区ACCTGGGGGAGTA序列，另一端包含第四响应区TGCGGAGGAAGGT序列。当该元件处于肿瘤特征微环境高ATP浓度条件下，第三响应和第四响应序列会与ATP相互作用形成发卡型结构，使ATP核酸适体从开关元件上脱离。应用在LYTAC结构上时，会释放出阳离子非依赖性甘露糖6-磷酸受体(CI-M6PR)核酸适体用于与CI-M6PR结合并恢复LYTAC的蛋白功能，从而起到肿瘤特异性地靶向蛋白降解。类似地，对于其他膜蛋白如HER2、EpCAM、Nucleolin、PTK7、VEGF、EGFR，也可以利用它们的分裂型核酸适体制备对肿瘤生物标志物具有响应性的开关元件。
本发明的第二个目的是提供一种特异性响应靶标的组合体，含有所述响应型开关元件和靶向溶酶体穿梭受体的核酸适体，此时构建得到肿瘤特异性溶酶体靶向嵌合体。因此：
本发明的第三个目的是提供一种肿瘤特异性溶酶体靶向嵌合体，包括至少一个溶酶体穿梭受体识别结构(该结构中可含有至少一个靶向溶酶体穿梭受体的核酸适体)和至少一个响应型开关元件，所述溶酶体穿梭受体识别结构可与响应型开关元件上的碱基互补区域结合。
进一步地，所述肿瘤特异性溶酶体靶向嵌合体还包括至少一个靶标蛋白识别结构，所述靶标蛋白识别结构和溶酶体穿梭受体识别结构相互连接。具体地，所述肿瘤特异性溶酶体靶向嵌合体是对肿瘤微环境具有响应性的溶酶体靶向嵌合体。开关元件与溶酶体穿梭受体适体结合，在肿瘤微环境中，特异性响应后解离，从而暴露溶酶体穿梭受体适体，提供LYTAC与溶酶体穿梭受体结合位点，引起靶标蛋白的降解，进一步诱导肿瘤细胞凋亡。
进一步地，所述靶标蛋白识别结构与溶酶体穿梭受体识别结构通过连接分子连接。
进一步地，所述靶标蛋白识别结构能够与靶标蛋白结合。
进一步地，所述靶标蛋白识别结构选自抗体、纳米抗体、核酸适体、多肽中的一种或几种。
具体地，所述肿瘤特异性溶酶体靶向嵌合体包括如下所述的结构：通过连接分子连接的靶标蛋白识别结构和溶酶体穿梭受体识别结构，且所述溶酶体穿梭受体识别结构与靶标蛋白识别结构的摩尔比n≥1；当n＞1时，具有多价效应。
进一步地，所述1≤n≤15。
进一步地，所述连接分子为在肿瘤微环境中不可降解的分子。
进一步地，所述溶酶体穿梭受体识别结构或靶标蛋白识别结构通过柔性链段与连接分子连接或直接与连接分子连接。
进一步地，所述柔性链段选自聚乙二醇、碳链、碱基链中的一种或多种。
进一步地，所述连接分子含有以下结构中的至少一种：酰胺键、三唑环、硫醚键、二硫键。
进一步地，所述连接分子含有如下结构中的至少一种：
进一步地，所述靶标蛋白识别结构选自市售或自制的单克隆或多克隆抗体。
进一步地，所述靶标蛋白识别结构包括PD-L1抗体、HER2抗体、血小板源性生长因子抗体、蛋白酪氨酸激酶7抗体、间质上皮转化因子抗体、G蛋白偶联受体65抗体、CD47抗体、CD71抗体、EGFR抗体、ABCG2抗体。
进一步地，所述溶酶体穿梭受体识别结构包括CI-M6PR核酸适体、ASGPR核酸适体、转铁蛋白受体核酸适体、整合素核酸适体、细胞因子受体核酸适体、多聚寡核苷酸中的一种或几种。
具体地，所述CI-M6PR核酸适体选自如下序列中的一种或几种：
5’-GGGCGCGTAGATGACGAGCAGTCCTAACATCGTTTAGGAC-3’(SEQ ID NO.53)；
5’-GGGGAGGCGATTCGGTGTGTCCTCCAGAAGATATTTCCGA-3’(SEQ ID NO.54)；
5’-CGGGCACGGATGGAGTCGTTGCAGGGGCCCTTCCCCTTGG-3’(SEQ ID NO.55)；
5’-CCGACCGTGACCAGACAAACGTTTGAGTAGGCGCCCGACA-3’(SEQ ID NO.56)；
所述ASGPR核酸适体为如下序列：
5’-UUGAUUGCGUGUCAAUCAUGGCGUAGUAAAAGACAAGUAGCUACGAGGUCAUGUGUAUGUUGGGGAUUAGGACCUGAUUGAGUUCAGCCCACAUAC-3’(SEQ ID NO.57)。
当选用以上核酸适体时，将表1中的碱基互补区域替换成与上述核酸适体5’端一段序列互补的序列，碱基互补区域的长度优选为5-20nt，如CGCCTCCCC(对应SEQ ID NO.54)。
进一步地，所述多价的溶酶体靶向嵌合体的结构包括但不限于如下所示结构中的任一种：
其中，X为靶标蛋白识别结构，R1、R2为柔性链段，Y为溶酶体穿梭受体识别结构。
其中，R1和R2独立地选自PEG、碳链或碱基链。
本发明还提供上述响应型开关元件或肿瘤特异性溶酶体靶向嵌合体在制备蛋白降解剂或制备免疫治疗药物中的应用。
进一步地，所述免疫治疗药物可制备成免疫检查点阻断药物等，用于抗肿瘤。
基于上述方案，本发明还提供了一种多价的溶酶体靶向嵌合体及其在蛋白降解和免疫治疗中的应用，具体方案如下：
免疫检查点阻断治疗是肿瘤免疫治疗的主要手段，已有多种抗体药物在临床上应用，但是现有的免疫检查点阻断治疗的实际临床响应率较低，需要开发高效且安全的免疫检查点阻断药物。
近年研究表明，PDL1除了具有免疫检查点的功能外，其跨膜结构的胞内部分还会广泛参与到肿瘤细胞的生长、增殖以及转移等相关信号通路中，促进肿瘤细胞的发生发展，因此单单通过抗体等手段阻断PDL1的免疫检查点功能，仅能在一定程度上实现肿瘤免疫治疗的目标。目前有几种下调肿瘤细胞表面PDL1表达水平的策略，例如基于PDL1表达的信号通路，通过引入一些小分子抑制剂来减少PDL1的表达，但这种策略仅能干扰其中一条表达通路，肿瘤细胞可能通过其他代偿性通路来增加PDL1的表达，导致难以实现预期的PDL1下调效果。除了干扰PDL1表达的策略，还可以通过沉默PDL1相关基因来实现PDL1的下调，比如使用siRNA或者基因编辑工具CRISPR等等来实现PDL1基因的沉默，但是这种涉及基因编辑的策略在实操上，仍然面临递送效率低等问题，导致其不能被广泛应用。
近年来，随着新兴技术的发展，蛋白降解靶向嵌合体(PROTAC)结构获得广泛关注。PROTAC分子的两端分别识别靶标蛋白和E3泛素连接酶，实现靶标蛋白和E3泛素连接酶的连接，通过一系列生物学过程，靶标蛋白上被标记上泛素标签，从而被细胞内的蛋白酶体识别，进一步被蛋白酶体降解。由于泛素化过程主要发生在细胞内，这种策略对于膜蛋白的降解效果有限。
针对于膜蛋白的降解，美国化学家Carolyn Bertozzi教授在2020年首创性开发一种溶酶体靶向嵌合体(LYTAC)技术，其一端通过抗体靶向目标蛋白，另一端通过寡糖肽基团靶向阳离子非依赖性甘露糖6-磷酸受体(CI-M6PR)。这种溶酶体靶向嵌合体可与靶标膜蛋白和溶酶体穿梭受体连接形成三元复合物，并通过CI-M6PR的溶酶体穿梭活动进一步地介导靶标膜蛋白至溶酶体中发生彻底降解，从而达到下调细胞膜上靶标蛋白水平的效果。
另外，现有技术主要聚焦于新型分子工具的开发和在细胞层面的应用，在生物体层面和实际应用中尚未实践和推广，现有技术的难点在于工艺难度大、生物体内稳定性和安全性隐患、生产成本高、降解效率不稳定等方面。
目前现有技术中，LYTAC分子的使用会受到连接稳定性、结合效率等的限制，影响降解效率。
因此，本发明又提供了一种多价的溶酶体靶向嵌合体，包括以下任一种结构：通过连接分子连接的靶标蛋白识别结构和溶酶体穿梭受体识别结构，且所述溶酶体穿梭受体识别结构与靶标蛋白识别结构的摩尔比n>1。
进一步地，所述1<n≤15。
进一步地，所述溶酶体穿梭受体识别结构或靶标蛋白识别结构通过柔性链段或直接与连接分子连接。
进一步地，所述连接分子为在肿瘤微环境中不可降解的分子。
进一步地，所述柔性链段选自聚乙二醇、碳链、碱基链中的一种或多种。
进一步地，所述连接分子含有以下结构中的至少一种：酰胺键、三唑环、硫醚键、二硫键。
进一步地，所述连接分子含有如下结构中的至少一种：
进一步地，所述靶标蛋白识别结构包括PDL1抗体、HER2抗体、血小板源性生长因子抗体、蛋白酪氨酸激酶7抗体、间质上皮转化因子抗体、G蛋白偶联受体65抗体、CD47抗体、CD71抗体、EGFR抗体、ABCG2抗体等。
进一步地，所述靶标蛋白识别结构选自市售或自制的单克隆或多克隆抗体。
进一步地，所述溶酶体穿梭受体识别结构包括CI-M6PR核酸适体、ASGPR核酸适体、转铁蛋白受体(CD71)核酸适体、多聚寡核苷酸中的一种或几种。
优选地，所述CI-M6PR核酸适体的序列选自SEQ ID NO.53-56中的任一种；所述ASGPR核酸适体的序列为SEQ ID NO.57。
进一步地，所述多价的溶酶体靶向嵌合体的结构包括但不限于如下所示结构中的任一种：
其中，X为靶标蛋白识别结构，R1、R2为柔性链段，Y为溶酶体穿梭受体识别结构。
其中，R1和R2独立地选自PEG、碳链或碱基链。
本发明还提供上述多价的溶酶体靶向嵌合体在制备蛋白降解剂或制备免疫治疗药物中的应用。
进一步地，所述免疫治疗药物可制备成免疫检查点阻断药物等，用于抗肿瘤。
本发明的有益效果：
1)本发明所述特异性溶酶体靶向嵌合体能够与肿瘤细胞表面的靶标膜蛋白结合，利用溶酶体降解途径，降解靶标蛋白，进一步导致肿瘤凋亡，释放的肿瘤特异性抗原进一步引起抗肿瘤免疫反应，实现高效的抗肿瘤免疫治疗。
2)本发明所述特异性溶酶体靶向嵌合体具有肿瘤微环境或特定标志物响应的特性，只有在肿瘤组织部位能够发挥降解膜蛋白的功能，克服了目前LYTAC不具有肿瘤选择性的问题。因此，经过静脉给药途径，在正常血液循环中，肿瘤特异性溶酶体靶向嵌合体也不会导致正常细胞表面蛋白的降解。降低对患者正常细胞的影响，避免在靶脱肿瘤毒性，从而具有更好的安全性，也有助于提高免疫细胞对肿瘤的杀伤效率，增强抗肿瘤免疫治疗的效果。
3)本发明所述特异性溶酶体靶向嵌合体能够将多个溶酶体穿梭受体适体与抗体结合，具有多价效应，增加溶酶体靶向嵌合体同时结合靶标蛋白、溶酶体穿梭受体的效率，降解靶标蛋白的概率更大、效率更高，进一步引起肿瘤细胞凋亡的概率增加，提高LYTAC技术在肿瘤治疗中的疗效，增加了LYTAC技术在肿瘤治疗中的应用潜力。
4)本发明所述特异性溶酶体靶向嵌合体具有结构可控、制备简便、产率高的优点，便于大批量生产和临床应用。
5)本发明的溶酶体靶向嵌合体具有多价效应，使靶标蛋白识别结构与多个溶酶体穿梭受体识别结构相连后，结合溶酶体穿梭受体的效率更高，从而提高靶标蛋白的降解效率，还能够在靶标部位持续作用，实现低剂量高疗效的有益效果。除了实现免疫检查点阻断的结果，因高效诱导细胞表面PDL1的降解会导致肿瘤细胞凋亡，释放肿瘤抗原，刺激免疫细胞增强抗肿瘤免疫反应，综合了免疫原性死亡-抗原呈递和免疫细胞激活-免疫检查点阻断效应的连续反应，从而实现连贯的抗肿瘤免疫反应，放大疗效。
附图说明
图1为本发明的开关元件响应示意图。
图2为不同的肿瘤特异性溶酶体靶向嵌合体随着pH值的变化，其荧光信号的变化统计图。
图3为不同组别中细胞表面FAM荧光信号强度统计图。
图4为不同组别细胞的PD-L1蛋白的蛋白质免疫印迹图。
图5为实施例5中不同组别细胞的PD-L1含量。
图6为实施例6中不同组DC细胞凋亡比例统计图。
图7为实施例7中小鼠外周血CD45+和CD11+细胞的PD-L1含量统计图。
图8为实施例8中荷瘤小鼠体重变化统计图。
图9为实施例8中小鼠外周血中CD45+和CD11+细胞数量统计图。
图10为肿瘤细胞表面PDL1蛋白的蛋白免疫印迹图谱。
图11为实施例9中小鼠肿瘤体积变化情况。
图12为实施例9中小鼠体重变化情况。
图13是实施例10中PC-3前列腺癌细胞系的细胞经不同处理后的PD-L1蛋白免疫印迹实验结果，以对照例为基准，计算其它组别PD-L1相对含量。
图14是实施例10中Hela宫颈癌细胞系的细胞经不同处理后的PD-L1蛋白免疫印迹实验结果，以对照例为基准，计算其它组别PD-L1相对含量。
图15是实施例11中小鼠4T1皮下肿瘤模型的肿瘤生长曲线。
图16是实施例11中小鼠4T1皮下肿瘤模型的小鼠体重变化统计图。
图17是实施例12中开关元件分别为序列SEQ ID NO.42～45的肿瘤特异性溶酶体靶向嵌合体在不同ATP浓度下的荧光信号强度统计图。
图18是实施例12中开关元件分别为序列SEQ ID NO.49～52的肿瘤特异性溶酶体靶向嵌合体在不同ATP浓度下的荧光信号强度统计图。
图19是实施例B1检测是不同样品中B19F10肿瘤细胞上含有的PDL1蛋白印迹图；
图20是实施例B2中不同组别肿瘤细胞PDL1的蛋白印迹图；
图21是实施例B3中不同样品浓度下细胞孵育24小时后的PDL1含量统计图；
图22是实施例B4中，样品与细胞共孵育过程中不同时间点细胞膜PDL1蛋白相对含量统计图；
图23是实施例B5中不同组别细胞凋亡比例统计图；
图24是实施例B6中不同组别细胞相对活性统计图；
图25是实施例B7中细胞表面钙网蛋白激光共聚焦捕捉的荧光信号图；
图26是实施例B7中不同组别细胞ATP释放量统计图；
图27是实施例B8中不同组别树突状细胞成熟比例的统计图；
图28是实施例C1中不同组别小鼠的肿瘤生长曲线图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
除特殊说明外，本申请文件中，所述A为腺嘌呤，T为胸腺嘧啶，C为胞嘧啶，G为鸟嘌呤。
本申请中，术语“开关元件”是指对溶酶体穿梭受体适体通常有屏蔽功能的结构，在特定环境下，开关元件能够解除屏蔽作用。特定环境包括特定范围的pH值，存在钾离子、ATP(腺嘌呤核苷三磷酸)或其他生物标志物。
本说明书中实施例用到的检测设备及方法有：
流式细胞仪，BD，型号FACSCalibur。
共聚焦显微镜，Leica,型号TCS SP8。
蛋白质印迹法，Western Blot，参考本领域技术人员已知的标准规程操作。
酶标仪，Synergy。
除特别说明外，实施例中所用非肿瘤特异性溶酶体靶向嵌合体有结构式：X-(R1-L-R2-Y)n。其中X为靶标蛋白的对应抗体，即PD-L1抗体；L为连接分子；Y为CI-M6PR的核酸适体，序列为SEQ ID NO.53；n＝6；R1和R2为柔性连段。
实施例中所用肿瘤特异性溶酶体靶向嵌合体为在上述非肿瘤特异性溶酶体靶向嵌合体的结构基础上，Y与开关元件结合，所述开关元件为Seq.1。
实施例1
一种肿瘤特异性溶酶体靶向嵌合体，包括靶标蛋白识别结构、溶酶体穿梭受体识别结构和特异性响应型开关结构开关元件。
其中特异性响应型开关元件的具体序列如下表所示，图1为肿瘤特异性溶酶体靶向嵌合体的示意图，其中以CI-M6PR核酸适体为例，所述开关元件拥有与M6PR的互补配对区域。下表中，pH响应的开关元件中，常规字体部分为响应区，斜体加粗部分为连接序列、加下划线部分为与适体碱基互补配对区域。
表1

实施例2：
pH响应型肿瘤特异性溶酶体靶向嵌合体能够在特定pH条件(肿瘤微环境的微酸条件)下打开开关结构开关元件，解除对溶酶体穿梭受体适体序列的屏蔽作用，即在特定pH条件下，溶酶体穿梭受体适体能够与溶酶体穿梭受体结合，使LYTAC引起靶标蛋白的溶酶体靶向降解。
样品编号及开关元件结构转变50％时对应的pH(pHT)如表所示：

实验方法：在M6PR核酸适体5’端标记荧光基团四甲基罗丹明(TAMRA)，开关元件上标记淬灭基团BHQ-2，核酸适体和开关元件两者在生理条件下互补配对时，不能检出荧光信号。当肿瘤特异性溶酶体靶向嵌合体暴露在低pH下时，开关元件打开，荧光基团和淬灭基团分离，从而暴露荧光信号，因此在pH变化过程中，荧光信号越强，表示溶酶体穿梭受体适体暴露量越高，从而能够结合细胞表面的溶酶体穿梭受体。pHT值通过分析开关元件的结构转变中点pHT，能够衡量设计的开关元件是否适用于本体系，能够使开关元件在生理条件pH 7.4和肿瘤微环境pH 6.5之间发生灵敏的构象转变。理论上，合适的pHT应处于pH 6.5-7.4之间，且能充分抑制CI-M6PR核酸适体或解除抑制。
图2为不同的样品随着pH值变化，荧光信号变化统计图。其中实施例2.1的样品，pH为7.4模拟生理环境pH条件时，荧光强度约90％被抑制，说明开关元件基本完全抑制核酸适体活性；随着pH值逐渐降低，荧光强度逐渐增加，而在pH约为6.6左右时，(肿瘤微环境pH约为6.5)荧光信号强度趋于平衡，释放出绝大部分约80％信号，说明Seq.1具有在pH不大于6.6左右的微酸环境下开关元件响应基本充分打开，解除对溶酶体穿梭受体适体的屏蔽作用。相较之下，实施例2.2～实施例2.5对应的开关元件结构发生约50％转变时对应的pH约为6.6、6.75、7.07、5.95。其中，实施例2.5的pHT值不符合从生理条件pH 7.4到肿瘤微环境pH 6.5的区间，实施例2.2～2.4对CI-M6PR核酸适体抑制/解除抑制效率偏低。实施例2.1为最佳序列用于构造开关元件。
因此，结合图2数据，进一步设计带有开关结构的肿瘤特异性溶酶体靶向嵌合体，用于降解肿瘤组织中特定靶蛋白。本申请所述肿瘤特异性溶酶体靶向嵌合体能够随着pH条件的变化，打开开关元件，暴露溶酶体穿梭受体适体，进一步说明本申请所述肿瘤特异性溶酶体靶向嵌合体能够在肿瘤部位微酸性的微环境条件下发挥携带靶蛋白靶向溶酶体的功能，而无法在正常的生理环境中，对其他表达同种蛋白的细胞造成影响。
实施例3：
改变pH条件，检测肿瘤特异性溶酶体靶向嵌合体的核酸适体端和肿瘤细胞表面溶酶体穿梭蛋白的结合能力。
在肿瘤特异性溶酶体靶向嵌合体的溶酶体穿梭受体适体(识别结合细胞表面CI-M6PR的核酸适体)5’端标记FAM荧光基团，将LYTAC与B16F10细胞共孵育，调节孵育体系pH值。注意在体外条件下不额外加IFN-γ刺激时，B16F10细胞表面PD-L1表达量低，因此，LYTAC的抗体端被近似视为无法与细胞结合，如果检测到细胞表面有FAM信号，只能由核酸适体端与CI-M6PR结合，因此通过流式细胞仪进行定量检测细胞表面的荧光信号强度，可以判断LYTAC上的开关结构是否在不同pH条件下抑制/解除抑制CI-M6PR结合活性，进而不能/能够与B16F10细胞连接。此外，B16F10细胞与不同样品结合后，细胞均用PBS洗3次后上机检测，FAM荧光基本不受pH影响。
分组如下：
结果分析：统计数据如图3所示，图3为不同组别中细胞表面FAM荧光信号强度统计图。结果显示，对比例3.1中，只有肿瘤细胞，几乎检测不到任何荧光信号，说明系统中背景荧光信号含量极低。实施例3.2中FAM荧光信号较弱，与对比例3.1接近，说明在pH为7.4的条件下，溶酶体靶向嵌合体并未与肿瘤细胞结合，即pH在7.4的条件下，肿瘤特异性溶酶体靶向嵌合体的开关元件对靶向基团有屏蔽作用。实施例3.3中，pH在6.5左右，模拟实体瘤部位肿瘤微环境中的pH条件，结果肿瘤特异性溶酶体靶向嵌合体成功与肿瘤细胞结合，说明肿瘤特异性溶酶体靶向嵌合体在微酸环境下，开关元件打开，暴露结合位点，即可与肿瘤细胞结合，且结合效率较高。实施例3.4中游离CI-M6PR核酸适体在7.4的pH环境下，能够成功与肿瘤细胞结合，为阳性对照组，显示实施例3.3在肿瘤微酸环境中开关元件能充分解除抑制作用，恢复肿瘤特异性溶酶体靶向嵌合体与CI-M6PR的相互作用，有利于进一步地促进靶标蛋白降解。综合本实施例中几组实验数据，说明本申请所述肿瘤特异性溶酶体靶向嵌合体能够在特定pH值的肿瘤微环境中发挥结合作用，在一般体液中性pH中不发挥作用，从而避免在循环过程中与其他携带相同受体的细胞结合，提高利用效率。
实施例4：
肿瘤细胞和不同样品共孵育，调整pH，检测肿瘤细胞表面PD-L1的蛋白含量。蛋白含量越低，表明肿瘤细胞表面的PD-L1蛋白被降解，即说明溶酶体靶向嵌合体发挥作用。所述肿瘤细胞为B16F10细胞系。
实验分组如下：
细胞与样品共孵育24小时后，用蛋白质免疫印迹表征肿瘤细胞表面的蛋白含量。结果如图4所示，图4为不同组别细胞的PD-L1蛋白的蛋白质免疫印迹图。结果显示，几组细胞均含有PD-L1条带，其中，实施例4.1和实施例4.2的不能降解肿瘤细胞表面的PD-L1蛋白。而实施例4.3中，在pH7.4的条件下，非肿瘤特异性溶酶体靶向嵌合体(与肿瘤特异性溶酶体靶向嵌合体相比，区别仅为不含有开关元件，下同)降解约57％PD-L1，说明溶酶体靶向嵌合体能够发挥降解PD-L1的功能。在实施例4.4和4.5中，均使用肿瘤特异性溶酶体靶向嵌合体(带有开关元件)，在pH＝7.4的条件下，肿瘤细胞表面的PD-L1不能被降解，但当调整pH为6.5左右时，肿瘤细胞表面的PD-L1含量明显下降约61％，接近非肿瘤特异性溶酶体靶向嵌合体的降解效果。验证本申请所述肿瘤特异性溶酶体靶向嵌合体具有pH响应型的特性，且开关元件基本能够完全抑制/恢复对PD-L1降解的活性。
实施例5：
肿瘤特异性溶酶体靶向嵌合体在正常生理环境条件下，不会对免疫细胞造成损伤。
将不同样品与树突状细胞DC2.4(以下简称DC细胞)共孵育24小时，检测DC细胞表面PD-L1的含量，用蛋白质免疫印迹法处理和检测样品，分组及pH条件如下：

结果如图5，图5为实施例5中不同组别细胞的PD-L1含量，与对比例5.1相比，实施例5.2中，由于样品没有肿瘤特异性，在pH为7.4的环境中，会导致DC细胞表面PD-L1蛋白的部分降解，而实施例5.3中，本发明所述特异性溶酶体靶向嵌合体，则不会影响DC细胞表面的PD-L1蛋白含量。降解DC细胞表面PD-L1的副作用将被进一步研究。
实施例6：
肿瘤特异性溶酶体靶向嵌合体在生理条件下不会导致DC细胞的凋亡。
将不同样品与DC细胞孵育72小时，然后Annexin V-FITC和PI染色，用流式检测细胞凋亡情况，分组如下：
统计不同组别中DC细胞凋亡的比例，图6是实施例6中不同组DC细胞凋亡比例统计图，结果显示，对比例6.1和对比例6.2中，细胞凋亡数量较少，基本维持在10％左右。但是，实施例6.3中DC细胞产生了显著凋亡(超过60％)，说明非肿瘤特异性溶酶体靶向嵌合体在会影响DC细胞的活性，可能带来副作用。相较之下，实施例6.4中肿瘤特异性溶酶体靶向嵌合体很好地避免了对DC细胞的副作用，对比例6.1相比无显著差别，说明本发明所述肿瘤特异性靶向嵌合体可以逆转对影响DC细胞的负面影响，从而对整体抗肿瘤治疗更有利。但由于细胞在体外和活体内的生存环境有差异，溶酶体靶向嵌合体在实际使用中对DC细胞影响的程度还需进一步探究和验证。
实施例7：
在体内层面验证肿瘤特异性溶酶体靶向嵌合体对DC细胞PD-L1的降解作用
实验方法：
给小鼠静脉输注样品溶液，20或40μg/只(于200μL生理盐水中)，样品剂量如下表所示，注射后第一天，取小鼠外周血，通过流式细胞仪检测不同组外周血中CD45+和CD11+细胞表面的PD-L1含量，制作统计图。
分组说明：
结果分析：
给药后第一天检测小鼠外周血中CD45和CD11c双阳性细胞(即为外周血中DC细胞)表面PD-L1的相对含量，结果如图7，图7是实施例7中小鼠外周血CD45+和CD11+细胞的PD-L1含量统计图。结果显示，实施例7.2中小鼠外周血的DC细胞表面PD-L1相对含量较高，但与对照组相比，有约15％的下降，而实施例7.3中小鼠外周血的DC细胞表面PD-L1含量有较大幅度～60％的降低，而实施例7.4和实施例7.5中小鼠外周血的DC细胞表面PD-L1含量处于较高水平，几乎与对照组一致。说明本发明所述肿瘤特异性溶酶体靶向嵌合体不会降低小鼠外周血中DC细胞表面的PD-L1含量，即肿瘤特异性溶酶体靶向嵌合体不会导致外周血DC细胞表面的PD-L1的降解，从而避免了对外周血DC细胞的潜在副作用风险，具有更好的安全性。
实施例8：小鼠外周血中CD45+CD11+细胞数量变化
给荷瘤小鼠静脉输注样品溶液，20或40μg/只(于200μL生理盐水中)，给药1针，样品剂量如下表所示。
分组说明：
分别在给药后第1、3、5、7天，检测小鼠外周血中CD45+CD11c+细胞(即外周血中DC细胞)在CD45+细胞(即总免疫细胞)的占比。如图8所示，实施例8.2中，非肿瘤特异性溶酶体靶向嵌合体对外周血DC细胞有波动性的影响，DC在给药7天后基本恢复至与实施例8.1一致的水平，但实施例8.3在给予更高剂量给药后，小鼠外周血中DC在一周内未完全恢复，显著下降约50％。相反地，在实施例8.4和实施例8.5中，肿瘤特异性溶酶体靶向嵌合体未见明显影响外周血DC，证明肿瘤特异性溶酶体靶向嵌合体在生理条件下降解外周血DC-PD-L1的活性基本能被完全抑制，可以推测肿瘤特异性溶酶体靶向嵌合体具有更高的利用度和安全性。
此外，在给药第7天，我们对小鼠淋巴结中DC进一步进行分析，发现实施例8.3高剂量非肿瘤特异性溶酶体靶向嵌合体显著降低了淋巴结中总免疫细胞中的DC占比(图9)，可能是通过影响外周血DC归巢至淋巴结带来的结果。淋巴结中DC对于抗肿瘤免疫具有重要作用。
同时，检测荷瘤小鼠肿瘤部位细胞表面PD-L1含量的相对值，采用WB检测，图10是肿瘤细胞表面PD-L1蛋白的蛋白免疫印迹图谱，并统计其相对于空白对照组的含量。结果显示，溶酶体靶向嵌合体不论是否为肿瘤特异性，均能够达到降解肿瘤细胞表面PD-L1含量的效果，并且，肿瘤特异性溶酶体靶向嵌合体相较于非特异性的，具有更优异的降解效果，等剂量的肿瘤特异性溶酶体靶向嵌合体比溶酶体靶向嵌合体更多地作用于肿瘤部位的细胞膜表面PD-L1的降解，利用度更好、效率更高、安全性更高。
实施例9：
治疗实验
在小鼠皮下肿瘤模型上验证本申请所述的肿瘤特异性溶酶体靶向嵌合体对实体瘤的治疗效果。分组如下：

实验方法：在小鼠背部接种B16细胞，建立小鼠皮下黑色素瘤模型，以第一次静脉输注样品为第0天，小鼠肿瘤体积平均约50立方毫米，第0天、第3天、第6天、第9天分别通过尾静脉注射上述剂量的样品，每两天记录一次肿瘤体积，统计结果如图11所示，每两天记录一次小鼠体重，统计结果如图12所示。
结果分析：
通过图11的数据分析，首先可以发现实施例9.2～9.4的肿瘤生长曲线均比对比例9.1缓慢，且效果较为显著，说明溶酶体靶向嵌合体具有肿瘤治疗的效果。其次，实施例9.2和实施例9.3相比，以及实施例9.4和实施例9.5相比，同种样品剂量增加时，治疗效果均有提高。最关键的是，明显观察到等剂量的肿瘤特异性溶酶体靶向嵌合体相对于非特异性的溶酶体靶向嵌合体有更好的治疗效果。一方面，说明肿瘤特异性溶酶体靶向嵌合体具有肿瘤治疗效果，另一方面，说明提高样品在肿瘤部位的剂量有助于疗效的增加，并且，结合体外实验数据和本实施例数据，可以合理推测肿瘤特异性溶酶体靶向嵌合体通过减少在非肿瘤部位的结合，从而增加整个体系中发挥肿瘤杀伤作用的分子有效量，从而进一步增加疗效，更有助于临床应用。
通过图12的数据分析，大部分小鼠的体重在整个治疗期间没有明显下降，说明样品的安全性较好，但是实施例9.3中小鼠的体重增长较为缓慢，可能是由于溶酶体靶向嵌合体与免疫细胞的非特异性吸附，对小鼠健康有轻微影响，特异性溶酶体靶向嵌合体具有更好的安全性。
实施例10：
人源细胞系中肿瘤特异性溶酶体靶向嵌合体的PD-L1降解能力检测。
实验方法与实施例4相同，区别在于，使用的细胞为人源Hela宫颈癌细胞系和PC-3前列腺癌细胞系。
分组：
图13是PC-3前列腺癌细胞系的细胞经不同处理后的PD-L1相对含量。图14是Hela宫颈癌细胞系的细胞经不同处理后的PD-L1相对含量。结果显示，本申请所述的肿瘤特异性溶酶体靶向嵌合体能够在6.5左右的pH值环境下降解肿瘤细胞表面的PD-L1，在pH约7.4的环境下不具有降解PD-L1的功能，说明本申请所述肿瘤特异性溶酶体靶向嵌合体具有pH响应的特点，且一般实体瘤内pH环境为弱酸性，说明本发明具有组织选择性，在体内其他环境中不会降解其他细胞表面的相同抗原，从而具有更好的安全性。
实施例11：
本申请所述肿瘤特异性溶酶体靶向嵌合体在小鼠4T1皮下肿瘤模型中的治疗效果。
实验方法与实施例9相同，区别在于肿瘤模型不同，前期建立小鼠4T1皮下肿瘤模型，随机分组后给药。
分组：
图15是实施例11中小鼠肿瘤生长曲线，图16是实施例11中小鼠体重变化曲线。结果显示，40μg剂量的肿瘤特异性溶酶体靶向嵌合体治疗的小鼠，其肿瘤体积增长速度较其他组别显著下降(**代表p≤0.01)，说明本申请所述肿瘤特异性溶酶体靶向嵌合体具有更好的疗效。并且与等剂量的非特异性溶酶体靶向嵌合体相比，疗效更佳。
实施例12：
ATP响应型肿瘤特异性溶酶体靶向嵌合体能够在ATP的作用下打开开关结构开关元件，解除对溶酶体穿梭受体适体序列的屏蔽作用，即在一定浓度的ATP环境中，溶酶体穿梭受体适体能够与溶酶体穿梭受体结合，使LYTAC引起靶标蛋白的溶酶体靶向降解。构建序列并进行ATP响应的测试。
实验方法：在M6PR核酸适体5’端标记荧光基团四甲基罗丹明(TAMRA)，开关元件上标记淬灭基团BHQ-2，核酸适体和开关元件两者在生理条件下互补配对时，不能检出荧光信号。当肿瘤特异性溶酶体靶向嵌合体暴露在含有ATP的环境中时，开关元件打开，荧光基团和淬灭基团分离，从而暴露荧光信号，因此在不同ATP浓度的环境中，检测得到的荧光信号越强，表示溶酶体穿梭受体适体暴露量越高，结合细胞表面的溶酶体穿梭受体的概率越高。外周血和健康组织间质中ATP是10～100nM，含量极低，而肿瘤微环境中ATP含量约为1～5mM，当ATP响应的开关元件能够在该浓度范围内打开且在nM级别中无响应时，说明该开关元件能够满足特异性响应的需求。
实验结果：图17是开关元件分别为序列SEQ ID NO.42～45的肿瘤特异性溶酶体靶向嵌合体在不同ATP浓度下的荧光信号强度统计图，结果显示，四条序列均能够满足在mM级别浓度的ATP环境中响应，打开开关元件，暴露核酸适体并触发荧光，且在较低的浓度中几乎没有触发开关元件，即说明这些序列满足ATP特异性响应开关元件的需求，能够在肿瘤区别于其他正常组织中的ATP浓度环境下实现溶酶体靶向降解的功能。
图18是开关元件分别为序列SEQ ID NO.49～52的肿瘤特异性溶酶体靶向嵌合体在不同ATP浓度下的荧光信号强度统计图。结果显示，四条序列均能够满足在mM级别浓度的ATP环境中响应，打开开关元件，暴露核酸适体并触发荧光，且在较低的浓度中几乎没有触发开关元件，其中SEQ ID NO.49作为开关元件，响应效率最高。说明这些序列满足ATP特异性响应开关元件的需求，能够在肿瘤特定的ATP环境下实现溶酶体靶向降解的功能。
本发明还提供了一种多价的溶酶体靶向嵌合体(LYTAC)，其中含有靶标蛋白识别结构和靶向溶酶体穿梭受体的配体结构，且一个靶标蛋白识别结构连接不止一个配体结构，这种多价的蛋白降解剂在体内稳定性更好，降解效率更高。
实施例A1：一种多价的溶酶体靶向嵌合体，包括靶标蛋白识别结构和溶酶体穿梭受体识别结构。具体结构组成如下表所示：

实施例A2：一种多价的溶酶体靶向嵌合体的制备方法：
PDL1抗体的氨基置换为叠氮，得到叠氮修饰的PDL1抗体；CI-M6PR核酸适体经DBCO修饰，得到DBCO修饰的CI-M6PR核酸适体；
叠氮修饰的PDL1抗体和DBCO修饰的CI-M6PR核酸适体通过点击化学反应连接。
所述叠氮修饰抗体和点击化学反应制备LYTAC的实验方法为：
1)将30μM的PDL1抗体与1.5mM双功能交联剂NHS-PEG4-N3在中性PBS缓冲液中混合，并至于温和条件下搅拌下反应1小时(300rpm,25℃)，得到产物，即叠氮修饰的PDL1抗体。
2)反应完成后，将产物用50kD超滤离心管过滤并用中性PBS缓冲液清洗，充分去除过量的双功能交联剂NHS-PEG4-N3。
3)将10μM带有叠氮基团的PDL1抗体与DBCO修饰的核酸适体混合，并在25℃温育1～24小时，得到溶酶体靶向嵌合体。为了去除过量的DBCO-寡聚核苷酸，需使用50kD超滤离心过滤器超滤溶酶体靶向嵌合体并经中性PBS缓冲液清洗。
4)最终收集溶酶体靶向嵌合体保存于4℃备用。
通过调节叠氮修饰的PDL1抗体和DBCO修饰的CI-M6PR核酸适体的投料比例，可得到多价的溶酶体靶向嵌合体。对应实施例A1.1～实施例A1.4，投料量如下表所示：
多价的溶酶体靶向嵌合体是一个抗体上连接多个核酸适体，能够增加抗体识别配位后，与溶酶体穿梭蛋白结合的亲和力(效率)，提高溶酶体靶向的效果。
实施例B1：溶酶体靶向嵌合体的PDL1降解效率检测实验。
将105个B19F10肿瘤细胞分别与10nM的实施例A1.1～实施例A1.4的溶酶体靶向嵌合体共孵育，然后使用western blot技术检测肿瘤细胞上PDL1的含量。另外增加溶酶体靶向嵌合体的用量，105个肿瘤细胞分别与50nM的实施例A1.1～实施例A1.4的溶酶体靶向嵌合体共孵育，同样用western blot技术检测肿瘤细胞PDL1的含量。
图19检测是不同样品中B19F10肿瘤细胞含有的PDL1蛋白印迹图，条带深浅代表PDL1含量，条带越深，PDL1含量约高，条带越浅，PDL1含量越低。从图19中可以看出，实施例A1.1～实施例A1.4的溶酶体靶向嵌合体均能降低肿瘤细胞中PDL1的含量，即说明本发明所述溶酶体靶向嵌合体具有下调PDL1水平的能力。其次，随着溶酶体靶向嵌合体用量的增加，降解PDL1的效率增加，其中实施例A1.1、实施例A1.2和实施例A1.3降解PDL1的效率和用量关联度更高，实施例A1.4的溶酶体靶向嵌合体，其用量增加，并不能相应程度地改善降解效率，猜测其原因可能是抗体上偶联过多的核酸适体，会影响抗体和PDL1之间的识别效率，从而干扰溶酶体靶向嵌合体和靶标蛋白的结合，进而影响溶酶体靶向溶解的效率。
总之，本发明所述溶酶体靶向嵌合体，通过增加抗体上连接核酸适体的数量，能够引起明显的多价效应，相对于连接单个核酸适体，显著提高了溶酶体靶向嵌合体降解靶标蛋白的效率，能够在更低剂量下实现下调靶标蛋白含量，增强抗肿瘤免疫治疗的效果。
实施例B2：通过添加不同抑制剂，验证本发明所述溶酶体靶向嵌合体降解PDL1的途径为溶酶体降解途径。
实验方法：将105个B16F10细胞与50nM实施例A1.2的溶酶体靶向嵌合体共孵育，同时加入5μM的MG132蛋白酶体抑制剂或加入100μM的Chloroquine溶酶体抑制剂，共孵育24小时后，用western blot检测肿瘤细胞上PDL1蛋白的含量。
空白对照：细胞不做任何处理；
对比例B2.1：B16F10细胞与实施例A1.2的溶酶体靶向嵌合体共孵育；
实施例B2.2：B16F10细胞与实施例A1.2的溶酶体靶向嵌合体、MG132蛋白酶体抑制剂共孵育；
实施例B2.3：B16F10细胞与实施例A1.2的溶酶体靶向嵌合体、Chloroquine溶酶体抑制剂共孵育。
图20是实施例B2中不同组别肿瘤细胞PDL1的蛋白印迹图。结果显示，在实施例B2.2中加入蛋白酶体抑制剂，对实施例A1.2的溶酶体靶向嵌合体降解PDL1蛋白的效率没有影响；而实施例B2.3中，加入溶酶体抑制剂，能够明显干扰实施例A1.2的溶酶体靶向嵌合体降解PDL1蛋白，几乎不能实现PDL1的降解，说明本发明所述溶酶体靶向嵌合体是通过溶酶体途径而非蛋白酶体途径实现PDL1的降解。
实施例B3：
实验方法：按照实施例B1的方法，区别在于，多价溶酶体靶向嵌合体来自实施例A1.2，浓度分别为2nM、5nM、10nM、50nM、100nM，每组样品与105个B16F10细胞共孵育，孵育时间24小时。
实验结果：图21是实施例B3中不同样品浓度下细胞孵育24小时后的PDL1含量统计图，其中横坐标为多价溶酶体靶向嵌合体浓度，纵坐标为细胞表面PDL1的相对含量，结果显示，随着多价溶酶体靶向嵌合体使用浓度的提高，相同时间内细胞表面PDL1蛋白被降解的越多，说明高浓度的多价溶酶体靶向嵌合体降解细胞膜表面PDL1蛋白的效率越高。并且说明本发明所述多价溶酶体靶向嵌合体均一性较好，才能保证降解效率和浓度的一致性。
实施例B4：实验验证本发明所述溶酶体靶向嵌合体降解PDL1的持续性作用。
将约105个B16F10细胞与50nM的实施例A1.2的溶酶体靶向嵌合体的样品共孵育，在不同时间点(4小时、8小时、12小时、20小时、30小时)检测细胞膜上PDL1蛋白的相对含量，图22是孵育过程中不同时间点细胞膜PDL1蛋白相对含量统计图，结果显示，12小时内，PDL1抗体的含量下调明显，12小时左右，PDL1抗体几乎全被降解，20小时之后，样品的膜表面PDL1蛋白含量下调速率降低，相对含量达到很低的水平，说明本发明所述溶酶体靶向嵌合体能够实现细胞膜表面PDL1抗体表达持续下调，并使PDL1的含量维持在较低水平，即本发明所述溶酶体靶向嵌合体能够下调生物体内肿瘤的细胞膜表面PDL1抗体，维持较长时间，从而实现抗肿瘤免疫治疗的效果。
实施例B5：本发明所述溶酶体靶向嵌合体下调肿瘤细胞表面PDL1蛋白水平后，会引起细胞凋亡。
由于PDL1蛋白的胞内部分还参与了细胞增殖、转移等信号通路，因此推测细胞膜PDL1被降解后，能够引起肿瘤细胞增殖减缓的结果，设计本实施例验证。
分组：
对比例B5.1：不做任何处理
实施例B5.2：PDL1抗体
实施例B5.3：未偶联的PDL1抗体和CI-M6PR核酸适体
实施例B5.4：实施例A1.2的溶酶体靶向嵌合体
实验方法：将约105个B16F10细胞与不同样品(待补充浓度)共孵育24小时，用流式细胞术检测细胞凋亡的比例。
图23是实施例B5中不同组别细胞凋亡比例统计图，结果显示，本发明所述溶酶体靶向嵌合体能够引起大部分肿瘤细胞的凋亡。即当细胞膜上PDL1蛋白被降解后，直接会导致肿瘤细胞的凋亡，该现象从未被现有技术报道，出乎意料的发现本发明所述溶酶体靶向嵌合体具有诱导肿瘤细胞凋亡的效果。本发明所述的具有多价效应的溶酶体靶向嵌合体具有比现有技术中溶酶体靶向嵌合体更强的靶向降解效率，也因此，除了具有免疫检查点阻断效应之外，还会引起肿瘤细胞自发的凋亡，本发明所述溶酶体靶向嵌合体具有意想不到的技术效果。
实施例B6：MTT实验
本实施例用于探究多价溶酶体靶向嵌合体的有效作用浓度。
将对数期的B16F10细胞接种至96孔板中，每孔接种数量10000个，培养过夜后加入不同浓度的不同样品进行孵育，孵育24小时后，移除培养基，更换新鲜培养基，并加入MTT溶液(1mg/mL)，孵育约4小时，移除培养基，每孔加入150μL的二甲基亚砜，充分溶解细胞中的结晶，然后使用酶联免疫检测仪检测OD值在570nm处的吸光值，细胞存活率计算公式为：存活率＝(实验组吸光值/对照组吸光值)×100％。
分组说明：
实验结果：
图24是实施例B6中不同组别细胞相对活性统计图，实验结果显示，其中空白对照组的细胞相对存活率为100％，相较于空白对照组，多价靶向嵌合体样品浓度的提高，相同时间内多价溶酶体靶向嵌合体对肿瘤细胞的杀伤作用逐渐增强。本发明所述的溶酶体靶向嵌合体的半抑制浓度(IC50)约为4.2nM。IC50越小，说明杀伤肿瘤细胞的能力越强，说明本发明所述的溶酶体靶向嵌合体具有诱导肿瘤细胞死亡的效果。
本发明所述溶酶体靶向嵌合体具有多价效应，大幅提高了PDL1蛋白的降解效率，大量PDL1降解导致肿瘤细胞的死亡。因此，本发明所述溶酶体靶向嵌合体不仅能够实现PDL1的彻底移除，引起免疫检查点阻断，增强免疫细胞识别和杀伤肿瘤细胞的效果，还能够直接导致肿瘤细胞的凋亡，暴露肿瘤相关抗原，刺激免疫细胞增强抗肿瘤免疫反应，具有多重效果，显著增强肿瘤治疗效果。
实施例B7：证明本发明所述溶酶体靶向嵌合体引起的肿瘤细胞的凋亡，能够增强肿瘤细胞的免疫原性，设计本实施例证明，经溶酶体靶向嵌合体作用后，肿瘤细胞表面钙网蛋白增加。
对比例B7.1：空白对照
对比例B7.2：PDL1抗体与肿瘤细胞共孵育
对比例B7.3：未偶联的PDL1抗体和CI-M6PR核酸适体，与肿瘤细胞共孵育
实施例B7.4：实施例A1.2的溶酶体靶向嵌合体与肿瘤细胞共孵育
实验方法：待补充用量、检测方法等。
不同组别中处理后的细胞用钙网蛋白的检测试剂盒对钙网蛋白进行标记，标记方法按照说明书进行。然后，用4％多聚甲醛固定细胞，并使用Hoechst染色以标记细胞核，然后将染色后的细胞用激光共聚焦显微镜。
肿瘤细胞表面钙网蛋白的增加会使得肿瘤细胞更容易被免疫细胞识别，促进肿瘤的抗原呈递，引起免疫活化效应，进而诱导抗肿瘤免疫反应。图25是实施例B7中细胞表面钙网蛋白激光共聚焦捕捉的荧光信号图。
图25显示，实施例B7.4的细胞，钙网蛋白表达量增加，说明溶酶体靶向嵌合体引起了肿瘤细胞的表面钙网蛋白的增加，从而有助于免疫细胞识别肿瘤细胞，增强抗肿瘤免疫治疗的疗效。
此外，ATP释放是肿瘤细胞的免疫原性的另一个指标，ATP释放越高，免疫原性越强。
图26是实施例B7中不同组别细胞ATP释放量统计图，结果显示，实施例A1.2的溶酶体靶向嵌合体作用于肿瘤细胞后，能够引起肿瘤细胞ATP释放量的显著增加。以上释放的ATP和暴露的盖网蛋白均为损伤相关分子模式(DAMP)，与肿瘤细胞免疫原性呈正相关，说明本发明所述溶酶体靶向嵌合体能够增强肿瘤细胞的免疫原性。肿瘤细胞的免疫原性增强会引起强烈抗肿瘤免疫反应的发生，因此，本发明所述溶酶体靶向嵌合体能够引起强烈抗肿瘤免疫反应，从而实现抗肿瘤免疫治疗的目的。
实施例B8：将实施例9中凋亡的细胞产物用于刺激骨髓来源的树突状细胞(BMDC)的成熟实验。
分组：
对比例B8.1：对比例B7.1的细胞凋亡产物与骨髓来源的树突状细胞共孵育
对比例B8.2：对比例B7.2的细胞凋亡产物与骨髓来源的树突状细胞共孵育
实施例B8.3：实施例B7.4的细胞凋亡产物与骨髓来源的树突状细胞共孵育
实验方法：
从雄性C57BL/6小鼠的股骨和胫骨中分离骨髓细胞，并加入粒细胞巨噬细胞集落刺激因子(GM-CSF，浓度20ng/mL)和白介素-4(IL-4，浓度10ng/mL)的培养基，培养分离得到的骨髓细胞，培养5天后取少量细胞通过CD11c流式抗体染色检验成熟度，确认髓源细胞中已分化为DC细胞。同时，将B16F10肿瘤细胞与不同样品(50nM)共孵育48小时，然后将不同样品处理过的肿瘤细胞分别与DC细胞1：1共孵育3天，最后收集DC细胞，用流式细胞术测定DC细胞的CD80和CD86的表达情况。CD80和CN86是DC细胞成熟的关键标志物，CD80和CD86双阳性的细胞为成熟的髓源DC细胞。
骨髓来源的树突状细胞是目前发现的功能最强的抗原递呈细胞，并且树突状细胞是唯一能够显著刺激初始T细胞增殖的抗原呈递细胞，因此树突状细胞是生物体适应性T细胞免疫应答启动的关键环节，在肿瘤免疫治疗中有非常重要的作用。树突状细胞的成熟比例会影响后续抗肿瘤免疫反应的程度，成熟度越高，预期引起的抗肿瘤免疫反应越强烈。
图27是实施例B8中不同组别树突状细胞成熟比例的统计图，结果显示，实施例B7.4的细胞凋亡产物能够引起骨髓来源的树突状细胞的成熟，说明本发明所述溶酶体靶向嵌合体能够引起抗肿瘤免疫反应，实现活体水平的抗肿瘤免疫治疗。
实施例C1：动物实验证明本发明所述溶酶体靶向嵌合体能够引起抗肿瘤免疫反应，抑制肿瘤生长和转移。
小鼠背部接种肿瘤细胞(约5×105B16F10个细胞)，待肿瘤生长到25mm3，随机分组，分组情况如下：
对比例C1.1：空白对照组，不对小鼠肿瘤进行任何处理
对比例C1.2：荷瘤小鼠采用PDL1抗体治疗
实施例C1.3：荷瘤小鼠采用PDL1抗体和M6PR核酸适体混合物治疗，PDL1抗体和M6PR核酸适体的剂量比例为1：6
实施例C1.4：荷瘤小鼠采用实施例A1.2的溶酶体靶向嵌合体治疗
实验方法：
对比例C1.2、实施例C1.3、实施例C1.4每次静脉注射给药20μg PDL1抗体或等浓度LYTAC(以PDL1定量)，每3天一次，共4次，记录肿瘤体积变化，并制作肿瘤生长曲线，结果如图28所示。
实验结果：图28是实施例C1中不同组别小鼠的肿瘤生长曲线图，结果显示，对比例C1.2所采用的传统免疫检查点阻断疗法并不能很好地抑制肿瘤生长，实施例C1.3中仅将溶酶体靶向受体的核酸适体与PDL1抗体混合后使用，其效果与单独的免疫检查点阻断疗法无异。而实施例C1.4中，采用本发明所述的溶酶体靶向嵌合体治疗，具有明显抑制肿瘤生长的效果。说明本发明所述的溶酶体靶向嵌合体具有在活体上诱导抗肿瘤治疗的应用前景，并且疗效较好。本发明所述溶酶体靶向嵌合体，可以通过静脉方式给药，给药方式便捷，效率高。
实施例D：其他连接分子的多价溶酶体靶向嵌合体及其降解蛋白的效率研究。
根据实施例A2的制备方法，制备得到多种多价溶酶体靶向嵌合体，均具有降解靶标蛋白的效果，且降解效率均优于单价的溶酶体靶向嵌合体。

以上实施例用到的检测设备及方法有：流式细胞仪，BD，型号FACSCalibur。共聚焦显微镜，Leica,型号TCS SP8。蛋白质印迹，Western Blot。
综上，以上具有多价效应的溶酶体靶向嵌合体使靶标蛋白识别结构与多个溶酶体穿梭受体识别结构相连后，结合溶酶体穿梭受体的效率更高，从而提高靶标蛋白的降解效率。通过共价偶联反应制备本发明的多价溶酶体靶向嵌合体，合成方法简单，反应效率高，利于纯化操作，整体制备成本低。
本发明的多价溶酶体靶向嵌合体在体内的稳定性好，无不利于生理活动的反应发生，且由于识别效率高，能更好地富集到肿瘤部位，提高使用安全性。
本发明的多价溶酶体靶向嵌合体能够在靶标部位持续作用，实现低剂量高疗效的有益效果。除了实现免疫检查点阻断的结果，还可通过高效诱导细胞表面PDL1的降解导致肿瘤细胞凋亡，释放肿瘤抗原，刺激免疫细胞增强抗肿瘤免疫反应，综合了免疫原性死亡-抗原呈递和免疫细胞激活-免疫检查点阻断效应的连续反应，从而实现连贯的抗肿瘤免疫反应，放大疗效。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。
尽管上述披露中通过各种示例讨论了一些目前认为有用的发明实施例，但应当理解的是，该类细节仅起到说明的目的，附加的权利要求并不仅限于披露的实施例，相反，权利要求旨在覆盖所有符合本申请实施例实质和范围的修正和等价组合。
同理，应当注意的是，为了简化本申请披露的表述，从而帮助对一个或多个发明实施例的理解，前文对本申请实施例的描述中，有时会将多种特征归并至一个实施例、附图或对其的描述中。但是，这种披露方法并不意味着本申请对象所需要的特征比权利要求中提及的特征多。实际上，实施例的特征要少于上述披露的单个实施例的全部特征。
一些实施例中使用了描述成分、属性数量的数字，应当理解的是，此类用于实施例描述的数字，在一些示例中使用了修饰词“大约”、“近似”或“大体上”来修饰。除非另外说明，“大约”、“近似”或“大体上”表明所述数字允许有±变化。相应地，在一些实施例中，说明书和权利要求中使用的数值参数均为近似值，该近似值根据个别实施例所需特点可以发生改变。
最后，应当理解的是，本申请中所述实施例仅用以说明本申请实施例的原则。其他的变形也可能属于本申请的范围。因此，作为示例而非限制，本申请实施例的替代配置可视为与本申请的教导一致。相应地，本申请的实施例不仅限于本申请明确介绍和描述的实施例。

Claims (19)

  1. 一种响应型开关元件，其特征在于，所述响应型开关元件为一条DNA序列，且其上设有相互连接的多个DNA功能区域，在未响应时与靶标核酸适体结合，响应时与靶标核酸适体分离，至少能够响应pH、离子、生物分子中的一种或几种。
  2. 根据权利要求1所述的响应型开关元件，其特征在于，所述响应型开关元件从DNA序列的5’端至3’端顺序中，至少含有以下Ⅰ-Ⅲ中的任一结构：
    Ⅰ.所述响应型开关元件上设有相互连接的响应区、连接序列、响应区、碱基互补区域、响应区、连接序列、响应区，所述响应区含有若干胞嘧啶；
    Ⅱ.所述响应型开关元件上设有相互连接的第一响应区、连接序列、碱基互补区域、连接序列、第二响应区，所述第一响应区和第二响应区由含有核心序列5’-GGGTAGGGCGGGTTGGG-3’的序列拆分得到；
    Ⅲ.所述响应型开关元件上设有相互连接的第三响应区、碱基互补区域、第四响应区，将生物分子核酸适体拆分为第一序列和第二序列，所述第三响应区包括第一序列，第四响应区包括第二序列，且所述第三响应区和第四响应区可与生物分子形成发卡结构；
    所述碱基互补区域用于与靶标核酸适体互补。
  3. 根据权利要求2所述的响应型开关元件，其特征在于，所述碱基互补区域与靶标核酸适体至少有任意5个连续的碱基参与互补配对。
  4. 根据权利要求2所述的响应型开关元件，其特征在于，所述连接序列的核苷酸选自A和T中的任意一种或两种，所述连接序列的核苷酸的数量为1-8个。
  5. 根据权利要求2所述的响应型开关元件，其特征在于，当响应pH时，所述响应区包含4-8个胞嘧啶。
  6. 根据权利要求2所述的响应型开关元件，其特征在于，当响应钾离子时，所述第一响应区和第二响应区通过含有核心序列5’-GGGTAGGGCGGGTTGGG-3’的序列拆分得到，且拆分位点位于所述核心序列上，拆分后第一响应区和第二响应区上核心序列的G碱基数目比为1：1或者3：1。
  7. 根据权利要求2所述的响应型开关元件，其特征在于，所述第三响应区和第四响应区还含有用于稳定第一序列和/或第二序列的延长序列，所述延长序列为1-3个碱基，其中每个碱基独立地选自G、C、A、T，所述延长序列位于第一序列的3’端和第二序列的5’端，且两个延长序列碱基是互补配对的。
  8. 根据权利要求2所述的响应型开关元件，其特征在于，所述生物分子核酸适体至少含有如下所示的序列：ACCTGGGGGAGTATGCGGAGGAAGGT，且第三响应区至少含有ACCTGGGGGAGTA，第四响应区至少含有TGCGGAGGAAGGT。
  9. 根据权利要求1所述的响应型开关元件，其特征在于，所述响应型开关元件包含SEQ ID NO.1-52任一所示的序列。
  10. 一种肿瘤特异性溶酶体靶向嵌合体，其特征在于，包括至少一个溶酶体穿梭受体识别结构和至少一个权利要求1-9任一项所述的响应型开关元件；所述溶酶体穿梭受体识别结构含有至少一个靶向溶酶体穿梭受体的核酸适体，且所述靶向溶酶体穿梭受体的核酸适体可与响应型开关元件上的碱基互补区域结合。
  11. 根据权利要求10所述的肿瘤特异性溶酶体靶向嵌合体，其特征在于，所述肿瘤特异性溶酶体靶向嵌合体包括靶标蛋白识别结构，且所述靶标蛋白识别结构和溶酶体穿梭受体识别结构通过连接分子连接。
  12. 根据权利要求11所述的肿瘤特异性溶酶体靶向嵌合体，其特征在于，所述连接分子为在肿瘤微环境中不可降解的分子。
  13. 根据权利要求10所述的肿瘤特异性溶酶体靶向嵌合体，其特征在于，所述肿瘤特异性溶酶体靶向嵌合体包括如下所述的结构：通过连接分子连接的靶标蛋白识别结构和溶酶体穿梭受体识别结构，且所述溶酶体穿梭受体识别结构与靶标蛋白识别结构的摩尔比n≥1。
  14. 根据权利要求10-13任一项所述的肿瘤特异性溶酶体靶向嵌合体，其特征在于，所述连接分子含有以下结构中的至少一种：酰胺键、三唑环、硫醚键、二硫键。
  15. 根据权利要求14所述的肿瘤特异性溶酶体靶向嵌合体，其特征在于，所述连接分子含有如下结构中的至少一种：
  16. 权利要求1-9任一项所述的响应型开关元件或权利要求10-15任一项所述的肿瘤特异性溶酶体靶向嵌合体在制备蛋白降解剂或制备免疫治疗药物中的应用。
  17. 一种抗肿瘤药物，其特征在于，所述抗肿瘤药物中含有权利要求1-9任一项所述的响应型开关元件或权利要求10-15任一项所述的肿瘤特异性溶酶体靶向嵌合体。
  18. 一种多价的溶酶体靶向嵌合体，其特征在于，所述多价的溶酶体靶向嵌合体包括如下所述的结构：通过连接分子连接的靶标蛋白识别结构和溶酶体穿梭受体识别结构，且所述溶酶体穿梭受体识别结构与靶标蛋白识别结构的摩尔比n>1。
  19. 权利要求18所述的多价的溶酶体靶向嵌合体在制备蛋白降解剂或制备免疫治疗药物中的应用。
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