WO2021068890A1 - 溶酶体靶向的抗体-药物偶联物及其应用 - Google Patents

溶酶体靶向的抗体-药物偶联物及其应用 Download PDF

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WO2021068890A1
WO2021068890A1 PCT/CN2020/119996 CN2020119996W WO2021068890A1 WO 2021068890 A1 WO2021068890 A1 WO 2021068890A1 CN 2020119996 W CN2020119996 W CN 2020119996W WO 2021068890 A1 WO2021068890 A1 WO 2021068890A1
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antibody
lysosomal
drug conjugate
tamra
group
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PCT/CN2020/119996
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English (en)
French (fr)
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蔡晓青
蒋先兴
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中山大学
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Priority to EP20874161.1A priority Critical patent/EP4043034A4/en
Priority to JP2022519150A priority patent/JP7549908B2/ja
Priority to CN202080013281.8A priority patent/CN113453718B/zh
Priority to US17/765,785 priority patent/US20230066474A1/en
Publication of WO2021068890A1 publication Critical patent/WO2021068890A1/zh

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Definitions

  • the invention belongs to the technical field of biomedicine, and relates to an antibody-drug conjugate and its application, in particular to a lysosome-targeted antibody-drug conjugate and its application.
  • ADC Antibody-drug conjugate
  • ADC drug has become a very promising treatment in anti-cancer therapy because of its combination of the targeting of antibody macromolecules and the cytotoxicity of chemotherapeutic drugs.
  • ADC drugs need antibodies to specifically recognize and bind to cell surface antigens to function, and then they undergo endocytosis in an antigen-mediated manner.
  • the ideal ADC drug will release the cytotoxic small molecule drug through the endosome-lysosome intracellular pathway, and then the cytotoxic small molecule drug will reach the target of action (common tubulin and DNA ) Play a role and cause cell death.
  • ADC drugs into eukaryotic cells is mainly through the following three different ways: (1) clathrin-mediated endocytosis, (2) caveolin-mediated endocytosis, (3) macropinocytosis.
  • clathrin-mediated endocytosis and caveolin-mediated endocytosis are antigen-dependent, and clathrin-mediated endocytosis is the most effective way to transport proteins to lysosomes.
  • antibodies that enter the cell through caveolin-mediated endocytosis tend to eventually accumulate in the endoplasmic reticulum or Golgi apparatus.
  • the protein that enters the cell through the non-selective giant pinocytosis may either enter the lysosome along with the fusion of the vesicle and the lysosome, or may rearrange outside the cell, but different cells are different, such as the human epidermal cancer cell A431 cell
  • the giant pinocytosis vesicles in the vesicles rarely fuse with lysosomes, and more are excreted.
  • the Fc segment of a full-length antibody can interact with FcRn in a pH-dependent manner, which facilitates the circulation of the antibody to the outside of the cell and prolongs the antibody half-life.
  • ADC drugs can effectively internalize and enter the lysosome is crucial for their efficacy. Only when ADC drugs are degraded by lysosomes and release cytotoxic small molecules, through the interaction of cytotoxic small molecules with the target (commonly tubulin and DNA), can they finally achieve the effect of effectively killing tumor cells. Studies have shown that cell surface antigens are in a dynamic back and forth between the inside and outside of the cell membrane, and part of the ADC drugs that enter the cytoplasm will be transported to the outside of the cell membrane. In fact, the amount of cytotoxic small molecule drugs released in the cytoplasm is very small.
  • the existing ADC drugs often have insignificant killing effects on cancer cells and cannot achieve ideal therapeutic effects.
  • the latest research results show that the reduction of ADC drug accumulation in lysosomes or the decrease of protease activity in lysosomes is also the main reason for the clinical resistance of ADC drugs.
  • the antibody part of ADC drugs used in clinical practice has not undergone any modification, and it cannot effectively reach the lysosome site after being endocytosed into tumor cells; therefore, there are often poor efficacy and drug resistance in clinical use. problem.
  • the purpose of the present invention is to solve the problems existing in the prior art, and provide a lysosomal targeted antibody-drug conjugate and its application.
  • This technology uses lysosomal targeted small molecules or functional polypeptides to target antibodies- Modification of the antibody part of the drug conjugate provides a simple and universal method for guiding the antibody-drug conjugate into the lysosome to effectively release cytotoxic small molecules, which can improve the efficacy of ADC drugs
  • Unique and efficient strategy to improve the enrichment of antibody-drug conjugates in lysosomes, enhance the cell internalization ability of antibody-drug conjugates, increase the internalization rate of antibody-drug conjugates and lysozyme The degree of body enrichment improves the therapeutic effect of the antibody-drug conjugate.
  • the present invention provides a lysosomal targeted antibody-drug conjugate, the structure of the antibody-drug conjugate is as follows:
  • Dr is a drug
  • Ab is an antibody
  • O is a lysosomal targeting small molecule or a functional polypeptide used to increase the lysosomal targeting ability of the antibody-drug conjugate;
  • n1 and n2 are integers greater than or equal to 1, and n1 and n2 are the same or different.
  • the antibody is a monoclonal antibody or a nanobody
  • the monoclonal antibody is one of Glembatumumab, Vandortuzumab, Tisotumab, Enfortumab, Cetuximab, Coltuximab, Lorvotuzumab, Gemtuzumab, Trastuzumab, and Ladiratuzumab
  • the Nanobody is 7D12 , EGA1, 9G8, C7b, 5F7, 2Rs15d.
  • the functional polypeptide is a lysosomal sorting peptide, a cell penetrating peptide, or a combination of a lysosomal sorting peptide and a cell penetrating peptide; the functional polypeptide is connected to the C-terminus of the antibody.
  • the lysosomal sorting peptide is NPXY, [DE]XXXL[LI], DXXLL, NPFXD, NPFXXD, where X is any amino acid, Is any bulky hydrophobic side chain amino acid, N is asparagine, P is proline, Y is tyrosine, E is glutamic acid, L is leucine, I is isoleucine, F is phenylalanine Acid, D is aspartic acid, [DE] represents one of D or E, and [LI] represents one of L or I.
  • the cell penetrating peptide is one of a cationic penetrating peptide, an amphipathic penetrating peptide, and a hydrophobic penetrating peptide.
  • the cationic penetrating peptide is one of polyarginine, HIV-TAT, DPV1047, and Penetratin;
  • the amphiphilic penetrating peptide is MPG, PVEC, MAP, Pept-1, Transportan, One of P28;
  • the hydrophobic penetrating peptide is one of C105Y, PFVYLI, and Pep-1.
  • the lysosomal targeting small molecule is connected to the amino acid side chain of the antibody.
  • the amino acid side chain is a natural amino acid side chain or a bioorthogonal group.
  • the natural amino acid side chain is one of a lysine side chain and a cysteine side chain;
  • the bioorthogonal group is an azide group, an alkynyl group, an aldehyde group, a ketone group, and a fluorine group.
  • One of the sulfonate groups is one of a lysine side chain and a cysteine side chain;
  • the lysosomal targeting small molecule is a compound containing a pH sensitive group or a compound containing a sugar group.
  • the pH sensitive group is one of a sulfonic acid group, a 4-morpholino group, a 2-morpholinoethylaminoethyl group, and a methoxypolyethylene glycol group.
  • the sugar group is one of glucose, mannose, 6-phosphate-mannose, and galactose.
  • the present invention also provides an application of the above-mentioned lysosome-targeted antibody-drug conjugate in anticancer drugs.
  • the lysosome-targeted antibody-drug conjugate of the present invention has the following advantages:
  • the lysosomal targeted antibody-drug conjugate of the present invention modifies the antibody in the antibody-drug conjugate through lysosomal targeting small molecules or functional polypeptides, so that the antibody-drug conjugate is
  • the drug has significant cell internalization ability, faster internalization rate and higher degree of lysosome enrichment, which increases the cell penetration of the antibody-drug conjugate, and at the same time the lysozyme of the antibody-drug conjugate Body targeting is excellent.
  • the lysosomal-targeted antibody-drug conjugates of the present invention have higher lysosomal targeting and show stronger inhibition of cancer cell proliferation.
  • Ability higher cancer cell death rate and better in vitro anti-tumor activity; therefore, it provides new possibilities for improving the efficacy of ADC drugs.
  • Figure 1 is a MALDI-TOF Mass chart of 7D12-TAMRA-LPETG-His 6 described in Example 1 of the present invention
  • Figure 3 is the sequence of the functional polypeptide described in Examples 2 and 3 of the present invention and the structural formula of the lysosomal targeting small molecule.
  • Compound 1 is sulfoacetic acid
  • compound 2 is 2-morpholinacetic acid
  • compound 3 is 2.
  • compound 4 is 2-(2-methoxyethoxy) ethoxyacetic acid
  • compound 5 is glucosanoic acid
  • Fig. 4a shows the results of polyacrylamide gel electrophoresis (SDS-PAGE) and His-tag antibody western blot (anti-His western blot) of the 7D12-polypeptide conjugate described in Example 2 of the present invention;
  • Figure 4b shows the polyacrylamide gel electrophoresis (SDS-PAGE) of Trastuzumab-polypeptide conjugate
  • FIG. 5 is a MALDI-TOF Mass characterization result of 7D12-TAMRA-SA according to Embodiment 3 of the present invention.
  • FIG. 6 is the MALDI-TOF Mass characterization result of 7D12-TAMRA-PEG3 according to Example 3 of the present invention.
  • FIG. 7 is the MALDI-TOF Mass characterization result of 7D12-TAMRA-morpholine according to Embodiment 3 of the present invention.
  • FIG. 8 is the MALDI-TOF Mass characterization result of 7D12-TAMRA-EtNH-morpholine according to Embodiment 3 of the present invention.
  • FIG. 9 is a MALDI-TOF Mass characterization result of 7D12-TAMRA-glucose according to Embodiment 3 of the present invention.
  • FIG. 10 is a MALDI-TOF Mass characterization result of 7D12-MMAF-SA according to Embodiment 3 of the present invention.
  • FIG. 11 is a MALDI-TOF Mass characterization result of 7D12-MMAF-PEG3 according to Example 3 of the present invention.
  • FIG. 12 is a MALDI-TOF Mass characterization result of 7D12-MMAF-morpholine according to Embodiment 3 of the present invention.
  • FIG. 13 is a MALDI-TOF Mass characterization result of 7D12-MMAF-EtNH-morpholine according to Embodiment 3 of the present invention.
  • FIG. 14 is a MALDI-TOF Mass characterization result of 7D12-MMAF-glucose according to Example 3 of the present invention.
  • Example 15 is the characterization result of the internalization characteristics of the 7D12 conjugate described in Example 4 of the present invention.
  • 16 is a curve of the average fluorescence intensity change of the 7D12 conjugate described in Example 4 of the present invention into A431 cells over time;
  • Figure 17a is the 7D12-TAMRA, 7D12-TAMRA-LSP, 7D12-TAMRA-R 9 , 7D12-TAMRA-R 9 -LSP, 7D12-TAMRA-TAT, 7D12-TAMRA-TAT-LSP according to Embodiment 5 of the present invention Confocal microscopy pictures;
  • Figure 17b is a confocal microscope of 7D12-TAMRA, 7D12-TAMRA-SA, 7D12-TAMRA-PEG3, 7D12-TAMRA-morpholine, 7D12-TAMRA-EtNH-morpholine, 7D12-TAMRA-glucose according to Example 5 of the present invention image;
  • Fig. 17c is a confocal microscope picture of Trastuzumab-TAMRA, Trastuzumab-TAMRA-R9, Trastuzumab-TAMRA-R9-LSP and Trastuzumab-TAMRA-LSP described in Example 5 of the present invention;
  • Figure 18a is the test result of the Pierce correlation coefficient between the conjugate and lysosome shown in Figure 17a;
  • Figure 18b is the test result of the Pierce correlation coefficient between the conjugate and lysosome shown in Figure 17b;
  • Figure 18c is a test result of the Pierce correlation coefficient between the conjugate and lysosome shown in Figure 17c;
  • Figure 19a is a graph of the linear relationship drawn between the concentration of TAMRA-labeled antibody and the measured fluorescence intensity
  • Figure 19b is the calculation result of the amount of antibody internalized into the cell
  • Figure 20 is the test results of the effects of temperature and endocytosis inhibitors on the internalization of 7D12 conjugates
  • Figure 21 is the test results of the colocalization of 7D12-TAMRA-LSP, 7D12-TAMRA-TAT and 7D12-TAMRA-R 9 with clathrin or caveolin-1;
  • Figure 22 is the test results of the Pierce correlation coefficient between 7D12-TAMRA-LSP, 7D12-TAMRA-TAT and 7D12-TAMRA-R 9 and clathrin or caveolin-1;
  • Figure 23 is a graph of cell viability relative to the concentration of MMAF, 7D12 or tested conjugate
  • Figure 24 is a confocal image of a three-dimensional cell tumor sphere
  • Figure 25 is the test results of the inhibition of tumor spheroid growth under different concentrations of 7D12 conjugate treatment
  • Figure 26 is a test image of tumor sphere size change
  • Figure 27 is a schematic diagram of the mechanism of action of the antibody-drug conjugate of the present invention.
  • the antibody of the present invention is a monoclonal antibody or a nanobody, and the monoclonal antibody is Glembatumumab, Vandortuzumab, Tisotumab, Enfortumab, Cetuximab (cetuximab), Coltuximab, Lorvotuzumab, Gemtuzumab (getuzumab), Trastuzumab ( Trastuzumab), one of Ladiratuzumab; the Nanobody is one of 7D12, EGA1, 9G8, C7b, 5F7, 2Rs15d.
  • the amino acid sequence of the above antibody is as follows:
  • the amino acid sequence of 7D12 is:
  • amino acid sequence of EGA1 is:
  • amino acid sequence of 9G8 is:
  • the amino acid sequence of C7b is:
  • amino acid sequence of 5F7 is:
  • the amino acid sequence of 2Rs15d is:
  • the amino acid sequence of Glembatumumab is:
  • the amino acid sequence of Vandortuzumab is:
  • the amino acid sequence of Tisotumab is:
  • the amino acid sequence of Enfortumab is:
  • the amino acid sequence of Cetuximab is:
  • the amino acid sequence of Coltuximab is:
  • the amino acid sequence of Lorvotuzumab is:
  • the amino acid sequence of Gemtuzumab is:
  • the amino acid sequence of Trastuzumab is:
  • the amino acid sequence of Ladiratuzumab is:
  • the present invention is illustrated by taking Nanobody 7D12 and monoclonal antibody Trastuzumab as examples. It should be noted that the mutated antibody of the present invention is not limited to the following antibodies or mutation sites. The present invention is also applicable to mutations at other sites of other antibodies. The selection of the mutated antibody does not affect the antibody-drug coupling of the present invention. The performance of things.
  • an anti-Epidermal Growth Factor Receptor (EGFR) single domain nanobody 7D12 was selected as the template antibody.
  • 7D12 antibody has the following characteristics: (1) 7D12 antibody has high affinity to EGFR and can cause internalization after binding to EGFR; (2) 7D12 antibody cannot cause antibody-dependent cell-mediated cytotoxicity and complement due to the lack of Fc fragments The dependent cytotoxicity simplifies the interference of protein modification on antibody-drug conjugates.
  • the amino acid sequence of the antibody after the above mutation is as follows:
  • the preferred mutation site of the 7D12 antibody of the present invention is position 85, and position 85 is selected for mutation because: (1) it is located in the middle region away from the antigen binding region near the N-terminus; (2) solvent is accessible and easy to modify; (3) It is located in the loop region to minimize the interference to the quaternary structure of the protein.
  • the monoclonal antibody Trastuzumab was selected as the template antibody.
  • the alanine (Ala121, A121) at position 121 of the heavy chain of the Trastuzumab antibody was mutated to cysteine (Cys, C).
  • Cys, C cysteine
  • the amino acid sequence of the heavy chain of the Trastuzumab antibody after mutation is:
  • the spatial structure of the antibody after mutation is easier to bind to the drug, but it should be noted that the mutation site and the amino acid after mutation of the present invention are not limited to the above methods. In other embodiments, other sites can be performed according to the requirements of use. Mutation or mutation to other amino acids.
  • the above-mentioned mutant antibody is coupled with anti-tumor toxicity small molecule drugs.
  • the anti-tumor toxicity small molecule drugs include tubulin inhibitors and DNA damaging agents.
  • Tubulin inhibitors such as maytansinoids (DM1 and DM4), MMAF (monomethyl auristatin F), MMAE (monomethyl auristatin E), paclitaxel, taxanes, tubulysin and the like.
  • DNA damaging agents such as doxorubicin, calicheamicin, DuoCarmycin, camptothecin, pyrrolobenzodiazepines, etc.
  • the mutant 7D12 antibody is selected as the antibody in this example, and the anti-tumor toxicity small molecule drug is maleimidohexanoic acid-MMAF (Mc-MMAF), but it should be understood that the following operation methods are also applicable to other Antibodies and anti-tumor toxicity small molecule drugs.
  • MMAF is connected to the mutant 7D12 antibody molecule through the orthogonal reaction of maleimide and sulfhydryl group.
  • the specific operation method of anti-tumor toxicity small molecule drug coupling is as follows:
  • the antibody-drug conjugate of the present invention can also be labeled with a fluorescent molecule.
  • the fluorescent molecule is Rhodamine, CY3 fluorescent dye (Cyanine 3). ), Texas Red (Texas Red) one of the fluorescent dyes.
  • the labeling method of the present invention is not limited to the application on the 7D12 antibody, and the labeling method is also applicable to other antibodies.
  • the fluorescent molecule is maleimide-PEG3-tetramethylrhodamine (TAMRA), and TAMRA is connected to the mutant 7D12 antibody through the orthogonal reaction of maleimide and sulfhydryl group.
  • TAMRA maleimide-PEG3-tetramethylrhodamine
  • the antibody-drug conjugate of the present invention is modified with a functional polypeptide, and the functional polypeptide is Lysosome-sorting peptide (LSP), cell-penetrating peptide (cell- penetrating peptide, CPP, such as TAT or R 9 ), or a combination of lysosomal sorting peptide and cell penetrating peptide TAT-LSP and R 9 -LSP, where LSP, TAT, R 9 , TAT-LSP and R The amino acid sequence of 9- LSP is shown in Figure 3.
  • LSP Lysosome-sorting peptide
  • CPP cell-penetrating peptide
  • CPP cell- penetrating peptide
  • TAT-LSP and R 9 cell penetrating peptide
  • the lysosomal sorting peptide is NPXY, [DE]XXXL[LI], DXXLL, NPFXD, NPFXXD, where X is any amino acid, Is any bulky hydrophobic side chain amino acid, N is asparagine, P is proline, Y is tyrosine, E is glutamic acid, L is leucine, I is isoleucine, F is phenylalanine Acid, D is aspartic acid, [DE] represents one of D or E, and [LI] represents one of L or I.
  • the cell penetrating peptide is one of cationic penetrating peptide, amphiphilic penetrating peptide, and hydrophobic penetrating peptide.
  • the cationic penetrating peptide and its amino acid sequence are any of the following:
  • R n , n is an integer greater than or equal to 1;
  • HIV-TAT its amino acid sequence is:
  • amphiphilic penetrating peptide and its amino acid sequence are any of the following:
  • MPG its amino acid sequence is:
  • PVEC its amino acid sequence is:
  • Pept-1 its amino acid sequence is:
  • Transportan whose amino acid sequence is:
  • P28 its amino acid sequence is:
  • hydrophobic penetrating peptide and its amino acid sequence are any of the following:
  • Pep-1 its amino acid sequence is:
  • the method for modifying the functional polypeptide includes using transpeptidase and gene recombination to connect to the C-terminus of the antibody.
  • the method of functional polypeptide modification includes the following steps:
  • a transpeptidase is used to bind the functional polypeptide to the C-terminus of the mutant antibody in the form of a peptide bond.
  • LETG transpeptidase recognition sequence
  • oligoglycine is added to the N-terminus of the functional polypeptide sequence Sequence (trimeric glycine GGG is used in this example).
  • mutant antibody connects the LPETG sequence and His 6 sequence
  • 7D12-S85C-LPETG-His 6 was expressed in E. coli BL21 (DE3) and purified with Ni-NTA beads.
  • Ni-NTA beads were first balance Ni-NTA beads with a balance buffer (400mM NaCl, 50mM Tris ⁇ HCl pH 8.0, 20mM imidazole), then load the supernatant of the bacterial lysate, and then use the balance buffer to wash away non-specific
  • the bound protein is finally eluted with an elution buffer containing 200 mM imidazole, and the protein concentration is determined by the BCA method for protein recovery and exchange. There was no significant change in the yield of the mutant protein compared with the wild-type protein.
  • the transpeptidase in this example is a truncated fragment SrtA ⁇ N59 of the transpeptidase A (Sortase A) from Staphylococcus aureus, and its amino acid sequence is: QAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKVKNEKH DYKHDYKHDYKVJKVKVKNEVKVKGVKNETGVKVKVKVKG
  • the nucleophilic substrates of the transpeptidation reaction (GGG-LSP, GGG-R 9 , GGG-R 9 -LSP, GGG-TAT, GGG-TAT-LSP) were synthesized by standard solid-phase peptide synthesis.
  • the transpeptidation reaction was carried out in SrtA ⁇ N59 working buffer (150mM NaCl, 50mM Tris ⁇ HCl pH 7.5), and 5eq SrtA ⁇ N59 , 1eq 7D12-TAMRA-LPETG-His 6 or 7D12-MMAF-LPETG-His 6 were added to the reaction system.
  • SrtA ⁇ N59 working buffer 150mM NaCl, 50mM Tris ⁇ HCl pH 7.5
  • 5eq SrtA ⁇ N59 1eq 7D12-TAMRA-LPETG-His 6 or 7D12-MMAF-LPETG-His 6 were added to the reaction system.
  • Or Trastuzumab-TAMRA-LPETG-His 6 or Trastuzumab-MMAF-LPETG-His 6 and 10eq of peptide substrate the whole transpeptidation reaction process is connected overnight at room temperature. After the reaction, the reaction mixture is purified with Ni-NT
  • the successfully transpeptidized antibody will remove the His 6 tag, so it cannot bind to the Ni column, and the unsuccessfully transpeptidized antibody and SrtA ⁇ N59 will be affinity adsorbed on On the Ni column, the reaction product can be obtained by eluting with a low concentration of imidazole.
  • the final recovered protein product is analyzed by SDS-PAGE and anti-His western blot. The result shows that the product after transpeptide is in the same sample amount. Almost no bands appeared on the anti-His western blot.
  • 7D12-TAMRA-LPETG-His 6 7D12-MMAF-LPETG-His 6
  • Trastuzumab-TAMRA-LPETG-His 6 and Trastuzumab -MMAF-LPETG-His 6 is connected with GGG to obtain 7D12-TAMRA-GGG, 7D12-MMAF-GGG, Trastuzumab-TAMRA-GGG and Trastuzumab-MMAF-GGG as controls.
  • Figure 4a shows the results of polyacrylamide gel electrophoresis (SDS-PAGE) and His tag antibody immunoblotting test (anti-His western blot) of the 7D12-polypeptide conjugate, where each group
  • the polypeptide conjugates corresponding to the number are as follows: M: marker; 1: 7D12-TAMRA-LPETG-His 6 ; 2: 7D12-TAMRA-GGG; 3: 7D12-TAMRA-LSP; 4: 7D12-TAMRA-TAT; 5: 7D12-TAMRA-TAT-LSP; 6: 7D12-TAMRA-R 9 ; 7: 7D12-TAMRA-R 9 -LSP; 8: 7D12-MMAF-LPETG-His 6 ; 9: 7D12-MMAF-GGG; 10: 7D12 -MMAF-LSP; 11: 7D12-MMAF-TAT; 12: 7D12-MMAF
  • the GAAAACCTGTACTTCCAGGGA sequence for TEV protease cleavage was added to the N-terminus.
  • the C-terminus was constructed by PCR with a sense primer (5'-GGAATTCCATATGGAAAACCTGTACTTCCAGGGACAGGTGAAACTGGAGGAAAGCG-3') and an antisense primer (5'-CCGCTCGAGTCAATAGCCCGGGTTGCCGCTGCCTGCTAACGGTCACTTGGGTACC-3') to construct pET28C-7D12-S85.
  • the broken solution was centrifuged at a high speed with a fixed-angle rotor to remove unbroken cells, cell residues and insoluble proteins (4°C, 18000 rpm, 30 min).
  • Use a nickel column to purify the 7D12-S85C protein, rinse the resin with approximately 20 column volumes of Ni-NTA Buffer A, and collect all the Ni-NTA Buffer A flowing through the nickel column to detect impurity proteins that are non-specifically bound to the nickel column.
  • the target protein on the nickel column resin was gradient eluted with Ni-NTA Buffer B (20mM, 50mM, 100mM, 200mM, 400mM, 800mM) containing different imidazole concentrations, and each component was collected separately, and SDS-PAGE was performed. Electrophoresis analysis.
  • Maleimide-PEG3-TAMRA specifically labels 7D12-S85C-Peptide.
  • PBS pH 7.4
  • 5mM TCEP a very effective thiol reducing agent that can reduce the disulfide bonds in or between proteins
  • PBS pH 7.4
  • 1mM EDTA 1mM EDTA (3800rpm, 4°C).
  • the total reaction volume is 1ml
  • the final protein concentration is 1mg/ml
  • maleimide-PEG3-TAMRA dissolved with appropriate amount of DMSO
  • the fluorescent molecules and protein are mixed, and incubated in a shaker at 4°C 2h (protect from light).
  • the solution was changed with PBS (pH 7.4) to remove small fluorescent molecules, and the protein concentration was measured with NanoDrop.
  • the reacted 7D12-TAMRA protein is stored in a refrigerator at -80°C.
  • 7D12-S85C-Peptide is coupled with mc-MMAF.
  • PBS pH 7.4
  • PBS pH 7.4
  • 1mM EDTA 1mM EDTA (3800rpm, 4°C).
  • the total reaction volume is 1ml
  • the final protein concentration is 1mg/ml
  • mc-MMAF dissolved in appropriate DMSO
  • 7D12-MMAF and Trastuzumab-MMAF in Example 1 are used as examples for description in this example, but the present invention is not limited to this, and the modification method of this example is also applicable to other antibodies and other anti-tumor Other antibody-drug conjugates formed by toxic small molecules.
  • the present invention does not particularly limit the sequence of functional polypeptide modification and drug coupling, that is, the mutant antibody can be first Modified by functional peptides and then coupled with anti-tumor toxicity small molecule drugs, or can be coupled with anti-tumor toxicity small molecule drugs first and then modified by functional peptides; functional peptide modification and drug coupling sequence does not affect The performance of the antibody-drug conjugate of the present invention.
  • the present invention requires that the mutant antibody be modified with a functional polypeptide and then coupled with an anti-tumor toxicity small molecule drug.
  • the antibody-drug conjugate of the present invention can be modified by attaching a functional polypeptide to the C-terminus of the antibody, and can also be modified with lysosomal targeting small molecules. Retouch.
  • the lysosomal targeting type small molecule is a compound containing a pH-sensitive group or a compound containing a sugar group, wherein the pH-sensitive group is a sulfonic acid group, a 4-morpholino group, or a 2-morpholine group.
  • the pH-sensitive group is a sulfonic acid group, a 4-morpholino group, or a 2-morpholine group.
  • the structural formula of the lysosomal targeting small molecule is as follows:
  • X is selected from:
  • Y is selected from:
  • Z is selected from:
  • the wavy line indicates the connection position
  • the asterisk indicates that the carbon atom at that position is one or a mixture of two epimers
  • n3, n4 are integers greater than or equal to 1;
  • n5, n6, n7, n8, n9 are integers greater than or equal to 0;
  • n5 and n6 are the same or different;
  • n7, n8 and n9 are the same or different.
  • the lysosomal targeting type small molecule is connected to the amino acid side chain of the antibody, the amino acid side chain is a natural amino acid side chain or a bio-orthogonal group, and the natural amino acid side chain is a lysine side chain or a cysteine side chain
  • the bioorthogonal group contains one of the azide group, alkynyl group, aldehyde group, ketone group, and fluorosulfonate group that can undergo a bioorthogonal reaction.
  • the bioorthogonal group can Introduced by gene amplification technology or enzymatic reaction.
  • the present invention is illustrated by taking the lysosomal targeting small molecule (1-9) in FIG. 3 as an example.
  • the lysosomal targeting small molecules are respectively attached to antibodies labeled with MMAF and TAMRA (7D12-MMAF and 7D12-TAMRA).
  • the present invention does not specifically limit the sequence of lysosomal targeted small molecule modification and drug coupling, that is, the mutant antibody of the present invention can be modified with lysosomal targeted small molecule first and then combined with the drug.
  • Coupling of anti-tumor toxicity small molecule drugs can also be first coupled with anti-tumor toxicity small molecule drugs and then modified by lysosomal targeting small molecules; sequence of lysosomal targeting small molecule modification and drug coupling It does not affect the performance of the antibody-drug conjugate of the present invention.
  • flow cytometry is used to monitor the internalization of the antibody-drug conjugates of the present invention modified with functional polypeptides and modified with lysosomal targeting small molecules, which specifically includes the following steps:
  • HEK293T cell lines purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (source: ATCC)
  • the cells were cultured in DMEM (Gibco, USA) medium supplemented with 10% fetal bovine serum, plus penicillin ( 100U/mL)-Streptomycin (100g/mL). All cells were cultured in a humidified environment containing 5% CO 2 at 37°C. The cell morphology is normal and the growth status is good.
  • the effectiveness of ADC drugs also depends on the intracellular sorting kinetics of the antigen.
  • flow cytometry the performance of 7D12 antibody modified with fluorescent molecule TAMRA during the internalization of cells at different time points was monitored.
  • 7D12 conjugates after using different kinds of modifications (7D12-TAMRA, 7D12-TAMRA-LSP, 7D12-TAMRA-R 9 , 7D12-TAMRA-R 9 -LSP, 7D12-TAMRA-TAT, 7D12-TAMRA-glucose)
  • 7D12-TAMRA, 7D12-TAMRA-LSP, 7D12-TAMRA-R 9 , 7D12-TAMRA-R 9 -LSP, 7D12-TAMRA-TAT, 7D12-TAMRA-glucose A431 cells were co-incubated, and several modified 7D12-TAMRA were diluted with serum-free medium to a final concentration of 2 ⁇ M. Acid wash is then used to remove the antibody bound on the cell membrane, and quantitative analysis is performed at different time points by flow cytometry.
  • FIG. 15 is the characterization result of the internalization characteristics of the 7D12 conjugate.
  • a representative flow cytometry chart shows the internalization of the 7D12 conjugate at different time points.
  • ADC drugs are combined with tumor antigens on their surface, ADC drugs are internalized into endosomes, and then mature and fuse with lysosomes.
  • the linker is cleaved by a specific protease (e.g. cathepsin B) or by ADC drug degradation to release the drug.
  • the released drug passes through the lysosome membrane and reaches the cytoplasm to bind to its target, such as tubulin or DNA, and ultimately induce cell death. Therefore, whether the ADC drug enters the lysosome after internalization and whether it can accumulate in the lysosome is an important factor that determines its efficacy.
  • Figures 17a and 17b The test results of the 7D12-conjugate are shown in Figures 17a and 17b.
  • Figures 17a and 17b are the results of the internalization of the 7D12 conjugate and its transport through the lysosome.
  • Figure 17a shows the 7D12-TAMRA and 7D12-TAMRA- Confocal microscope pictures of LSP, 7D12-TAMRA-R 9 , 7D12-TAMRA-R 9 -LSP, 7D12-TAMRA-TAT, 7D12-TAMRA-TAT-LSP, the left column is lysosomes, the middle column is 7D12 Coupled signals, the right column is the image after the left column and the middle column overlap.
  • Figure 17b shows the confocal microscope pictures of 7D12-TAMRA, 7D12-TAMRA-SA, 7D12-TAMRA-PEG3, 7D12-TAMRA-morpholine, 7D12-TAMRA-EtNH-morpholine, and 7D12-TAMRA-glucose.
  • the middle column is the 7D12 coupling signal
  • the right column is the image after the left and middle columns overlap.
  • Figure 18a is the Pearson's correlation coefficients between the conjugate and lysosome shown in Figure 17a
  • Hoechst 33342 was also used for staining in the test of Trastuzumab conjugate.
  • the test results of Trastuzumab conjugates are shown in Figure 17c, 17c is the internalization of Trastuzumab conjugates and the results of their transport through lysosomes, and Figure 17c is Trastuzumab-TAMRA, Trastuzumab-TAMRA-R9, Trastuzumab-TAMRA- Confocal microscope pictures of R9-LSP and Trastuzumab-TAMRA-LSP.
  • the first column is the result of Hoechst33342 staining of the nucleus
  • the second column is the lysosome
  • the third column is the Trastuzumab coupling signal
  • the fourth column is The image after the first row to the third row are overlapped.
  • the 7D12-conjugate showed antigen specificity for EGFR.
  • the 7D12 conjugate did not show specific binding on the cell membrane.
  • the use of cell-penetrating peptide R 9 plus LSP modification can best help the 7D12 conjugate specifically target lysosomes in living cells. With or without the addition of LSP, there is a significant difference in the degree of overlap between the cell-penetrating peptide-modified conjugate and the lysosome.
  • Cellular endocytosis is a complex mechanism involving different pathways and large networks of protein-protein and protein-lipid interactions.
  • the first and most characteristic pathway is clathrin-dependent endocytosis, which starts from the plasma membrane and forms clathrin-coated invaginations, which are clamped to form clathrin-coated vesicles.
  • clathrin-independent pathway which includes caveolin-mediated endocytosis.
  • caveolin-mediated internalization makes it easier for early vesicles to enter the Golgi apparatus or the endoplasmic reticulum to fuse with it, and finally part of it is transferred through the circulation of the membrane and is discharged outside the cell.
  • the cellular uptake of the 7D12 conjugate at 4°C was compared with the cell uptake at 37°C.
  • the uptake of 7D12-TAMRA-functional polypeptide and 7D12-TAMRA-lysosomal targeting small molecules was significantly inhibited at 4°C, indicating that the internalization of Nanobodies is ATP-dependent.
  • endocytosis inhibitors were used.
  • the cationic amphoteric drug chlorpromazine (CPZ) is used to inhibit clathrin-mediated endocytosis by inducing the loss of clathrin.
  • EIPA Ethyl isopropyl amide
  • Nystatin Na + /H + exchange inhibitor
  • Nystatin can inhibit the endocytosis mediated by caveolae.
  • Caveolin-mediated endocytosis is considered to be one of the possible mechanisms that cause insensitivity to ADC drugs targeting its antigen.
  • the prior art shows that the internalization induced by TAT modification is mainly through the caveolin-mediated internalization pathway. It also indicates that LSP is internalized by the binding of clathrin AP2. Therefore, this example evaluated the co-localization of 7D12 modified with several functional polypeptides with caveolin-1 or clathrin.
  • A431 cells were incubated with several 7D12 conjugates, using rabbit-derived antibodies anti-clathrin and anti-cavolin-1 as primary antibodies. Then incubate FITC-labeled anti-rabbit secondary antibody for immunofluorescence analysis.
  • Figure 21 shows the test results of the colocalization of 7D12 conjugates (7D12-TAMRA-LSP, 7D12-TAMRA-TAT and 7D12-TAMRA-R 9 ) with clathrin or caveolin-1.
  • the nucleus was stained with Hoechst 33342. Shown as an image of representative cells.
  • the test found that the intracellular 7D12-TAMRA-LSP does not co-localize with caveolin, but partially co-localizes with clathrin.
  • 7D12-TAMRA-TAT did not co-localize with clathrin, but with caveolin.
  • the difference is that 7D12-TAMRA-R 9 co-localizes with clathrin and caveolin.
  • the A431 cells of human epidermal carcinoma cells were selected to evaluate the ability of 7D12-MMAF conjugate to inhibit the proliferation of cancer cells.
  • different concentrations of ADC drugs were added to a 96-well plate seeded with A431 cells. After 96 hours of treatment, the cck-8 kit was used to obtain the survival rate of the cells.
  • 7D12-MMAF-R 9 -LSP conjugate showed the highest lethality with an EC 50 value of 53.74 nM.
  • 7D12-MMAF-EtNH-morpholine modified by a small molecule conjugates several 7D12 has the lowest EC 50 is 117.9nM.
  • the wild-type 7D12 was used as the isotype control in the A431 cell line, and no significant cytotoxicity was found to determine the ADC activity.
  • 7D12-MMAF-R 9 -LSP was found to have certain cytotoxicity.
  • CPP cell penetrating peptide
  • a laser confocal test was performed to test whether CPP could carry the 7D12 conjugate into EGFR-negative cells after a long incubation period. And the results showed that after 6 hours or even longer incubation, R 9 modified 7D12 conjugate is internalized into negative cells, which explains why the modification results in CPP nonspecific cytotoxicity.
  • conjugated CPP is used to increase the internalization ability of Nanobodies, it is also necessary to consider its impact on the antigen specificity of Nanobodies.
  • the modified 7D12-MMAF conjugate exhibits a higher efficiency in mediating cell death.
  • the cytotoxicity of the modified 7D12-MMAF conjugate is related to its internalization and lysosomal targeting ability.
  • 7D12-MMAF-R 9 -LSP has the highest cancer cell growth inhibitory activity, followed by 7D12-MMAF-R 9 and 7D12-MMAF-LSP, respectively.
  • TAT or TAT-LSP modified conjugates have lower cancer cell killing activity.
  • 7D12-MMAF-R 9 -glucose has the highest cancer cell growth inhibitory activity, followed by 7D12-MMAF-EtNH-mophorline. There was no significant difference in the cell killing effect of SA, PEG and morpholine modified 7D12 conjugates.
  • the three-dimensional spheroid tumor model was further used to evaluate the permeability and efficacy of ADC drugs: the penetration of the three-dimensional tumor sphere was measured after 6 hours of incubation with various 7D12-TAMRA conjugates And co-localization capabilities. The diameter of all tumor spheroids is about 100 ⁇ m.
  • Figure 24 is a 3D tumor sphere model to evaluate the anti-tumor activity of 7D12 conjugates targeting lysosomes. From top to bottom, they were treated with 7D12-TAMRA, 7D12-TAMRA-R 9 -LSP and 7D12-TAMRA-glucose.
  • the 3D tissue model was used for growth inhibition test determination.
  • Three cell lines expressing high to low EGFR levels were selected: A431, MCF-7, HEK293T.
  • ADC drugs are added.
  • the 3D spheroids were incubated in a medium containing ADC drug (0.25nM to 2.5 ⁇ M) for 10 days, and the daily changes in the diameter of the spheroids were recorded.
  • Figures 25-26 The changes in the average diameter of all groups can be seen in Figures 25-26.
  • Figure 26 is a representative image of A431 tumor spheroid size changes under exposure to a specified concentration of 7D12 conjugate over time. This indicates that 7D12-MMAF-R 9 -LSP has the strongest growth inhibition on 3D spheres of A431 cells compared with 7D12-MMAF. At the same time, 7D12-MMAF-glucose also showed a strong growth inhibitory effect on tumor spheres. This is consistent with the results observed in the 2D tumor culture model.
  • MCF-7 and HEK293T are used to prove the specificity of these ADC drugs.
  • these cellular 3D spheres were treated with the same concentration of ADC, the growth of MCF-7 3D spheres with moderate EGFR expression was slightly inhibited.
  • the growth state of HEK293T 3D spheres is not affected by the experimental group.
  • ADCs lysosomal-targeted antibody-drug conjugates
  • ADCs are prepared by two modification methods: (1) connect a functional polypeptide to the C-terminus of the antibody; (2) connect the antibody to the lysosome Targeted small molecules. Whether it is modified by functional peptides or modified by lysosomal targeted small molecules, it can improve the enrichment of ADC drugs in lysosomes.
  • ADC drugs modified with R 9 -LSP functional polypeptide sequence show significantly enhanced cell internalization ability, faster internalization rate and higher degree of lysosomal enrichment.
  • ADC drugs modified with R 9 -LSP functional peptides will increase clathrin-mediated endocytosis, while the modification of lysosomal targeted small molecules will change the intracellular transport pathway of ADC drugs.
  • the modification of lysosomal targeted small molecules can improve the anti-tumor activity of ADC drugs in vitro.
  • the schematic diagram is shown in Figure 27.
  • the present invention provides a simple and universal method for guiding ADC drugs into lysosomes to effectively release cytotoxic small molecules, which can be a unique and efficient method for improving the efficacy of ADC drugs.

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Abstract

本发明公开了一种溶酶体靶向的抗体-药物偶联物及其应用，所述抗体-药物偶联物的结构为Dr n1AbO n2；其中，Dr为药物，Ab为抗体，O为溶酶体靶向型小分子或用于增加所述抗体-药物偶联物的溶酶体靶向能力的功能性多肽；n1、n2为大于或等于1的整数，n1和n2相同或不同。

Description

溶酶体靶向的抗体-药物偶联物及其应用 技术领域
本发明属于生物医药技术领域，涉及一种抗体-药物偶联物及其应用，具体地说涉及一种溶酶体靶向的抗体-药物偶联物及其应用。
背景技术
抗体-药物偶联物(Antibody-drug conjugate,ADC，以下简称ADC药物)因其兼具抗体大分子的靶向性和化疗药物小分子的细胞毒性成为抗癌治疗中十分有前景的一种治疗手段。ADC药物发挥作用需要抗体与细胞表面抗原特异性识别并结合，然后通过抗原介导的方式发生内吞。当ADC药物内化进入细胞后，理想的ADC药物会通过内涵体-溶酶体胞内途径释放出细胞毒性小分子药物，然后细胞毒性小分子药物到达作用靶标(常见的有微管蛋白和DNA)发挥作用并且引发细胞死亡。
ADC药物内化进入真核细胞主要通过以下三种不同的途径：(1)网格蛋白介导的内吞，(2)小窝蛋白介导的内吞，(3)巨胞饮。在这三种方式中，网格蛋白介导的内吞和小窝蛋白介导的内吞都是抗原依赖性的，网格蛋白介导的内吞最能有效的将蛋白运输至溶酶体中，而通过小窝蛋白介导的内吞进入细胞内的抗体往往最终会聚集在内质网或者高尔基体。通过非选择性的巨胞饮进入胞内的蛋白既可能随着囊泡与溶酶体融合进入溶酶体也可能重排至胞外，但不同细胞有所不同，比如人表皮癌细胞A431细胞中的巨胞饮囊泡就很少与溶酶体融合，更多的是排出细胞。而全长抗体的Fc段可以与FcRn发生pH依赖性作用，因此有利于抗体循环至细胞外，延长抗体半衰期。
ADC药物能否有效内化并进入溶酶体对其发挥药效至关重要。只有当ADC药物经溶酶体降解并释放出细胞毒性小分子，通过细胞毒性小分子与靶标(常见的有微管蛋白和DNA)相互作用，最终才能达到有效杀死肿瘤细胞的效果。研究表明，细胞表面抗原处于往返于细胞膜内外的动态中，进入胞浆内的ADC药物有一部分又会被运输到细胞膜外，实际上在胞浆内被释放的细胞毒性小分子药物的量很少；尤其在抗原表达量本身就很低的癌细胞中，进入细胞内有效作用的细胞毒小分子药物的量更少。因此，现有的ADC药物往往对癌细胞的杀伤效果不明显，不能起到理想的治疗效果。此外，最新研究结果表明，ADC药物在溶酶体内富集减少或者溶酶体内蛋白酶的活性降低，也是导致ADC药物出现临床耐药性问题的主要原因。目前，临床中使用的ADC药物中的抗体部分并没有经过任何修饰，其内吞进肿瘤细胞后未能有效到达溶酶体部位；因此，在临床使用中往往存在药效差和耐药性等问题。
因此，实有必要提供一种新的ADC药物以解决现有技术存在的问题。
发明内容
本发明的目的在于解决现有技术存在的问题，提供一种溶酶体靶向的抗体-药物偶联物及其应用，该技术通过溶酶体靶向型小分子或功能性多肽对抗体-药物偶联物中的抗体部分进行修饰，提供了一种简单且具有普适性用于引导抗体-药物偶联物进入溶酶体从而有效释放细胞毒小分子的方法，能成为提高ADC药物疗效的独特且高效的策略，以提高抗体-药物偶联物在溶酶体内的富集、增强抗体-药物偶联物的细胞内化能力、提高抗体-药物偶联物的内化速率以及溶酶体富集程度，从而提高了抗体-药物偶联物的治疗效果。
为达上述目的，本发明提供一种溶酶体靶向的抗体-药物偶联物，所述抗体-药物偶联物的结构如下所示：
Dr n1AbO n2；
其中，Dr为药物；
Ab为抗体；
O为溶酶体靶向型小分子或用于增加所述抗体-药物偶联物的溶酶体靶向能力的功能性多肽；
n1、n2为大于或等于1的整数，n1和n2相同或不同。
优选地，所述抗体为单克隆抗体或纳米抗体，所述单克隆抗体为Glembatumumab、Vandortuzumab、Tisotumab、Enfortumab、Cetuximab、Coltuximab、Lorvotuzumab、Gemtuzumab、Trastuzumab、Ladiratuzumab中的一种；所述纳米抗体为7D12、EGA1、9G8、C7b、5F7、2Rs15d中的一种。
优选地，所述功能性多肽为溶酶体分选肽、细胞穿膜肽或溶酶体分选肽与细胞穿膜肽的组合物；所述功能性多肽连接于所述抗体的C末端。
优选地，所述溶酶体分选肽为NPXY、
Figure PCTCN2020119996-appb-000001
[DE]XXXL[LI]、DXXLL、NPFXD、NPFXXD中的一种，其中X为任意氨基酸，
Figure PCTCN2020119996-appb-000002
为任意大体积疏水侧链氨基酸，N为天冬酰胺，P为脯氨酸，Y为酪氨酸，E为谷氨酸，L为亮氨酸，I为异亮氨酸，F为苯丙氨酸，D为天冬氨酸，[DE]表示为D或E其中之一，[LI]表示为L或I其中之一。
优选地，所述细胞穿膜肽为阳离子型穿膜肽、两亲型穿膜肽、疏水型穿膜肽中的一种。
优选地，所述阳离子型穿膜肽为多聚精氨酸、HIV-TAT、DPV1047、Penetratin中的一种；所述两亲型穿膜肽为MPG、PVEC、MAP、Pept-1、Transportan、P28中的一种；所述疏水型穿膜肽为C105Y、PFVYLI、Pep-1中的一种。
优选地，所述溶酶体靶向型小分子连接于所述抗体的氨基酸侧链。
优选地，所述氨基酸侧链为天然氨基酸侧链或生物正交基团。
优选地，所述天然氨基酸侧链为赖氨酸侧链、半胱氨酸侧链中的一种； 所述生物正交基团为叠氮基团、炔基、醛基、酮基、氟代磺酸酯基团中的一种。
优选地，所述溶酶体靶向型小分子为含pH敏感性基团的化合物或含糖基团的化合物。
优选地，所述pH敏感性基团为磺酸基、4-吗啉基、2-吗啉乙氨基乙基、甲氧基聚乙二醇基团中的一种。
优选地，所述糖基团为葡萄糖、甘露糖、6-磷酸-甘露糖、半乳糖中的一种。
本发明还提供一种上述溶酶体靶向的抗体-药物偶联物在抗癌药物中的应用。
本发明的溶酶体靶向的抗体-药物偶联物相比现有技术具有以下优点：
本发明所述的溶酶体靶向的抗体-药物偶联物，通过溶酶体靶向型小分子或功能性多肽对抗体-药物偶联物中的抗体进行修饰，使得抗体-药物偶联物具有显著的细胞内化能力、更快的内化速率和更高的溶酶体富集程度，增加了抗体-药物偶联物的细胞穿透性，同时抗体-药物偶联物的溶酶体靶向性优异。相比未经修饰的传统抗体-药物偶联物，本发明的溶酶体靶向的抗体-药物偶联物具有更高的溶酶体靶向性，并显示更强的抑制癌细胞增殖的能力、更高的癌细胞致死率以及更优异的体外抗肿瘤活性；因此，为提高ADC药物的疗效提供了新的可能。
附图说明
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明，其中：
图1是本发明实施例1所述的7D12-TAMRA-LPETG-His 6的MALDI-TOF Mass图；
图2是本发明实施例1所述的7D12-MMAF-LPETG-His 6的 MALDI-TOF Mass图；
图3是本发明实施例2和3所述的功能性多肽的序列和溶酶体靶向型小分子的结构式，化合物1是磺基乙酸、化合物2是2-吗啉乙酸、化合物3是2-吗啉乙氨基丙酸、化合物4是2-(2-甲氧基乙氧基)乙氧基乙酸、化合物5是葡糖乙酸；
图4a是本发明实施例2所述的7D12-多肽偶联物的聚丙烯酰胺凝胶电泳(SDS-PAGE)以及His标签抗体免疫印迹试验(anti-His western blot)表征结果；
图4b为Trastuzumab-多肽偶联物的聚丙烯酰胺凝胶电泳(SDS-PAGE)；
图5是本发明实施例3所述的7D12-TAMRA-SA的MALDI-TOF Mass表征结果；
图6是本发明实施例3所述的7D12-TAMRA-PEG3的MALDI-TOF Mass表征结果；
图7是本发明实施例3所述的7D12-TAMRA-morpholine的MALDI-TOF Mass表征结果；
图8是本发明实施例3所述的7D12-TAMRA-EtNH-morpholine的MALDI-TOF Mass表征结果；
图9是本发明实施例3所述的7D12-TAMRA-glucose的MALDI-TOF Mass表征结果；
图10是本发明实施例3所述的7D12-MMAF-SA的MALDI-TOF Mass表征结果；
图11是本发明实施例3所述的7D12-MMAF-PEG3的MALDI-TOF Mass表征结果；
图12是本发明实施例3所述的7D12-MMAF-morpholine的MALDI-TOF Mass表征结果；
图13是本发明实施例3所述的7D12-MMAF-EtNH-morpholine的MALDI-TOF Mass表征结果；
图14是本发明实施例3所述的7D12-MMAF-glucose的MALDI-TOF  Mass表征结果；
图15是本发明实施例4所述的7D12偶联物内化特性的表征结果；
图16是本发明实施例4所述的7D12偶联物随着时间内化到A431细胞中的平均荧光强度变化曲线；
图17a是本发明实施例5所述的7D12-TAMRA、7D12-TAMRA-LSP、7D12-TAMRA-R 9、7D12-TAMRA-R 9-LSP、7D12-TAMRA-TAT、7D12-TAMRA-TAT-LSP的共聚焦显微镜图片；
图17b是本发明实施例5所述的7D12-TAMRA、7D12-TAMRA-SA、7D12-TAMRA-PEG3、7D12-TAMRA-morpholine、7D12-TAMRA-EtNH-morpholine、7D12-TAMRA-glucose的共聚焦显微镜图片；
图17c是本发明实施例5所述的Trastuzumab-TAMRA、Trastuzumab-TAMRA-R9、Trastuzumab-TAMRA-R9-LSP和Trastuzumab-TAMRA-LSP的共聚焦显微镜图片；
图18a是图17a所示的偶联物和溶酶体之间的皮尔斯相关系数测试结果；
图18b是图17b所示的偶联物和溶酶体之间的皮尔斯相关系数测试结果；
图18c为图17c所示的偶联物和溶酶体之间的皮尔斯相关系数测试结果；
图19a是在TAMRA标记的抗体浓度和测量得到的荧光强度之间绘制的线性关系图；
图19b是内化到细胞中的抗体量计算结果；
图20是温度和内吞作用抑制剂对7D12偶联物内化的影响测试结果；
图21是7D12-TAMRA-LSP，7D12-TAMRA-TAT和7D12-TAMRA-R 9与网格蛋白或小窝蛋白-1共定位的测试结果；
图22是7D12-TAMRA-LSP，7D12-TAMRA-TAT和7D12-TAMRA-R 9和网格蛋白或小窝蛋白-1之间的皮尔斯相关系数测试结果；
图23是细胞活力相对于MMAF，7D12或测试的偶联物的浓度的关系 图；
图24是三维细胞肿瘤球体的共聚焦图像；
图25是不同浓度的7D12偶联物处理下测试对肿瘤球体生长的抑制作用测试结果；
图26是肿瘤球体大小变化的测试图像；
图27是本发明所述的抗体-药物偶联物的作用机制原理图。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
抗体-药物偶联物的制备
(1)抗体的选择
本发明的抗体为单克隆抗体或纳米抗体，所述单克隆抗体为Glembatumumab、Vandortuzumab、Tisotumab、Enfortumab、Cetuximab(西妥昔单抗)、Coltuximab、Lorvotuzumab、Gemtuzumab(吉妥珠单抗)、Trastuzumab(曲妥珠单抗)、Ladiratuzumab中的一种；所述纳米抗体为7D12、EGA1、9G8、C7b、5F7、2Rs15d中的一种。上述抗体的氨基酸序列如下所示：
①纳米抗体
7D12的氨基酸序列为：
Figure PCTCN2020119996-appb-000003
EGA1的氨基酸序列为：
Figure PCTCN2020119996-appb-000004
Figure PCTCN2020119996-appb-000005
9G8的氨基酸序列为：
Figure PCTCN2020119996-appb-000006
C7b的氨基酸序列为：
Figure PCTCN2020119996-appb-000007
5F7的氨基酸序列为：
Figure PCTCN2020119996-appb-000008
2Rs15d的氨基酸序列为：
Figure PCTCN2020119996-appb-000009
②单克隆抗体
Glembatumumab的氨基酸序列为：
Figure PCTCN2020119996-appb-000010
Figure PCTCN2020119996-appb-000011
Vandortuzumab的氨基酸序列为：
Figure PCTCN2020119996-appb-000012
Tisotumab的氨基酸序列为：
Figure PCTCN2020119996-appb-000013
Figure PCTCN2020119996-appb-000014
Enfortumab的氨基酸序列为：
Figure PCTCN2020119996-appb-000015
Figure PCTCN2020119996-appb-000016
Cetuximab的氨基酸序列为：
Figure PCTCN2020119996-appb-000017
Coltuximab的氨基酸序列为：
Figure PCTCN2020119996-appb-000018
Figure PCTCN2020119996-appb-000019
Lorvotuzumab的氨基酸序列为：
Figure PCTCN2020119996-appb-000020
Figure PCTCN2020119996-appb-000021
Gemtuzumab的氨基酸序列为：
Figure PCTCN2020119996-appb-000022
Trastuzumab的氨基酸序列为：
Figure PCTCN2020119996-appb-000023
Figure PCTCN2020119996-appb-000024
Ladiratuzumab的氨基酸序列为：
Figure PCTCN2020119996-appb-000025
(2)抗体的突变
为方便理解，本发明以纳米抗体7D12和单克隆抗体Trastuzumab为例进行阐述。需注意的是，本发明的突变后抗体并不限于以下抗体或突变位点，本发明同样适用于其他抗体的其他位点的突变，突变后抗体的选择并不影响本发明抗体-药物偶联物的性能。
①纳米抗体7D12的突变
本实施例选择抗-表皮生长因子受体(Epidermal Growth Factor Receptor，EGFR)的单域纳米抗体7D12作为模板抗体。其中，7D12抗体具有如下特点：(1)7D12抗体对EGFR具有高亲和力并且结合EGFR后能够引起内化；(2)7D12抗体由于缺乏Fc片段不能引起抗体依赖的细胞介导的细胞毒性作用和补体依赖的细胞毒性作用，简化了蛋白修饰对抗体-药物偶联物的干扰。为了得到位点特异性的标记抗体，将7D12抗体上第85位的丝氨酸(Ser85，S85)或第40位的丙氨酸(Ala40，A40)、第42位的甘氨酸(Gly42，G42)、第15位的甘氨酸(Gly15，G15)、第75位的丙氨酸(Ala75，A75)、第66位的甘氨酸(Gly66，G66)中的一种，突变为半胱氨酸(Cys，C)。上述突变后抗体的氨基酸序列如下所示：
S85突变为C：
Figure PCTCN2020119996-appb-000026
A40突变为C：
Figure PCTCN2020119996-appb-000027
G42突变为C：
Figure PCTCN2020119996-appb-000028
G15突变为C：
Figure PCTCN2020119996-appb-000029
A75突变为C：
Figure PCTCN2020119996-appb-000030
G66突变为C：
Figure PCTCN2020119996-appb-000031
本发明的7D12抗体优选的突变位点为第85位，选择第85位进行突变是因为：(1)其处于中间区域远离靠近N末端的抗原结合区域；(2)溶剂可接触，容易修饰；(3)其位于loop区，尽量减小对蛋白四级结构的干扰。
②单克隆抗体Trastuzumab的突变
本实施例选择单克隆抗体Trastuzumab作为模板抗体。为了得到位点特异性的标记抗体，将Trastuzumab抗体上重链第121位的丙氨酸(Ala121，A121)突变为半胱氨酸(Cys，C)。上述突变后抗体的氨基酸序列如下所示：
突变后Trastuzumab抗体的重链氨基酸序列为：
H-GAMMA1(A121突变为C)：
Figure PCTCN2020119996-appb-000032
Figure PCTCN2020119996-appb-000033
突变后抗体的空间结构更易与药物进行结合，但需注意的是，本发明的突变位点及突变后的氨基酸并不限于上述方式，在其他实施例中还可根据使用需求对其他位点进行突变或突变为其他氨基酸。
(3)突变后的抗体与药物偶联
将上述突变后的抗体与抗肿瘤毒性小分子药物偶联，抗肿瘤毒性小分子药物包括微管蛋白抑制剂和DNA损伤剂。微管蛋白抑制剂例如美登素(Maytansinoids，DM1和DM4)、MMAF(单甲基澳瑞他汀F)、MMAE(一甲基澳瑞他汀E)、紫杉醇、紫杉烷类、tubulysin等。DNA损伤剂，如阿霉素(doxorubicin)、卡奇霉素(calicheamicin)、倍癌霉素(Du℃armycin)、喜树碱、pyrrolobenzodiazepines等。
为方便阐述，本实施例的抗体选用突变后的7D12抗体、抗肿瘤毒性小分子药物选用马来酰亚胺己酸-MMAF(Mc-MMAF)，但应理解，后述操作方法同样适用于其他抗体和抗肿瘤毒性小分子药物。MMAF通过马来酰亚胺与巯基的正交反应连接在突变后的7D12抗体分子上。抗肿瘤毒性小分子药物偶联的具体操作方法如下：
首先，用5mM Tris(2-carboxyethyl)phosphine(TCEP；赛默飞世尔科技公司)在室温下还原突变后的抗体7D12上的-SH约30min，然后用含有1mM EDTA的PBS换液去除TCEP，在蛋白溶液中加入5eq的mc-MMAF在4℃下连接过夜，最后换液去除多余抗肿瘤毒性小分子药物即可得到蛋白质混合液。得到的蛋白质混合液通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF Mass)确定分子量。如图2所示，为本实施例所述的7D12-MMAF-LPETG-His 6的MALDI-TOF Mass图，从图中可以看出突变后 的抗体7D12几乎已经全部与MMAF偶联形成7D12-MMAF抗体-药物偶联物。
(4)标记抗体-药物偶联物
优选地，为方便监测抗体-药物偶联物的内化情况，本发明的抗体-药物偶联物还可进行荧光分子标记，所述荧光分子为罗丹明(Rhodamine)、CY3荧光染料(Cyanine 3)、德克萨斯红(Texas Red)荧光染料中的一种。
为方便说明本发明的标记方法，继续使用7D12抗体为例进行阐述，当然本发明的标记方法并不只限于7D12抗体上的应用，该标记方法同样适用于其他抗体中。
本实施例中，所述荧光分子选用马来酰亚胺(maleimide)-PEG3-四甲基罗丹明(TAMRA)，TAMRA通过马来酰亚胺与巯基的正交反应连接在突变后的7D12抗体分子上。荧光分子进行标记的具体操作方法如下：
首先，用5mM Tris(2-carboxyethyl)phosphine(TCEP；赛默飞世尔科技公司)在室温下还原突变后的7D12抗体上的-SH约30min，然后用含有1mM EDTA的PBS换液去除TCEP，在蛋白溶液中加入5eq的maleimide-PEG3-TAMRA在4℃下连接过夜，最后换液去除多余小分子即可得到7D12-TAMRA。得到的蛋白通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF Mass)确定分子量。如图1所示，为本实施例所述的7D12-TAMRA-LPETG-His 6的MALDI-TOF Mass图，从图中可以看出突变后的抗体7D12几乎已经全部标记上TAMRA。
实施例2
功能性多肽进行修饰
在一实施例中，本发明的抗体-药物偶联物经功能性多肽进行修饰，所述功能性多肽为溶酶体分选肽(Lysosome-sorting peptide，LSP)、细胞穿膜肽(cell-penetrating peptide，CPP，例如TAT或R 9)，或溶酶体分选肽与细胞穿 膜肽的组合物TAT-LSP和R 9-LSP，其中，LSP、TAT、R 9、TAT-LSP以及R 9-LSP的氨基酸序列如图3所示。
所述溶酶体分选肽为NPXY、
Figure PCTCN2020119996-appb-000034
[DE]XXXL[LI]、DXXLL、NPFXD、NPFXXD中的一种，其中X为任意氨基酸，
Figure PCTCN2020119996-appb-000035
为任意大体积疏水侧链氨基酸，N为天冬酰胺，P为脯氨酸，Y为酪氨酸，E为谷氨酸，L为亮氨酸，I为异亮氨酸，F为苯丙氨酸，D为天冬氨酸，[DE]表示为D或E其中之一，[LI]表示为L或I其中之一。
所述细胞穿膜肽为阳离子型穿膜肽、两亲型穿膜肽、疏水型穿膜肽中的一种。
所述阳离子型穿膜肽及其氨基酸序列为以下任一种：
多聚精氨酸，其氨基酸序列为：
R n，n为大于或等于1的整数；
HIV-TAT，其氨基酸序列为：
Figure PCTCN2020119996-appb-000036
DPV1047，其氨基酸序列为：
Figure PCTCN2020119996-appb-000037
Penetratin，其氨基酸序列为：
Figure PCTCN2020119996-appb-000038
所述两亲型穿膜肽及其氨基酸序列为以下任一种：
MPG，其氨基酸序列为：
Figure PCTCN2020119996-appb-000039
PVEC，其氨基酸序列为：
Figure PCTCN2020119996-appb-000040
MAP，其氨基酸序列为：
Figure PCTCN2020119996-appb-000041
Pept-1，其氨基酸序列为：
Figure PCTCN2020119996-appb-000042
Transportan，其氨基酸序列为：
Figure PCTCN2020119996-appb-000043
P28，其氨基酸序列为：
Figure PCTCN2020119996-appb-000044
所述疏水型穿膜肽及其氨基酸序列为以下任一种：
C105Y，其氨基酸序列为：
Figure PCTCN2020119996-appb-000045
PFVYLI，其氨基酸序列为：
Figure PCTCN2020119996-appb-000046
Pep-1，其氨基酸序列为：
Figure PCTCN2020119996-appb-000047
所述功能性多肽修饰的方法包括利用转肽酶和基因重组连接于所述抗体的C末端。所述功能性多肽修饰的方法包括以下步骤：
(1)利用转肽酶将功能性多肽连接于所述抗体的C末端
为减少功能性多肽的引入对抗体结构的影响，采用转肽酶将功能性多肽以肽键的形式结合在突变后抗体的C末端。上述结合的实现需要具备如下两个重要元素：(1)需要通过基因工程在抗体的C末端添加一段转肽酶识别序列(LPETG)，(2)在功能性多肽序列的N末端添加寡聚甘氨酸序列(本实施例中采用三聚甘氨酸GGG)。
①突变后的抗体连接LPETG序列和His 6序列
首先在突变后的抗体7D12-S85C的C末端连接一段LPETG序列和His 6tag序列，连接后的氨基酸序列为：
Figure PCTCN2020119996-appb-000048
在E.coli BL21(DE3)中表达得到了7D12-S85C-LPETG-His 6，并用Ni-NTA beads进行纯化。简言之，先用平衡缓冲溶液(buffer)(400mM NaCl，50mM Tris·HCl pH 8.0，20mM咪唑)平衡Ni-NTA beads，然后上样菌体裂解液上清，再用平衡buffer洗去非特异性结合的蛋白，最后用含200mM咪唑的洗脱buffer洗脱目的蛋白，蛋白回收换液采用BCA法确定蛋白浓度。突变后蛋白与野生型蛋白相比产率没有明显变化。
②功能性多肽修饰
本实施例的转肽酶是来自金黄色葡萄球菌的转肽酶A(Sortase A)的截短片段SrtA ΔN59，其氨基酸序列为：QAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVKLEHHHHHH，表达方法采用常规现有技术。同时，转肽反应的亲核底物(GGG-LSP,GGG-R 9,GGG-R 9-LSP,GGG-TAT,GGG-TAT-LSP)采用标准的固相多肽合成法合成。最后，转肽反应在SrtA ΔN59工作buffer(150mM NaCl，50mM Tris·HCl pH 7.5)中进行，反应体系中加入5eq SrtA ΔN59，1eq 7D12-TAMRA-LPETG-His 6或者7D12-MMAF-LPETG-His 6、或者Trastuzumab-TAMRA-LPETG-His 6或者Trastuzumab-MMAF-LPETG-His 6以及10eq的多肽底物，整个转肽反应过程在室温下进行过夜连接。反应结束后，将反应混合液采用Ni-NTA beads纯化，转肽成功的抗体会脱去His 6tag，因此不能结合在Ni柱上，而未转肽成功的抗体和SrtA ΔN59会亲和吸附 在Ni柱上，采用低浓度咪唑洗脱即可得到反应产物，最终回收的蛋白产物通过SDS-PAGE以及anti-His western blot进行分析，结果显示在相同上样量的情况下，转肽后产物在anti-His western blot上几乎没有条带显现出来。为了统一变量和去除His 6tag对抗体-药物偶联物的影响，按照上述方法在7D12-TAMRA-LPETG-His 6、7D12-MMAF-LPETG-His 6、Trastuzumab-TAMRA-LPETG-His 6和Trastuzumab-MMAF-LPETG-His 6上连接上GGG得到7D12-TAMRA-GGG、7D12-MMAF-GGG、Trastuzumab-TAMRA-GGG和Trastuzumab-MMAF-GGG作为对照。如图4a和图4b所示，图4a为7D12-多肽偶联物的聚丙烯酰胺凝胶电泳(SDS-PAGE)以及His标签抗体免疫印迹试验(anti-His western blot)表征结果，其中各组号对应的多肽偶联物如下：M:marker；1:7D12-TAMRA-LPETG-His 6；2:7D12-TAMRA-GGG；3:7D12-TAMRA-LSP；4:7D12-TAMRA-TAT；5:7D12-TAMRA-TAT-LSP；6:7D12-TAMRA-R 9；7:7D12-TAMRA-R 9-LSP；8:7D12-MMAF-LPETG-His 6；9:7D12-MMAF-GGG；10:7D12-MMAF-LSP；11:7D12-MMAF-TAT；12:7D12-MMAF-TAT-LSP；13:7D12-MMAF-R 9；14:7D12-MMAF-R 9-LSP；图4b为Trastuzumab-多肽偶联物的聚丙烯酰胺凝胶电泳(SDS-PAGE)，15:Trastuzumab；16:Trastuzumab-TAMRA-LSP；17:Trastuzumab-MMAF-LSP；18:Trastuzumab-TAMRA-R 9；19:Trastuzumab-MMA F-R 9；20:Trastuzumab-TAMRA-R 9-LSP；21:Trastuzumab-MMAF-R 9-LSP。
(2)利用基因重组将功能性多肽连接于所述抗体的C末端
①质粒构建
以质粒pET28a-7D12-S85C-LPETG-His 6为模板，在N末端添加用于TEV蛋白酶切割的GAAAACCTGTACTTCCAGGGA序列。同时，在C处添加融合多肽序列代替LPETG-His 6。C-末端通过与有义引物(5'-GGAATTCCATATGGAAAACCTGTACTTCCAGGGACAGGTGAAACTGGAGGAAAGCG-3')和反义引物 (5'-CCGCTCGAGTCAATAGCCCGGGTTGCCGCTGCCTGCTAACGGTCACTTGGGTACC-3')的PCR反应来构建pET28a-7D12-S85C-Peptide。
②蛋白质表达
将10μl含有pET28a-7D12-S85C-Peptide质粒的E.coli BL-21甘油菌加入到10ml Luria broth(LB)培养基(含50μg/ml卡那霉素)中，于恒温摇床中培养过夜(37℃，220rpm)。作为大量表达的种子液。将10ml种子液转接到1L新鲜的液体LB培养基(含50μg/ml卡那霉素)，置于37℃摇床振荡培养约8小时，用紫外可见分光光度计读取菌液在600nm处的吸收值，当OD600达到0.5-0.6时，加入诱导剂IPTG(终浓度为1mM)，于25℃继续培养约16小时。
7D12-S85C-Peptide蛋白的纯化、收集。将培养后的菌液置于落地式高速离心机，使用水平转子离心收集菌体(4℃，5000rpm，30min)，弃去上清，加入适量Ni-NTA Buffer A重新悬浮菌体(每升培养物大约需要15ml Ni-NTA Buffer A)。菌体悬浮液置于冰浴中，使用超声波细胞破碎仪破碎菌体(探头直径为6mm，功率260W)，直至细菌悬浮液不再粘稠并且呈现半透明的均一溶液状态。破碎后的溶液使用定角转子高速离心除去未破碎的菌体、菌体残渣以及不溶的蛋白质(4℃，18000rpm，30min)。使用镍柱纯化7D12-S85C蛋白，用约20倍柱体积的Ni-NTA Buffer A漂洗树脂，收集所有流过镍柱的Ni-NTA Buffer A用于检测和镍柱非特异性结合的杂质蛋白质。随后，分别用含不同咪唑浓度的Ni-NTA Buffer B(20mM，50mM，100mM，200mM，400mM，800mM)对镍柱树脂上的目的蛋白进行梯度洗脱，分别收集各个组分，进行SDS-PAGE电泳分析。
依据SDS-PAGE电泳分析的结果，合并含有较纯目的蛋白组分，转移至蛋白质浓缩管中，使用含5mMβ-ME的PBS(pH 7.4)置换含有咪唑的高盐Ni-NTA Buffer，确保将蛋白质溶液中的咪唑含量降至1mM以下，换液四次并将蛋白浓缩到1.5ml，并用NanoDrop测溶液中7D12-S85C蛋白的浓度。
(3)蛋白质与荧光小分子或毒性小分子的偶联
Maleimide-PEG3-TAMRA特异性标记7D12-S85C-Peptide。在7D12-S85C蛋白中加入含有5mM TCEP(一种非常有效的硫醇类还原剂可将蛋白内或蛋白间的二硫键还原)的PBS(pH 7.4)，室温下孵育30分钟。然后将蛋白转移到蛋白质浓缩管中(截断值为10kDa)，用含有1mM EDTA的PBS(pH 7.4)换液(3800rpm，4℃)。将换液后的蛋白保存于1.5ml EP管中，并用NanoDrop测定浓度。紧接着进行标记反应，总共反应体积1ml，蛋白终浓度为1mg/ml，maleimide-PEG3-TAMRA(用适量DMSO溶解)为蛋白五倍量，荧光分子与蛋白混合后，在4℃摇床中孵育2h(避光)。反应完成后用PBS(pH 7.4)换液除去荧光小分子，并用NanoDrop测定蛋白浓度。反应后的7D12-TAMRA蛋白保存于-80℃冰箱中。
7D12-S85C-Peptide与mc-MMAF偶联。在7D12-S85C蛋白中加入含有5mM TCEP的PBS(pH 7.4)，室温下孵育30分钟。然后将蛋白转移到蛋白质浓缩管中(截断值为10kDa)，用含有1mM EDTA的PBS(pH 7.4)换液(3800rpm，4℃)。将换液后的蛋白保存于1.5ml EP管中，并用NanoDrop测定浓度。紧接着进行标记反应，总共反应体积1ml，蛋白终浓度为1mg/ml，mc-MMAF(用适量DMSO溶解)为蛋白五倍量，荧光分子与蛋白混合后，在4℃摇床中孵育2h(避光)。反应完成后用PBS(pH 7.4)换液除去药物小分子，并用NanoDrop测定蛋白浓度。反应后的7D12-MMAF蛋白保存于-80℃冰箱中。
为方便理解，在本实施例中选用了实施例1中7D12-MMAF、Trastuzumab-MMAF为例进行说明，但本发明并不限于此，本实施例的修饰方法同样适用于其他抗体与其他抗肿瘤毒性小分子形成的其他抗体-药物偶联物。
需注意的是，当利用转肽酶将功能性多肽连接于所述抗体的C末端时，本发明并不特别限制功能性多肽修饰与偶联药物的顺序，也就是，突变后的 抗体可以先经功能性多肽修饰再与抗肿瘤毒性小分子药物进行偶联，也可以先与抗肿瘤毒性小分子药物偶联再经功能性多肽进行修饰；功能性多肽修饰与药物偶联的顺序并不影响本发明抗体-药物偶联物的性能。当利用基因重组将功能性多肽连接于所述抗体的C末端时，本发明需先将突变后的抗体经功能性多肽修饰再与抗肿瘤毒性小分子药物进行偶联。
实施例3
溶酶体靶向型小分子修饰抗体药物偶联物
在另一实施例中，本发明的抗体-药物偶联物除采用在抗体的C末端连接功能性多肽的方法修饰外，还可以采用溶酶体靶向型小分子对抗体药物偶联物进行修饰。
所述溶酶体靶向型小分子为含pH敏感性基团的化合物或含糖基团的化合物，其中，所述pH敏感性基团为磺酸基、4-吗啉基、2-吗啉乙氨基乙基、甲氧基聚乙二醇基团中的一种；所述糖基团为葡萄糖、甘露糖、6-磷酸-甘露糖、半乳糖中的一种。溶酶体靶向型小分子的结构式如下所示：
X-Y-Z；
其中，
X选自：
Figure PCTCN2020119996-appb-000049
Figure PCTCN2020119996-appb-000050
Y选自：
Figure PCTCN2020119996-appb-000051
Figure PCTCN2020119996-appb-000052
Z选自：
Figure PCTCN2020119996-appb-000053
波浪线表示连接位置；
星号表示该位置的碳原子为差向异构体的一种或两种的混合物；
n3,n4为大于或等于1的整数；
n5,n6,n7,n8,n9为大于或等于0的整数；
n5和n6相同或不同；
n7,n8和n9相同或不同。
所述溶酶体靶向型小分子连接于抗体的氨基酸侧链，氨基酸侧链为天然氨基酸侧链或生物正交基团，天然氨基酸侧链为赖氨酸侧链、半胱氨酸侧链中的一种；生物正交基团含有能发生生物正交反应的叠氮基团、炔基、醛基、酮基、氟代磺酸酯基团中的一种，生物正交基团可以通过基因扩增技术或酶促反应引入。
为方便理解，本发明以图3中溶酶体靶向型小分子(1-9)为例进行阐述。
本实施例的溶酶体靶向型小分子修饰抗体-药物偶联物的具体步骤如下：
本实施例中，所述溶酶体靶向型小分子分别连接于经MMAF和TAMRA标记的抗体上(7D12-MMAF和7D12-TAMRA)。
为将溶酶体靶向型小分子连接在抗体上，采用具体如下步骤：首先用DMF溶解小分子，并加入2eq的EDC/NHS进行活化，室温下活化6小时后在真空浓缩仪中去除DMF。待溶剂蒸干后，加入PBS溶解，同时加入7D12-TAMRA、7D12-MMAF，4℃下过夜连接，通过超滤浓缩去除多余小分子，得到的蛋白用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF Mass表征)，结果如图5-14所示。根据图5-14的结果显示，因为不是位点特异性连接，最终得到的蛋白质混合物，但每个蛋白平均连接1个小分子。最后共得到10种偶联物，包括 7D12-MMAF-小分子偶联物(7D12-MMAF-SA，图10),7D12-MMAF-吗啉(morpholine)(图12),7D12-MMAF-EtNH-morpholine(图13),7D12-MMAF-PEG3(图11)，7D12-MMAF-葡萄糖(glucose)(图14)，7D12-TAMRA-小分子偶联物(7D12-TAMRA-SA,图5)，7D12-TAMRA-morpholine(图7),7D12-TAMRA-EtNH-morpholine(图8),7D12-TAMRA-PEG3(图6)和7D12-TAMRA-glucose(图9)。
需注意的是，本发明并不特别限制溶酶体靶向型小分子修饰与药物偶联的顺序，也就是，本发明突变后的抗体可以先经溶酶体靶向型小分子修饰再与抗肿瘤毒性小分子药物进行偶联，也可以先与抗肿瘤毒性小分子药物偶联再经溶酶体靶向型小分子进行修饰；溶酶体靶向型小分子修饰与药物偶联的顺序并不影响本发明抗体-药物偶联物的性能。
实施例4
在本实施例中，采用流式细胞术监测本发明分别经功能性多肽修饰和经溶酶体靶向型小分子修饰的抗体-药物偶联物的内化情况，具体包括以下步骤：
(1)细胞培养
采用A431、BT474、MCF-7、HEK293T细胞系(购买自中科院上海细胞库(来源：ATCC))，将细胞培养在添加10％胎牛血清的DMEM(Gibco，USA)培养基中，外加青霉素(100U/mL)-链霉素(100g/mL)。所有的细胞均培养在37℃含有5％CO 2的湿润环境下。细胞形态正常，生长状态良好。
(2)采用流式细胞术监测经修饰的抗体-药物偶联物的内化情况：
除了抗体本身的特异性、选择性和药代动力学性质外，ADC药物的有 效性同时也依赖于抗原的细胞内分选动力学。通过流式细胞术，监测经荧光分子TAMRA修饰的7D12抗体在不同时间点下细胞内化过程中的表现。
使用不同种修饰(7D12-TAMRA，7D12-TAMRA-LSP，7D12-TAMRA-R 9，7D12-TAMRA-R 9-LSP，7D12-TAMRA-TAT，7D12-TAMRA-glucose)后的7D12偶联物与A431细胞共孵育，用无血清的培养基将几种修饰后的7D12-TAMRA稀释至终浓度为2μM。随后使用酸洗除去细胞膜上结合的抗体，通过流式细胞术在不同时间点定量分析。
通过流式细胞术的分析和平均细胞荧光值的分析结果清楚地表明，不同的7D12偶联物具有相似的内化终点并在4小时达到终点。因此，我们在后续的实验中选择孵育4小时后观察其与溶酶体的共定位。特别之处在于，与具有或不具有修饰的偶联物相比，R 9修饰的偶联物具有明显的、更强及更快的内化能力。相反，几种其他的功能性肽在同一时间显示相对较小的峰移位，这也表明较少的抗体药物偶联物被内化到细胞中，存在较低的平均细胞荧光值，之后达到平台。此外R 9和R 9-LSP之间没有差异，证明与R 9的连接是一种强大的细胞穿透修饰，不会受到外加其他功能肽的影响。测试结果如图15和图16所示，图15为7D12偶联物内化特性的表征结果，代表性的流式细胞术图显示7D12偶联物在不同时间点的内化情况，图16为7D12偶联物随着时间内化到A431细胞中的平均荧光强度变化曲线。数据表示为平均值±SEM(n＝3)。
实施例5
经功能性多肽修饰的抗体-药物偶联物和经溶酶体靶向型小分子修饰的抗体-药物偶联物的溶酶体靶向效率的研究：
通常ADC药物内吞途径中的运输和加工过程具体为：ADC药物与其表面肿瘤抗原结合，ADC药物被内化成内体，随后成熟并与溶酶体融合。在 溶酶体中，通过特异性蛋白酶(例如组织蛋白酶B)或通过ADC药物降解来切割接头以释放药物。释放的药物穿过溶酶体膜并到达细胞质与其靶标结合，例如微管蛋白或DNA，最终诱导细胞死亡。因此，ADC药物内化后是否进入溶酶体，并且能否在溶酶体累积，是决定其发挥药效的重要因素。
基于上述流式观察内化动力学的结果，选择4小时作为终点，观察修饰后的抗体与溶酶体共定位的程度。在共孵育4小时后，通过激光共聚焦显微镜观察几种不同的经修饰的7D12偶联物和Trastuzumab偶联物靶向溶酶体的情况。用溶酶体绿色荧光探针(LysoTracker Green)染色细胞中的溶酶体，用荧光分子TAMRA标记7D12-偶联物和Trastuzumab偶联物。在任何可比较的图像集中，图像缩放和拍照条件是相同的。在定量分析中，相同的阈值用于所有图像。
7D12-偶联物的测试结果如图17a和17b所示，图17a和17b为7D12偶联物的内化和其通过溶酶体转运研究结果，其中图17a为7D12-TAMRA、7D12-TAMRA-LSP、7D12-TAMRA-R 9、7D12-TAMRA-R 9-LSP、7D12-TAMRA-TAT、7D12-TAMRA-TAT-LSP的共聚焦显微镜图片，其中，左列为溶酶体，中列为7D12偶联信号，右列为左列和中列重合后的图像。图17b为7D12-TAMRA、7D12-TAMRA-SA、7D12-TAMRA-PEG3、7D12-TAMRA–morpholine、7D12-TAMRA-EtNH-morpholine、7D12-TAMRA-glucose的共聚焦显微镜图片，其中，左列为溶酶体，中列为7D12偶联信号，右列为左列和中列重合后的图像。图18a为图17a所示的偶联物和溶酶体之间的皮尔斯相关系数测试结果(Pearson’s correlation coefficients)，图18b是图17b所示的偶联物和溶酶体之间的皮尔斯相关系数测试结果，平均值±SEM。误差棒代表SEM(n＝20)。ns：没有显著性。**P<0.01；***P<0.001；****P<0.0001。
Trastuzumab偶联物的测试中还使用Hoechst33342进行染色。Trastuzumab偶联物的测试结果如图17c所示，17c为Trastuzumab偶联物的 内化和其通过溶酶体转运研究结果，其中图17c为Trastuzumab-TAMRA、Trastuzumab-TAMRA-R9、Trastuzumab-TAMRA-R9-LSP和Trastuzumab-TAMRA-LSP的共聚焦显微镜图片，其中，第一列为细胞核用Hoechst33342染色后的结果，第二列为溶酶体，第三列为Trastuzumab偶联信号，第四列为第一列至第三列重合后的图像。图18c为图17c所示的偶联物和溶酶体之间的皮尔斯相关系数测试结果(Pearson’s correlation coefficients)，平均值±SEM。误差棒代表SEM(n＝20)。ns：没有显著性。***P<0.001；****P<0.0001。
7D12-偶联物显示出对EGFR的抗原特异性。在具有低表达EGFR的阴性对照HEK293T细胞系中，7D12偶联物在细胞膜上不显示特异性结合。其次，通过比较几种修饰方法对共定位程度的影响后，使用细胞穿膜肽R 9加LSP修饰可以最好地帮助7D12偶联物特异性靶向活细胞中的溶酶体。在添加或不添加LSP的情况下，细胞穿膜肽修饰的偶联物与溶酶体之间的重叠程度存在显著差异。表明细胞穿膜肽有助于纳米抗体进入细胞，而LSP有助于它识别溶酶体。此外，已被证明靶向溶酶体的小分子修饰的结合物也显示出良好的溶酶体共定位，包括EtNH-morpholine，glucose。目前还没有人尝试将这些小分子应用于纳米抗体以增加其溶酶体靶向性。
实施例6
定量分析经功能性多肽和经溶酶体靶向型小分子修饰对抗体-药物偶联物内化水平的影响：
通过实施例4确定与溶酶体的共定位情况后，通过具体数值衡量内化进入细胞的几种7D12偶联物的浓度。从而反映不同修饰下，抗体被内化程度的改变，并尝试解释内化后抗体-药物偶联物在溶酶体累积的表现。
为了量化进入细胞的抗体-药物偶联物，如图19a所示，在TAMRA标记的抗体浓度和测量得到的荧光强度之间绘制线性关系图，测试结果表 明，标准曲线成高度线性关系(R 2＝0.9936)用于后续将原始荧光强度数据转换为7D12-TAMRA浓度。n＝3，误差棒代表SD。随后进行修饰的7D12内化定量，将几种7D12偶联物与A431细胞在12孔板中以2μM的终浓度孵育4小时，酸洗，洗去细胞表面结合的抗体后，裂解细胞得到上清然后通过荧光测定法进行荧光测量。从标准曲线获得线性关系后，计算内化到细胞中的抗体量，其结果如图19b所示，数据表示为平均值±SEM(n＝3)，**P<0.01。
结果表明，与未经修饰的7D12相比，用R 9-LSP修饰的7D12具有更高的内化浓度，这再次证明R 9增加了抗体的内化能力。另一方面，glucose的修饰对7D12的内化浓度没有太大影响。
实施例7
经功能性多肽修饰的抗体-药物偶联物和经溶酶体靶向型小分子修饰的抗体-药物偶联物内化机制的研究：
细胞的内吞作用是一种复杂的机制，涉及不同的途径和蛋白质-蛋白质和蛋白质-脂质相互作用的大型网络。第一个也是最具特征的途径是网格蛋白依赖的内吞作用，它从质膜开始，形成网格蛋白包被的内陷，夹住以构成网格蛋白包被的囊泡。较不明确的是非经典的，网格蛋白非依赖性途径，其中包括小窝蛋白介导的内吞作用。以往的研究表面网格蛋白介导的内化，更倾向与溶酶体融合，最终内吞物被溶酶体环境消化，释放进入细胞。而小窝蛋白介导的内化使得早期囊泡更容易进入高尔基体或内质网与之融合，最后部分通过膜的循环转移，被排出细胞外。
本实施例中，首先为了测试7D12偶联物的细胞摄取是否是能量依赖性的，将7D12偶联物在4℃下的细胞摄取与37℃下的细胞摄取进行比较。7D12-TAMRA-功能性多肽和7D12-TAMRA-溶酶体靶向型小分子的摄取均在4℃时被显著抑制，表明纳米抗体的内化是具有ATP依赖性的。然后，为了进一步表征摄取机制，使用胞吞作用抑制剂。阳离子两性药物氯丙嗪 (CPZ)用于通过诱导网格蛋白的损失来抑制网格蛋白介导的内吞作用。乙基异丙基酰胺(EIPA)，一种Na +/H +交换抑制剂，在血浆膜上引起胆固醇螯合，用于阻断巨胞饮作用。以及制霉菌素(Nystatin)，能够抑制细胞膜穴样内陷介导的内吞作用。在不同种抑制剂推荐使用浓度和孵育时间分别处理后，将细胞与不同的7D12偶联物进行共孵育。通过观察与不处理组的内化情况相比的变化，来判断7D12偶联物的内化方式具体以哪一种方式为主。
三种小分子抑制剂分别显著抑制了7D12偶联物的内化。这些抑制剂之间在7D12-TAMRA，7D12-TAMRA-LSP和7D12-TAMRA-morpholine的内化方面没有显著差异。然而，7D12-TAMRA-R 9-LSP的摄取主要受CPZ抑制，而7D12-TAMRA-TAT-LSP主要受制霉菌素抑制。测试结果如图20所示，图20为温度和内吞作用抑制剂对7D12偶联物内化的影响，7D12-TAMRA-R 9-LSP和7D12-TAMRA-TAT-LSP的吸收主要分别为网格蛋白介导的内吞作用和小窝蛋白介导的内吞作用，这与溶酶体共定位的结果一致。
实施例8
经功能性多肽修饰的抗体-药物偶联物和经溶酶体靶向型小分子修饰的抗体-药物偶联物的胞内转运研究：
小窝蛋白介导的内吞作用被认为是造成靶向其抗原的ADC药物不敏感的可能机制之一。现有技术表明TAT修饰诱导的内化主要通过小窝蛋白介导的内化途径。同时也指明LSP被网格蛋白AP2的结合内化。因此，本实施例评估了几种功能性多肽修饰后的7D12与小窝蛋白-1或网格蛋白的共定位。
首先，将A431细胞与几种7D12偶联物一起孵育，使用兔源抗体抗网格蛋白和抗小窝蛋白-1作为一抗。然后孵育FITC标记的抗兔二抗用于免疫 荧光分析。
图21为7D12偶联物(7D12-TAMRA-LSP，7D12-TAMRA-TAT和7D12-TAMRA-R 9)与网格蛋白或小窝蛋白-1共定位的测试结果，细胞核用Hoechst 33342染色，所显示为代表性细胞的图像。测试发现细胞内7D12-TAMRA-LSP不与小窝蛋白共定位，但与网格蛋白部分共定位。相反，发现7D12-TAMRA-TAT与网格蛋白没有共定位，而是与小窝蛋白共定位。不同的是，7D12-TAMRA-R 9与网格蛋白和小窝蛋白均有共定位。测试结果如图22所示：7D12偶联物(7D12-TAMRA-LSP，7D12-TAMRA-TAT和7D12-TAMRA-R 9)和网格蛋白或小窝蛋白-1之间的皮尔斯相关系数测试(Pearson’s correlation coefficients，平均值±SEM)。误差棒代表SEM(n＝20)。*P<0.05；****P<0.0001。这表明在7D12-TAMRA-R 9的内化过程中，网格蛋白介导的内吞作用和小窝蛋白介导的内吞作用都有涉及。
实施例9
经功能性多肽修饰的抗体-药物偶联物和经溶酶体靶向型小分子修饰的抗体-药物偶联物的体外药效学研究：
(1)通过2D肿瘤细胞培养模型观察溶酶体靶向的ADC药物的细胞毒性，具体步骤如下：
选择在人表皮癌细胞A431细胞，评估7D12-MMAF偶联物抑制癌细胞增殖的能力。使用2D肿瘤细胞培养模型，不同浓度的ADC药物被加入种有A431细胞的96孔板中，处理96h后，使用cck-8试剂盒，得到细胞的存活率。
如图23所示的测试结果(通过2D肿瘤细胞培养模型评估7D12偶联物的体外细胞毒性。用MMAF，7D12或所示的不同浓度的7D12-MMAF偶联物处理A431细胞96小时。以细胞活力相对于MMAF，7D12或测试的偶联物的浓度作图)表明，靶向溶酶体修饰的7D12-MMAF偶联物具有显著的杀伤能力。结果与溶酶体共定位实验一致，具有强溶酶体靶向的7D12偶联物 显示出更好的癌细胞致死率。值得注意的是，7D12-MMAF-R 9-LSP偶联物显示出最高的杀伤力，EC 50值为53.74nM。同时，由小分子修饰的几种7D12偶联物中的7D12-MMAF-EtNH-morpholine具有最低的EC 50为117.9nM。采用野生型7D12作为A431细胞系中的同种型对照，并且没有发现显著的细胞毒性来确定ADC的活性。在阴性对照HEK293T细胞系中，发现7D12-MMAF-R 9-LSP有一定的细胞毒性。这可能是细胞穿膜肽(CPP)携带药物进入细胞的能力太强，导致一些非特异性杀伤。随后通过激光共聚焦测试长时间孵育后CPP是否可以将7D12偶联物携带到EGFR阴性细胞中。并且结果表明，在用6小时或甚至更长时间孵育后，用R 9修饰的7D12偶联物被内化到阴性细胞中，这解释了CPP修饰导致非特异性细胞毒性的原因。另外也可以表明如果使用缀合的CPP来增加纳米抗体的内在化能力，同时也需要考虑其对纳米抗体的抗原特异性的影响。
MMAF，7D12或测试的偶联物的EC 50值(nM)如表1所示，数据表示为平均值±SEM(n＝3)。
表1
Figure PCTCN2020119996-appb-000054
Figure PCTCN2020119996-appb-000055
由表1的结果可以看出，与MMAF和7D12-MMAF相比，修饰后的7D12-MMAF偶联物在介导细胞死亡方面表现出更高的效率。总结来说，修饰后的7D12-MMAF偶联物的细胞毒性与其内化和溶酶体靶向能力相关。对于功能性多肽修饰的7D12偶联物，7D12-MMAF-R 9-LSP的癌细胞生长抑制活性最高，其次分别是7D12-MMAF-R 9和7D12-MMAF-LSP。相对于LSP、R 9或R 9-LSP修饰的偶联物，TAT或TAT-LSP修饰的偶联物的癌细胞杀伤活性较低。对于溶酶体靶向型小分子修饰的7D12偶联物，7D12-MMAF-R 9-glucose的癌细胞生长抑制活性最高，其次是7D12-MMAF-EtNH-mophorline。SA、PEG和morpholine修饰的7D12偶联物的细胞杀伤效果没有显著差异。
(2)通过3D肿瘤球体培养模型观察溶酶体靶向的ADC药物的抗肿瘤活性，具体步骤如下：
除单层肿瘤培养模型用于体外毒性评估外，还进一步利用三维球体肿瘤模型评估ADC药物的渗透性和功效：在用各种7D12-TAMRA偶联物孵育6小时后测定对三维肿瘤球体的渗透和共定位能力。所有肿瘤球状体的直径约为100μm。图24为通过3D肿瘤球体模型评估靶向溶酶体的7D12偶联物的抗肿瘤活性，由上至下分别为用7D12-TAMRA，7D12-TAMRA-R 9-LSP和7D12-TAMRA-glucose处理的A431三维细胞肿瘤球体的共聚焦图像。表明没有额外修饰的7D12-TAMRA仅显示出主要为弥漫性的表面染色，没有渗透到A431肿瘤球体的核心中并且几乎不显示与溶酶体的共定位。7D12-TAMRA-R 9-LSP显示出对A431球状体的高渗透性。7D12-TAMRA-glucose的渗透性略弱于7D12-TAMRA-R 9-LSP。它们都具有良好的溶酶体靶向作用。
之后，将3D组织模型用于生长抑制实验测定。选择表达高至低EGFR 水平的三个细胞系：A431，MCF-7，HEK293T。当3D球状体具有直径约200μm的对称和球形形状时，添加不同修饰后的ADC药物。将3D球状体在含有ADC药物(0.25nM至2.5μM)的培养基中孵育10天，同时记录每天球体直径的变化情况。
所有组的平均直径的变化可以在图25-26中看到，图25为不同浓度的7D12偶联物处理下测试对肿瘤球体生长的抑制作用，数据表示为平均值±SEM(n＝3)。图26为随着时间的推移暴露于指定浓度的7D12偶联物下A431肿瘤球体大小变化的代表性图像。这表明与7D12-MMAF相比，7D12-MMAF-R 9-LSP对A431细胞3D球体具有最强的生长抑制。同时，7D12-MMAF-glucose也显示出对肿瘤球体的较强的生长抑制作用。这与2D肿瘤培养模型中观察到的结果一致。除了具有高表达EGFR的A431细胞外，MCF-7和HEK293T用于证明这些ADC药物的特异性。当用相同浓度的ADC处理这些细胞3D球体时，具有中度EGFR表达的MCF-7 3D球体的生长受到轻微抑制。同时，HEK293T 3D球体的生长状态不受实验组的影响。这些结果再次证明了ADC药物靶向特异性抗原的能力，在模拟体内环境的3D球体肿瘤生长中，起到明显的抑制生长作用，并且通过使用靶向溶酶体的修饰来实现ADC药物更好的活性。
上述测试表明，通过两种修饰方法制备溶酶体靶向的抗体-药物偶联物(ADCs)：(1)在抗体的C末端连接功能性多肽；(2)在抗体上偶联溶酶体靶向型小分子。无论是经功能性多肽修饰还是经溶酶体靶向型小分子修饰，均可提高ADC药物在溶酶体内的富集。并且修饰有R 9-LSP功能性多肽序列的ADC药物呈现出显著增强的细胞内化能力、更快的内化速率和更高的溶酶体富集程度。机理研究表明修饰R 9-LSP功能性多肽后的ADC药物会增加由网格蛋白介导的内吞，而经溶酶体靶向型小分子的修饰会改变ADC药物的胞内运输途径。同时还确定了经溶酶体靶向型小分子修饰可以提高ADC药物的体外抗肿瘤活性，原理图如图27所示。本发明提供了一种 简单且具有普适性用于引导ADC药物进入溶酶体从而有效释放细胞毒小分子的方法，可成为提高ADC药物疗效的独特且高效的方法。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (13)

  1. 一种溶酶体靶向的抗体-药物偶联物，其特征在于，所述抗体-药物偶联物的结构如下所示：
    Dr n1AbO n2；
    其中，Dr为药物；
    Ab为抗体；
    O为溶酶体靶向型小分子或用于增加所述抗体-药物偶联物的溶酶体靶向能力的功能性多肽；
    n1、n2为大于或等于1的整数，n1和n2相同或不同。
  2. 根据权利要求1所述的溶酶体靶向的抗体-药物偶联物，其特征在于，所述抗体为单克隆抗体或纳米抗体，所述单克隆抗体为Glembatumumab、Vandortuzumab、Tisotumab、Enfortumab、Cetuximab、Coltuximab、Lorvotuzumab、Gemtuzumab、Trastuzumab、Ladiratuzumab中的一种；所述纳米抗体为7D12、EGA1、9G8、C7b、5F7、2Rs15d中的一种。
  3. 根据权利要求1所述的溶酶体靶向的抗体-药物偶联物，其特征在于，所述功能性多肽为溶酶体分选肽、细胞穿膜肽或溶酶体分选肽与细胞穿膜肽的组合物；所述功能性多肽连接于所述抗体的C末端。
  4. 根据权利要求3所述的溶酶体靶向的抗体-药物偶联物，其特征在于，所述溶酶体分选肽为NPXY、
    Figure PCTCN2020119996-appb-100001
    [DE]XXXL[LI]、DXXLL、NPFXD、NPFXXD中的一种，其中X为任意氨基酸，
    Figure PCTCN2020119996-appb-100002
    为任意大体积疏水侧链氨基酸，N为天冬酰胺，P为脯氨酸，Y为酪氨酸，E为谷氨酸，L为亮氨酸，I为异亮氨酸，F为苯丙氨酸，D为天冬氨酸，[DE]表示为D或E其中之一，[LI]表示为L或I其中之一。
  5. 根据权利要求3所述的溶酶体靶向的抗体-药物偶联物，其特征在于， 所述细胞穿膜肽为阳离子型穿膜肽、两亲型穿膜肽、疏水型穿膜肽中的一种。
  6. 根据权利要求5所述的溶酶体靶向的抗体-药物偶联物，其特征在于，所述阳离子型穿膜肽为多聚精氨酸、HIV-TAT、DPV1047、Penetratin中的一种；所述两亲型穿膜肽为MPG、PVEC、MAP、Pept-1、Transportan、P28中的一种；所述疏水型穿膜肽为C105Y、PFVYLI、Pep-1中的一种。
  7. 根据权利要求1所述的溶酶体靶向的抗体-药物偶联物，其特征在于，所述溶酶体靶向型小分子连接于所述抗体的氨基酸侧链。
  8. 根据权利要求7所述的溶酶体靶向的抗体-药物偶联物，其特征在于，所述氨基酸侧链为天然氨基酸侧链或生物正交基团。
  9. 根据权利要求8所述的溶酶体靶向的抗体-药物偶联物，其特征在于，所述天然氨基酸侧链为赖氨酸侧链、半胱氨酸侧链中的一种；所述生物正交基团为叠氮基团、炔基、醛基、酮基、氟代磺酸酯基团中的一种。
  10. 根据权利要求1所述的溶酶体靶向的抗体-药物偶联物，其特征在于，所述溶酶体靶向型小分子为含pH敏感性基团的化合物或含糖基团的化合物。
  11. 根据权利要求10所述的溶酶体靶向的抗体-药物偶联物，其特征在于，所述pH敏感性基团为磺酸基、4-吗啉基、2-吗啉乙氨基乙基、甲氧基聚乙二醇基团中的一种。
  12. 根据权利要求10所述的溶酶体靶向的抗体-药物偶联物，其特征在于，所述糖基团为葡萄糖、甘露糖、6-磷酸-甘露糖、半乳糖中的一种。
  13. 一种如权利要求1-12任一项所述的溶酶体靶向的抗体-药物偶联物在抗癌药物中的应用。
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