Liposome delivery system of p53 agonistic peptide mediated by humanized cd33 antibody

Abstract

The application provides a liposome drug delivery system, which comprises a liposome, a targeting molecule and a polypeptide drug encapsulated in the liposome, wherein the liposome is prepared from hydrogenated soy phosphatidylcholine, cholesterol, polyethylene glycol-distearyl phosphatidyl ethanolamine and maleimide-polyethylene glycol-distearyl phosphatidyl ethanolamine; the targeting molecule is an antibody coupled to the surface of the liposome, the antibody is a humanized CD33 antibody with targeting effect, the light chain sequence of the humanized CD33 antibody is SEQ ID NO: 1, and the heavy chain sequence of the humanized CD33 antibody is SEQ ID NO: 2; and the polypeptide drug is a p53 agonistic peptide.

Description

Technical Field

[0001] This invention relates to the fields of pharmacology and biology, and specifically to a liposome drug delivery system for humanized CD33 antibody-mediated p53 agonist peptide for the treatment of AML. Background Technology

[0002] Acute myeloid leukemia (AML) is the most common type of acute leukemia, and its incidence and mortality rates are gradually increasing in my country. However, the current clinical treatment options for AML in China are mainly hematopoietic stem cell transplantation and chemotherapy. The high cost and limited matching bone marrow sources have become bottlenecks for the widespread clinical application of hematopoietic stem cell transplantation. Traditional chemotherapy regimens, such as cytarabine combined with daunorubicin "7+3" induction chemotherapy, have significant side effects and also face the challenge of high relapse rates caused by cancer cell mutations and clonal evolution.

[0003] One of the most promising molecular targets in anticancer drug research is the transcription factor p53, which can induce growth inhibition and apoptosis responses to cellular stress and plays a key role in preventing damaged cells from becoming carcinogenic. Impairment of the p53 signaling pathway is widespread in almost all human cancers. The mechanisms of impairment include (1) TP53 gene mutations, and (2) excessive ubiquitination and degradation of p53 protein caused by the E3 ubiquitin ligase MDM2 and / or its homologue MDMX. Furthermore, the proportions of p53 inhibition by MDM2 and MDMX may differ, exhibiting mutual promotion and compensation. Clinical data indicate that approximately 95% of AML patients are TP53 wild-type, with elevated expression of MDM2 and / or MDMX, which synergistically inhibit p53 transactivation activity and target p53 for degradation. Therefore, the MDM2 / MDMX-p53 protein interaction can serve as a broad and highly effective anti-AML therapeutic target, and targeting and blocking the negative regulation of p53 by MDM2 / MDMX has become a feasible strategy for anti-AML drug development.

[0004] A number of small molecule drugs targeting MDM2-p53 or MDMX-p53 with single-effect action have been developed and put into clinical trials. For example, Roche's Nutlin 3 and RG7112, and Amgen's AMG232 have completed Phase I clinical trials, demonstrating significant inhibitory effects on tumor growth in p53 wild-type patients, directly confirming the feasibility of treatment strategies targeting MDM2-p53. However, small molecule inhibitors, including Nutlin 3, RG7112, and AMG232, exhibit strong hematologic toxicity in almost all patients, with more serious side effects including thrombocytopenia and / or neutropenia. Small molecule drugs have fewer sites of action and poorer specificity to target proteins; in addition to binding to MDM2, they may interact with various other non-target proteins, causing unpredictable "off-target toxicity." To improve the selectivity of Nutlin-3a, a suitable drug delivery system is needed to achieve targeted delivery of Nutlin-3a.

[0005] Studies have utilized biochemical and biophysical research tools such as peptide and protein chemistry, structural biology, tumor biology, phage display technology, and molecular design to design a series of highly active novel MDM2 / MDMX dual antagonists to activate p53-PMI peptides (such as PMI-N8A, PMI-M3, and D-PMI-ω with KD values ​​of 0.49 / 2.4 nM, 21 / 253 pM, and 0.16 / 28.7 nM for MDM2 / MDMX, respectively), as well as stapled PMI peptides (sPMI) synthesized using stapling peptide modification technology. Although p53 agonist peptides have strong binding activity and good specificity to MDM2 / MDMX, they also exhibit the following disadvantages: (1) because the target proteins MDM2 and MDMX are located intracellularly, they do not specifically recognize the surface of tumor cells; (2) they have poor diffusion into cells; (3) the L-peptide is degraded by in vivo enzyme barriers; and (4) it is excreted by glomerular filtration (<20 kDa) with a short half-life. Therefore, a suitable drug delivery system is needed to deliver p53 agonist peptide.

[0006] Liposomes are currently widely used in the formulation development of chemotherapy drugs, gene therapy drugs, and diagnostic drugs (radioactive, paramagnetic, etc.). Polyethylene glycol-modified liposomes have a long-term circulating effect in vivo. Modifying the surface of liposomes with functional targeting molecules can endow them with active targeting capabilities. CD33 is a myeloid cell differentiation antigen, expressed on tumor cells in more than 90% of AML patients, but its expression level is very low in hematopoietic stem cells, mature granulocytes, and other tissues. CD33 provides a target basis for recognizing AML tumor cells, and CD33 monoclonal antibodies have become the preferred target head for drug delivery.

[0007] This invention provides a novel humanized CD33 antibody-mediated liposome drug delivery system, comprising liposomes, an antibody conjugated to the surface of the liposomes, and a peptide drug encapsulated within the liposomes. Because the liposome drug delivery system of this invention modifies the surface of the liposomes with a humanized CD33 antibody, it can target and deliver the drug to tumor cells, thereby reversing the drawback of drugs lacking tumor cell targeting and recognition, and simultaneously achieving the therapeutic effect of peptide drugs in AML. Summary of the Invention

[0008] On one hand, the present invention provides a liposome drug delivery system, comprising liposomes, a targeting molecule, and a polypeptide drug encapsulated within the liposomes.

[0009] The liposomes are prepared using hydrogenated soybean phosphatidylcholine, cholesterol, polyethylene glycol-distearate phosphatidylethanolamine, and maleimide-polyethylene glycol-distearate phosphatidylethanolamine.

[0010] The target molecule is an antibody conjugated to the surface of a liposome, the antibody is a humanized CD33 antibody with targeting activity, the light chain sequence of the humanized CD33 antibody is SEQ ID NO:1, and the heavy chain sequence of the humanized CD33 antibody is SEQ ID NO:2.

[0011] The polypeptide drug is p53 agonist peptide.

[0012] In a preferred embodiment, the molar ratio of hydrogenated soybean phosphatidylcholine, cholesterol, polyethylene glycol-distearate phosphatidylethanolamine, and maleimide-polyethylene glycol-distearate phosphatidylethanolamine in the liposome delivery system of the present invention is 50:38.5:3:2.

[0013] In a preferred embodiment, the p53 agonist peptide is selected from: TSFAEYWALLSP (SEQ ID NO:3), LTFLEYWAQLMQ (SEQ ID NO:4), efwyvef(p-Cl)ekllr (SEQ ID NO:5), wherein uppercase letters represent L-configured amino acids and lowercase letters represent D-configured amino acids.

[0014] In a preferred embodiment, the molar ratio of the antibody to maleimide-polyethylene glycol-distearate phosphatidylethanolamine is 1:36-1:200.

[0015] On the other hand, the present invention also provides a method for preparing a liposome drug delivery system, comprising the following steps:

[0016] a) A solution containing hydrogenated soybean phosphatidylcholine, cholesterol, polyethylene glycol-distearate phosphatidylethanolamine and maleimide-polyethylene glycol-distearate phosphatidylethanolamine as lipid excipients;

[0017] b) A solution providing the polypeptide drug;

[0018] c) Mix the solution from step a) with the solution from step b), wherein the weight ratio of lipid excipient to peptide is 10:1 to 20:1, and the volume ratio of the solution from step a) to the solution from step b) is 4:1 to 5:1.

[0019] d) Liposomes loaded with polypeptide drugs were prepared by reverse evaporation method;

[0020] e) The antibody is mixed with tris(2-carboxyethyl)phosphine to obtain a reduced antibody;

[0021] f) The reduced antibody is mixed with the liposomes loaded with peptide drugs prepared in step d) to obtain an antibody-conjugated liposome drug delivery system.

[0022] In a preferred embodiment, the weight ratio of lipid excipient to peptide in step c) is 15.3:1, and the volume ratio of the solution in step a) to the solution in step b) is 4.5:1.

[0023] In a preferred embodiment, step d) includes: adding 10 mM phosphate buffer solution with pH = 6.5 to make the volume ratio of organic phase to aqueous phase 9:1. In a preferred embodiment, step d) further includes: sequentially extruding the membrane through 400 nm, 200 nm and 100 nm.

[0024] In a preferred embodiment, the molar ratio of antibody to tris(2-carboxyethyl)phosphine in step e) is 1:5 to 1:10.

[0025] In a preferred embodiment, the molar ratio of antibody to maleimide-polyethylene glycol-distearate phosphatidylethanolamine in step f) is 1:147. Attached Figure Description

[0026] To provide a more complete understanding of the present invention, the following description is provided in conjunction with the accompanying drawings.

[0027] Figure 1These are the HPLC and ESI-MS chromatograms of PMI-N8A. Chromatographic methods: Column (YMC, C18): 150 × 4.6 mm; Mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid); Elution program: 2-32 min 5% B-65% B; Flow rate: 0.7 mL / min; Column temperature: 40℃; Detection: UV 280 nm, retention time: 22.415 min. ESI-MS: 1383.6, consistent with the theoretical molecular weight.

[0028] Figure 2 These are the HPLC and ESI-MS chromatograms of PMI-M3. The chromatographic method was the same as above, with a retention time of 25.651 min. The ESI-MS value was 1542, consistent with the theoretical molecular weight.

[0029] Figure 3 These are the HPLC and ESI-MS chromatograms of D-PMI-ω. The chromatographic method was the same as above, with a retention time of 23.704 min. The ESI-MS value was 1692.4, consistent with the theoretical molecular weight.

[0030] Figure 4 This is the elution pattern of humanized CD33 antibody on a Protein A antibody affinity chromatography column. After the eukaryotic cell protein expression system produced the antibody, the cell culture medium was collected and loaded onto a Protein A antibody affinity chromatography column (loading buffer: PBS), followed by elution with 100 mM pH=3.0 Gly-HCl. The antibody eluted in approximately 37 minutes.

[0031] Figure 5 SDS-PAGE was used to characterize antibody purity and molecular weight. After reduction with loading buffer (containing β-mercaptoethanol), SDS-PAGE showed heavy chain bands at 55–70 kDa and light chain bands at approximately 25 kDa, consistent with the theoretical molecular weight, and no other significant contaminants. When the antibody was loaded with native loading buffer (without β-mercaptoethanol) onto SDS-PAGE, only one protein band was observed, significantly higher than either the heavy or light chain. The efflux buffer did not contain the corresponding antibody band, indicating that the antibody would not efflux onto the affinity chromatography column, and the purification method was appropriate.

[0032] Figure 6The HPLC-MS characterization of the antibody molecular weight is shown. As shown in the figure, the antibody molecular weight is 148,128, the heavy chain molecular weight is 50,190, and the light chain molecular weight is 23,759. Chromatographic method: Column (Agilent, C4): 150 × 2.1 mm; Mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: 70% isopropanol + 20% acetonitrile + 10% water (containing 0.1% trifluoroacetic acid); Elution program: 2-10 min 100% B; Flow rate: 0.7 mL / min; Column temperature: 25℃; Detection: UV 280 nm, consistent with the theoretical molecular weight.

[0033] Figure 7 Native-PAGE was used to characterize the antibody-liposome coupling reaction. Ab-Lip 1-3 were designed with low (1 / 92.24), medium (1 / 36.89), and high (1 / 18.48) antibody / maleimide-polyethylene glycol-distearate phosphatidylethanolamine molar ratios. The uncoupled residual antibody in the reaction solution showed stronger translocation ability on Native-PAGE than the antibody already coupled to liposomes. Therefore, Native-PAGE can be used to characterize the residual antibody in the reaction. As shown in the figure, the amount of uncoupled residual antibody in Ab-Lip 1-2 was significantly lower than that in Ab-Lip 3, indicating that the antibody / maleimide-polyethylene glycol-distearate phosphatidylethanolamine molar ratio of 1 / 36.89 in Ab-Lip 2 represents the maximum antibody modification ratio.

[0034] Figure 8 The Sepharose CL-4B size exclusion gel chromatography (MSG) was used to characterize the antibody-liposome coupling reaction. Liposomes eluted in Sepharose CL-4B MSG at 6–8 min, while uncoupled free antibody eluted in Sepharose CL-4B at 10–25 min. The results were consistent with the above; Ab-Lip 1 and 2 showed virtually no uncoupled residual antibody elution, while Ab-Lip 3 showed significant free antibody elution. This indicates that the antibody / maleimide-polyethylene glycol-distearate phosphatidylethanolamine dosage ratio (molar ratio) of 1 / 36.89 in Ab-Lip 2 represents the maximum antibody modification ratio.

[0035] Figure 9 The BCA method was used to determine the antibody-conjugated amount on liposomes. After the liposomes were conjugated with antibodies, the unconjugated free antibodies were removed using a Sepharose CL-4B. The amount of antibody conjugated on the liposomes was then determined using the BCA method. The antibody conjugation amount of Ab-Lip 2-3 was the highest, more than twice that of Ab-Lip 1, indicating that the antibody / maleimide-polyethylene glycol-distearate phosphatidylethanolamine dosage ratio (molar ratio) of Ab-Lip 2 was 1 / 36.89, which is the maximum antibody modification ratio.

[0036] Figure 10 Showing Goat Anti-Mouse IgG (Alexa) 488) Detection of antibody-conjugated liposomes. After conjugation of liposomes with antibodies, unconjugated free antibodies were removed using Sepharose CL-4B, and then the antibody levels were measured using goat anti-mouse IgG (Alexa). 488) The antibody conjugated on the liposomes was identified and bound, and the amount of antibody conjugation was determined based on the fluorescence intensity. Ab-Lip 2 had the highest antibody conjugation amount, while Ab-Lip 3 had a similar amount, both greater than Ab-Lip 1. This indicates that the maleimide-polyethylene glycol-distearate phosphatidylethanolamine dosage ratio (molar ratio) represents the maximum proportion of antibody modification.

[0037] Figure 11 Particle size characterization of liposomes with different antibody conjugation amounts. The particle sizes of Ab-Lip 1-3 with antibody / maleimide-polyethylene glycol-distearate phosphatidylethanolamine dosage ratios (molar ratios) of low (1 / 92.24), medium (1 / 36.89), and high (1 / 18.48) were determined using a laser particle size analyzer. The particle size of the unconjugated antibody liposomes (Mal-Lip) was 122.53±0.68 nm, with a PDI of 0.098±0.026; the particle size of Ab-Lip 1 was 133.97±1.44 nm, with a PDI of 0.126±0.016; the particle size of Ab-Lip 2 was 149.43±0.76 nm, with a PDI of 0.117±0.005; and the particle size of Ab-Lip 3 was 142.50±1.35 nm, with a PDI of 0.093±0.030. The antibody conjugation amount of Ab-Lip 2 was significantly greater than that of Ab-Lip 1, and the increase in liposome size was significantly greater for Ab-Lip 2 than for Ab-Lip 1. The antibody conjugation amount of Ab-Lip 2 was basically the same as that of Ab-Lip 3, and the increase in liposome size for both was also similar.

[0038] Figure 12The SDS-PAGE data characterizes the antibody-liposome conjugation method. Mal-Lip represents liposomes without antibody conjugation, Anti-CD33 antibody represents free antibody, and Ab-Lip 2a-d represents conjugation using different concentrations of reducing agent and different treatment methods at the Ab-Lip 2 antibody / liposome dosage ratio. Specifically, Ab-Lip 2a represents an antibody / Tcep molar ratio of 7.5:1 where Tcep is not removed after reduction; Ab-Lip 2b represents an antibody / Tcep molar ratio of 7.5:1 where Tcep is removed by ultrafiltration after reduction; Ab-Lip 2c represents an antibody / Tcep molar ratio of 10:1 where Tcep is not removed after reduction; and Ab-Lip 2d represents an antibody / Tcep molar ratio of 10:1 where Tcep is removed by ultrafiltration after reduction. The SDS-PAGE results show that the light chain band shifts unchanged, while the heavy chain band shifts upward. Except for the case where one heavy chain in the conjugated antibody did not react, the uppermost impurity band in the Ab-Lip 2b group is the lightest, indicating that the reduction method of removing Tcep by ultrafiltration centrifugation after the reaction with an antibody / Tcep molar ratio of 7.5:1 is optimal.

[0039] Figure 13 This study demonstrates the optimization of the antibody / maleimide-polyethylene glycol-distearate phosphatidylethanolamine dosage ratio based on antibody targeting efficiency. Liposomes Ab-Lip@DiD①-③, loaded with the red fluorescent probe DiD, were designed with low (1 / 590.24), medium (1 / 147.56), and high (1 / 36.89) antibody / maleimide-polyethylene glycol-distearate phosphatidylethanolamine dosage ratios (molar ratios). Lip@DiD represents DiD-loaded liposomes without antibody conjugation. Each group of liposomes was applied to AML cells HL-60 (CD33). + MOLM-13 (CD33) + SUP-B15 (CD33) - Figure A shows the quantitative analysis of DiD red fluorescence by flow cytometry; Figure B shows the average fluorescence intensity during uptake; Figure C shows the median fluorescence intensity during uptake; and Figure D shows the percentage of positive cells. HL-60 and MOLM-13 showed the highest uptake of Ab-Lip@DiD② and exhibited strong cell selectivity, while SUP-B15 showed uptake of Ab-Lip@DiD①-③ comparable to Lip@DiD. These results indicate that a molar ratio of 1 / 147.56 for Ab-Lip@DiD② antibody to maleimide-polyethylene glycol-distearate phosphatidylethanolamine is the optimal ratio for antibody modification, maximizing targeting efficiency after antibody modification to liposomes.

[0040] Figure 14The results show that liposomes conjugated with humanized CD33 antibody and loaded with p53 agonist peptide were eluted using Sepharose CL-4B. Liposomes loaded with p53 agonist peptide were prepared by reverse evaporation and then conjugated with antibody at an antibody / liposome ratio of 1 / 147.56. Finally, unloaded peptides and unconjugated antibodies were removed using Sepharose CL-4B, and the dispersion medium was replaced with PBS. The results show that Ab-Lip@PMI-N8A, Ab-Lip@D-PMI-ω, and Ab-Lip@PMI-M3 all eluted within approximately 10 minutes.

[0041] Figure 15 Particle size characterization of liposomes conjugated with humanized CD33 antibody and loaded with p53 agonist peptide is shown. The particle size of Ab-Lip@PMI-N8A is 115.1 nm with a PDI of 0.151, the particle size of Ab-Lip@D-PMI-ω is 166.6 nm with a PDI of 0.210, and the particle size of Ab-Lip@PMI-M3 is 123.1 nm with a PDI of 0.157.

[0042] Figure 16 The results show the quantitative analysis of peptide drug content in liposomes using RP-HPLC. After demulsification and dilution with acetonitrile, the supernatant was injected into RP-HPLC to determine the drug content. The drug concentration was approximately 280–425 μg / mL, meeting experimental requirements. The encapsulation efficiency of PMI peptide reached 40–80%, which is a satisfactory encapsulation effect for large molecule drugs. Chromatographic methods: Column (YMC, C18): 150 × 4.6 mm; Mobile phase A: water (containing 0.1% trifluoroacetic acid), Mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid); Elution program: 2–32 min 15% B–90% B; Flow rate: 0.7 mL / min; Column temperature: 40℃; Detection: UV 214 nm.

[0043] Figure 17 The particle size stability of liposomes conjugated with humanized CD33 antibody and loaded with p53 agonist peptide was demonstrated. The prepared liposomes were incubated at 37°C, and particle size changes were detected at different time points. The results showed that Ab-Lip@PMI-N8A, Ab-Lip@D-PMI-ω, and Ab-Lip@PMI-M3 all exhibited good particle size stability, with only a 10% change in particle size within 72 hours. The particle size index (PDI) increased slightly after 24 hours, and increased by approximately 10–40% at 72 hours.

[0044] Figure 18The particle size stability of liposomes conjugated with humanized CD33 antibody and loaded with p53 agonist peptide was demonstrated in mouse serum. Each prepared liposome was mixed with an equal volume of mouse serum, and particle size changes were detected at different time points. The results showed that Ab-Lip@PMI-N8A, Ab-Lip@D-PMI-ω, and Ab-Lip@PMI-M3 all exhibited good particle size stability, with particle size changes of only 10–35% within 48 h, and a sudden increase in PDI of 20–40% at 24 h.

[0045] Figure 19 The binding of antibody-conjugated liposomes to the surface of AML cells was demonstrated. The antibody and antibody-conjugated liposomes were administered to AML cells HL-60 (CD33). + MOLM-13 (CD33) + SUP-B15 (CD33) - Incubation at 4°C inhibited energy absorption and cell entry, allowing the antibody to continue binding to the cell surface. Lip represents a simple liposome, Ab-Lip represents an antibody-conjugated liposome, and Ab represents a free antibody. The figure shows that the antibody and antibody-conjugated liposomes exhibit excellent recognition ability on the surfaces of HL-60 and MOLM-13 cells. The Ab-Lip positive signal is higher than Ab, possibly due to the amplification effect of multiple antibodies conjugated to a single liposome. Furthermore, the antibody and antibody-conjugated liposomes showed no significant binding to SUP-B15, demonstrating good targeting performance.

[0046] Figure 20 The uptake of antibody-coupled liposomes by AML cells was demonstrated. Antibodies and antibody-coupled liposomes were administered to AML cells HL-60 (CD33). + MOLM-13 (CD33) + SUP-B15 (CD33) - After incubation at 37°C to disrupt the cell membrane, goat anti-mouse IgG (Alexa) was used. 488) Detection of intracellular antibody distribution. Lip represents simple liposomes, Ab-Lip represents antibody-conjugated liposomes, and Ab represents free antibody. As shown in the figure, humanized anti-CD33 antibodies can be well taken up by HL-60 and MOLM-13 cells, and modifying them onto the surface of liposomes also endows the liposomes with CD33 targeting capabilities. + The formulation enables cell entry and maintains the cell selectivity of the humanized anti-CD33 antibody; SUP-B15 shows no visible fluorescence distribution within cells.

[0047] Figure 21 Competitive inhibition assay was demonstrated. Flow cytometry was used to measure MOLM-13 (CD33). +Fluorescent signals of Ab-Lip@DiD and Lip@DiD uptake before and after pre-incubation with a large amount of CD33 antibody were observed. The results show that MOLM-13 uptake of Ab-Lip@DiD can be competitively inhibited by CD33 antibody, indicating that the cellular pathway of Ab-Lip@DiD is mediated by CD33 protein.

[0048] Figure 22 The results demonstrate that the acidic fluorescent probe HPTS characterizes the acidic environment of lysosomes. The fluorescence intensity of HPTS decreased with decreasing pH upon excitation at 454 nm, while it remained unchanged with pH upon excitation at 413 nm. Therefore, I... 454 / I 413 This can characterize pH changes in the environment in which HPTS reside. Antibody-conjugated liposomes loaded with HPTS were administered to MOLM-13 (CD33). + I was measured at each time point. 454 / I 413 Value. The results show that within 0.25 h to 4 h after drug administration to cells, I... 454 / I 413 The value continued to decrease, reaching its lowest point after 4 hours, indicating that the liposomes entered the cell and entered the acidic intracellular environment, i.e., the lysosomes. After the lysosomes were destroyed using NH4Cl, I 454 / I 413 The value rebounded.

[0049] Figure 23 This demonstrates lysosomal colocalization. Ab-Lip@DiD was administered to MOLM-13 (CD33). + After incubation at 37℃ for 0.5 h and 4 h, lysosomes were labeled with a Lyso-sensor, and lysosomal co-localization was observed using a laser confocal microscope. The figures show that Lip@DiD was not taken up by MOLM-13, while Ab-Lip@DiD at 0.5 h had mostly just crossed the membrane and entered the cell, showing poor co-localization with lysosomes. At 4 h, most of Ab-Lip@DiD entered the lysosome, showing good co-localization, indicating that Ab-Lip@DiD is degraded within the lysosome after entering the cell.

[0050] Figure 24 This is a cytotoxicity assay. Liposomes conjugated with CD33 antibody and loaded with sPMI peptide were prepared, diluted to gradient concentrations using cell culture medium, and then administered to CD33 antibody-dependent cells. + , p53 depleted), MOLM-13 (CD33 + (p53 wild), SUP-B15 (CD33) -(p53 wild). After 72 hours, Ab-Lip@sPMI showed the best killing effect on MOLM-13, followed by SUP-B15. Due to the p53 deficiency in HL-60, HL-60 was insensitive to Ab-Lip@sPMI. The killing effect of Ab-Lip@sPMI on MOLM-13 was better than that of free sPMI, better than Lip@sPMI, but less effective than the small molecule p53 activator Nutlin-3a. However, Nutlin-3a has lower cell selectivity than peptide drugs, and at high concentrations, it still exhibits strong toxicity to p53-deficient HL-60.

[0051] Figure 25 This is an evaluation of apoptosis. Liposomes conjugated with CD33 antibody and loaded with sPMI peptide were prepared, diluted to a drug concentration of 30 μM using cell culture medium, and administered to CD33 antibody-dependent cells. + , p53 depleted), MOLM-13 (CD33 + (p53 wild), SUP-B15 (CD33) - After 8 hours, the drug solution was aspirated and replaced with fresh culture medium for 48 hours. As shown in the figure, due to the p53 deficiency in HL-60 cells, Ab-Lip@sPMI, Lip@sPMI, sPMI, and Nutlin-3a did not induce significant apoptosis. For MOLM-13 cells, due to antibody-mediated infiltration, the amount of Ab-Lip@sPMI absorbed into cells was greater than that of Lip@sPMI in a short time, resulting in a significantly better apoptosis effect. For SUP-B15 cells, neither Ab-Lip@sPMI nor Lip@sPMI was taken up, thus no significant apoptosis was observed. In conclusion, the liposome-encapsulated, antibody-conjugated delivery method, compared to free peptide drugs, exhibits better cell selectivity, which is beneficial for targeted therapy.

[0052] Figure 26 This section assesses the cell cycle. The method is the same as above, where the apoptosis body represents the apoptotic body. The results show that due to the p53 deficiency in HL-60, Ab-Lip@sPMI, Lip@sPMI, sPMI, and Nutlin-3a have no significant effect on the cell cycle. For MOLM-13 cells, the influx of Ab-Lip@sPMI into cells was greater than that of Lip@sPMI in a short period. Due to antibody mediation, the proportion of cells in the S+G2 phase was significantly lower than that of Lip@sPMI, and cell proliferation was inhibited. For SUP-B15, neither Ab-Lip@sPMI nor Lip@sPMI was taken up; therefore, compared to sPMI and Nutlin-3a, there was no significant change in the cell cycle. Detailed Implementation

[0053] The term "an" or "a kind" as used herein may refer to one or more kinds. As used in the claims herein, when used in conjunction with the word "comprising," "an" or "a kind" may refer to one or more kinds. The term "another kind" as used herein may refer to at least a second kind or more kinds. In certain embodiments, aspects of the invention may "consist substantially of" or "compose of" one or more elements or steps of the invention, for example. Some embodiments of the invention may consist of or consist substantially of one or more elements, method steps, and / or methods of the invention. It is important to consider that any method or composition described herein may be practiced relative to any other method or composition described herein.

[0054] Those skilled in the art will understand that the disclosed concepts and specific embodiments can be readily used as the basis for improving or designing other structures that achieve the same purpose as the present invention. Those skilled in the art will also recognize that such equivalent structures do not depart from the spirit and scope of the invention as set forth in the appended claims. New features considered characteristic of the invention, as well as other objects and advantages, regarding its construction and operation, can be described in detail below and in the appendix. Figure 1 This will allow for a better understanding. However, it should be understood that the accompanying drawings are for illustration and explanation only and are not intended to limit the scope of the invention.

[0055] This invention pertains to the pharmaceutical field and relates to liposomes conjugated with humanized CD33 antibodies and containing a drug (p53 agonist peptide) for the treatment of acute myeloid leukemia (AML), as well as their preparation methods and uses. Specifically, it relates to liposomes conjugated with humanized CD33 antibodies and containing a series of polypeptides (amino acid sequences: TSFAEYWALLSP, LTFLEYWAQLMQ, efwyvef(p-Cl)ekllr; uppercase letters represent L-configuration amino acids, lowercase letters represent D-configuration amino acids), their preparation methods, and their uses.

[0056] The drug delivery system of this invention comprises a targeting molecule (e.g., an antibody conjugated to the surface of the liposome, such as a humanized CD33 antibody), lipid excipients (hydrogenated soybean phosphatidylcholine, cholesterol, and polyethylene glycol-distearate phosphatidylethanolamine), and a drug (p53 agonist peptide). Results show that liposomes loaded with p53 agonist peptide can be efficiently prepared using a reverse evaporation method. Modifying the surface of the liposomes with a humanized CD33 antibody allows for targeted delivery of the drug to tumor cells, thereby reversing the drawbacks of drugs lacking tumor cell targeting recognition. Through relevant experiments, this invention confirms the formulation process of liposomes loaded with p53 agonist peptide and modified with a humanized CD33 antibody, demonstrating that this liposome drug delivery system can successfully deliver p53 agonist peptide into tumor cells, achieving targeted therapy.

[0057] Targeted molecules

[0058] The drug delivery system of this invention includes a targeting molecule. The targeting molecule can be an antibody conjugated to the surface of a liposome. In a specific embodiment, the targeting molecule can be a humanized CD33 monoclonal antibody. For example, in one specific embodiment, the targeting molecule is a humanized CD33 antibody, the light chain sequence of which is SEQ ID NO:1, and the heavy chain sequence of which is SEQ ID NO:2.

[0059] This invention relates to the preparation and purification of humanized CD33 antibodies. Based on experimental requirements, a humanization scheme for anti-CD33 antibodies was established. Using molecular cloning techniques, pCDNA3.1+ eukaryotic expression vectors carrying the light and heavy chain coding sequences of the humanized anti-CD33 antibody were successfully constructed. The expression vectors were then amplified using competent DH5α cells to obtain a large number of endotoxin-free plasmid vectors, which were then introduced into a eukaryotic cell expression system using transfection reagents. Since the antibody is a secretory protein, it was extracted from the culture medium and purified using a Protein A antibody affinity chromatography column to obtain the humanized anti-CD33 antibody. The purity and molecular weight of the antibody were verified using SDS-PAGE and HPLC-MS.

[0060] This invention also relates to screening the conjugation amount of humanized CD33 antibodies to liposomes. During the humanization of CD33 antibodies, a Cys group is added to the C-terminus of the heavy chain to facilitate conjugation with the maleimide group on the liposome surface. Considering the large volume of the antibody and liposomes, resulting in significant steric hindrance and difficulty in complete reaction, it is necessary to screen for the optimal antibody / maleimide-polyethylene glycol-distearate phosphatidylethanolamine dosage ratio to reduce antibody loss and achieve quantitative antibody conjugation. Conjugation reactions with low, medium, and high antibody / maleimide-polyethylene glycol-distearate phosphatidylethanolamine dosage ratios were designed. The unconjugated free antibody was characterized using Native-PAGE and size exclusion gel chromatography. The results were obtained using a BCA kit and goat anti-mouse IgG (Alexa). 488) The antibody conjugated to the liposome was quantified, its particle size was measured by a laser particle size analyzer, and the conjugation mode of the antibody to the liposome was characterized by SDS-PAGE.

[0061] This invention further relates to optimizing the conjugation amount of humanized CD33 antibody to liposomes. After determining the optimal antibody-liposome reaction ratio in formulation, the targeting efficiency of the antibody conjugated to liposomes was measured to optimize the optimal antibody conjugation amount. Conjugation reactions were designed with low, medium, and high antibody / maleimide-polyethylene glycol-distearate phosphatidylethanolamine dosage ratios. Liposomes labeled with the fluorescent probe DiD of the conjugated antibody were then administered to AML cells HL-60 (CD33). + MOLM-13 (CD33) + SUP-B15 (CD33) - The efficiency of liposomes targeting individual cells was quantified using a flow cytometer.

[0062] In some embodiments of the present invention, the molar ratio of antibody to maleimide-polyethylene glycol-distearate phosphatidylethanolamine in the liposome drug delivery system of the present invention is 1:36-1:200. In a preferred embodiment, the molar ratio of antibody to maleimide-polyethylene glycol-distearate phosphatidylethanolamine in the liposome drug delivery system of the present invention is 1:100-1:200. In a more preferred embodiment, the molar ratio of antibody to maleimide-polyethylene glycol-distearate phosphatidylethanolamine in the liposome drug delivery system of the present invention is 1:147.

[0063] In optional embodiments, the targeting molecules described in this invention are not limited to humanized CD33 monoclonal antibodies, but also include antibodies that specifically bind to cancer cells and target cells, such as anti-HER2; small molecule ligands, such as folic acid and vitamin C; polypeptide ligands, such as RGD cyclic peptides; and nucleic acid aptamers, such as A10 PSMA Apt.

[0064] peptide drugs

[0065] The drug delivery system of this invention includes a polypeptide drug. In a specific embodiment, the polypeptide drug is a p53 agonist peptide.

[0066] The p53 agonist peptides described in this invention are not limited to PMI peptides (amino acid sequences: TSFAEYWALLSP (SEQ ID NO:3), LTFLEYWAQLMQ (SEQ ID NO:4), efwyvef(p-Cl)ekllr (SEQ ID NO:5); uppercase letters represent L-configured amino acids, and lowercase letters represent D-configured amino acids), but also include peptide derivatives, such as p53 agonist peptides linked to charged amino acid fragments, such as polyaspartic acid, polyarginine, etc.; p53 agonist peptides linked to fatty acid fragments, such as palmitic acid, myristic acid, etc.; p53 agonist peptides linked to small molecule drugs, such as doxorubicin, etc.; and stereoenantiomers of p53 agonist peptides.

[0067] This invention uses solid-phase synthesis technology to prepare a series of p53 peptides (PMI-N8A, PMI-M3, D-PMI-ω), which are purified by preparative RP-HPLC, and their purity and molecular weight are confirmed by HPLC and MS, respectively.

[0068] In some embodiments of the present invention, a solution of a lipid excipient and a solution of a polypeptide drug are prepared, and then the two are mixed, wherein the mass ratio of the lipid excipient to the polypeptide drug is 10:1 to 20:1, and the volume ratio of the lipid excipient solution to the polypeptide drug solution is 4:1 to 5:1. In a preferred embodiment, a solution of a lipid excipient and a solution of a polypeptide drug are prepared, and then the two are mixed, wherein the mass ratio of the lipid excipient to the polypeptide drug is 15.3:1, and the volume ratio of the lipid excipient solution to the polypeptide drug solution is 4.5:1.

[0069] In one specific embodiment, the lipid excipient is dissolved in chloroform, the polypeptide drug is dissolved in methanol, and then the two are mixed, wherein the mass ratio of the lipid excipient to the polypeptide is 15.3:1, and the volume ratio of chloroform to methanol is 4.5:1.

[0070] Liposomes encapsulating peptide drugs

[0071] This invention employs a reverse evaporation method to prepare liposomes loaded with peptide drugs. After obtaining a peptide-loaded liposome suspension via reverse evaporation, the suspension is extruded through a membrane and purified by size exclusion gel chromatography to obtain p53 agonist peptide-loaded liposome nanoparticles. The particle size distribution is characterized using a laser particle size analyzer.

[0072] The lipid excipients described in this invention are selected from single polyethylene glycol-phospholipid materials, such as polyethylene glycol-distearate phosphatidylethanolamine (PEG-DSPE), polyethylene glycol-dipalmitoylphosphatidylcholine (PEG-DPPC), or mixtures of multiple polyethylene glycol-phospholipids.

[0073] The polyethylene glycol-phospholipid material of this invention has fatty acid chains on its phospholipids that may be identical or different, with each fatty acid chain containing 12-20 carbon atoms. The number-average molecular weight of the polyethylene glycol is 500-20000 Da, preferably 2000-5000 Da in this invention. One end of the polyethylene glycol is an active group that can be coupled to molecules that specifically recognize cancer cells or target cells. The active group of the polyethylene glycol is selected from maleimide, thiol, amide, amino, carboxyl, biotin, or avidin.

[0074] The lipid excipients described in this invention are selected from negatively charged phospholipid materials such as phosphatidylglycerol (PG) and phosphatidylserine (PS), electrically neutral phospholipid materials such as phosphatidylcholine (PC) and cholesterol, and positively charged phospholipid materials such as phosphatidylethanolamine (PE) and 1,2-dioleoyl-3-trimethylaminolactone (DOTAP).

[0075] In one specific embodiment, the liposomes are prepared using hydrogenated soybean phosphatidylcholine, cholesterol, polyethylene glycol-distearate phosphatidylethanolamine, and maleimide-polyethylene glycol-distearate phosphatidylethanolamine. For example, the molar ratio of hydrogenated soybean phosphatidylcholine, cholesterol, polyethylene glycol-distearate phosphatidylethanolamine, and maleimide-polyethylene glycol-distearate phosphatidylethanolamine is 50:38.5:3:2.

[0076] Antibody-conjugated liposome drug delivery system

[0077] The purpose of this invention is to overcome the inherent drawbacks in the drug development process of p53 agonist peptides and to compensate for the shortcomings of existing technologies for encapsulating peptides in liposomes. This invention provides a liposomal drug containing p53 agonist peptides with active targeting capabilities. Specifically, it relates to liposomes conjugated with humanized CD33 antibodies and encapsulating a drug (p53 agonist peptide) for the treatment of AML, as well as their preparation methods and uses.

[0078] Specifically, the humanized CD33 antibody-conjugated liposome drug delivery system of the present invention includes a target molecule (humanized CD33 antibody), a lipid excipient, and a drug (p53 agonist peptide). The lipid excipient can efficiently encapsulate the drug to form stable and controllable liposomes. The humanized CD33 antibody is site-specifically and quantitatively modified onto the surface of the liposomes. By specifically binding to CD33 on the surface of leukemia cells, the drug is released postcellularly via endocytosis and acts on the MDM2 / MDMX target, thereby activating the p53 anticancer signaling pathway and achieving the purpose of targeted cancer therapy.

[0079] In a specific implementation, the humanized CD33 antibody was incubated with tris(2-carboxyethyl)phosphine (Tcep) at room temperature at a molar ratio of 1:7.5 for 1 hour, followed by removal of Tcep by ultrafiltration centrifugation. The reaction of the humanized CD33 antibody with Tcep at a molar ratio of 1:7.5 maximizes the reduction of disulfide bonds at the terminal of the antibody's Fc fragment, without affecting disulfide bonds at other positions. Tcep may affect the subsequent antibody-liposome coupling reaction; its presence may allow the antibody to continue reducing other reaction sites beyond the terminal thiol group of the Fc fragment coupling with the liposome. Therefore, to achieve quantitative antibody coupling, ultrafiltration centrifugation can also be used to remove Tcep from the reaction system. The reduced antibody was mixed with the extruded liposomes at a molar ratio of antibody / maleimide-polyethylene glycol-distearate phosphatidylethanolamine of 1:147. The mixture was stirred at low speed for 3 hours at 4°C. The free peptide and uncoupled antibody were then separated by Sephadex CL-4B size exclusion gel chromatography to obtain an antibody-conjugated liposome formulation loading p53 agonist peptide with a particle size distribution of 100–170 nm. Because antibodies are large molecules with a certain nanoscale, while liposomes have a particle size of 100–200 nm and significant steric hindrance, it is difficult to achieve a 1:1 quantitative reaction between the thiol groups on the antibody and the maleimide groups on the liposomes. A molar ratio of antibody / liposome maleimide-polyethylene glycol-distearate phosphatidylethanolamine of 1 / 36.89 represents the maximum antibody modification ratio. If the antibody dosage is increased further, the antibody cannot continue to modify the liposome surface. Because antibodies need to overcome steric hindrance to recognize target proteins, if the antibody density on the liposome surface is too high, the steric hindrance is too great, which is not conducive to the antibody's function; if the antibody density on the liposome surface is too low, the probability of the antibody contacting the target protein is low, and the targeting efficiency is affected. After screening to obtain the maximum antibody modification amount, the optimal antibody modification ratio needs to be determined by the targeting efficiency of antibody-conjugated liposomes. The experimental results show that when the antibody / maleimide-polyethylene glycol-distearate phosphatidylethanolamine dosage ratio (molar ratio) is 1 / 147.56, the target efficiency of the prepared liposomes is the best.

[0080] After determining the formulation and process flow, the prepared humanized CD33 antibody-conjugated liposomes carrying p53 agonist peptide were subjected to formulation characterization. The particle size change of the liposomes over time in PBS and mouse serum was monitored using a laser particle size analyzer, and the peptide concentration was quantified and the encapsulation efficiency calculated using HPLC. In a specific embodiment, the liposome drug delivery system of the present invention achieves an encapsulation efficiency of 50-80% for the p53 agonist peptide.

[0081] To target CD33 with antibodies +The ability of the antibody to bind to CD33 protein was studied, and the ability of the antibody to bind to CD33 protein was verified after conjugation to liposomes. CD33 antibody and CD33 antibody-conjugated liposomes were designed and tested at 4°C with HL-60 (CD33) AML cells. + MOLM-13 (CD33) + SUP-B15 (CD33) - The binding experiment used goat anti-mouse IgG (Alexa) 488) The secondary antibody recognizes CD33 antibody, and the fluorescence intensity and percentage of positive cells bound to the cell surface are quantified by flow cytometry.

[0082] To evaluate the ability of the formulation to be recognized, taken up, and incorporated into target cells, a CD33 antibody and CD33 antibody-conjugated liposomes were designed and inoculated into AML cells HL-60 (CD33) at 37°C. + MOLM-13 (CD33) + SUP-B15 (CD33) - The experiment on ingestion used goat anti-mouse IgG (Alexa) 488) The secondary antibody recognizes CD33 antibody, and the intracellular antibody distribution is observed by laser confocal microscopy.

[0083] To verify that the cellular entry pathway of CD33 antibody-conjugated liposomes is mediated by CD33 protein, the change in the uptake capacity of CD33 antibody-conjugated liposomes by MOLM-13 before and after CD33 antibody supersaturation incubation was designed, and the uptake intensity of liposomes was quantified by flow cytometry.

[0084] To investigate whether CD33 antibody-conjugated liposomes enter lysosomes after cell entry, the fluorescence intensity changes of CD33 antibody-conjugated liposomes loaded with the acid-sensitive fluorescent probe HPTS after cell entry were used to characterize the lysosomes after the liposomes have passed through an acidic environment. Lysosomes were labeled with Lyso-Sensor and the colocalization of lysosomes was observed using laser confocal microscopy.

[0085] To conduct in vitro pharmacodynamic evaluation, liposomes conjugated with humanized CD33 antibody and loaded with p53 agonist peptide at different concentrations were applied to AML cells HL-60 (CD33). + , p53 depleted), MOLM-13 (CD33 + (p53 wild), SUP-B15 (CD33) - p53 (wild), viable cells were measured using CCK-8 assay, and IC50 was calculated. 50 value.

[0086] Liposomes conjugated with humanized CD33 antibody and loaded with p53 agonist peptide were applied to AML cells HL-60 (CD33). + , p53depleted), MOLM-13 (CD33 + (p53 wild), SUP-B15 (CD33) - (p53 wild), the number of apoptotic cells was quantified by flow cytometry using Annexin V-FITC / PI apoptosis staining.

[0087] Liposomes conjugated with humanized CD33 antibody and loaded with p53 agonist peptide were applied to AML cells HL-60 (CD33). + , p53depleted), MOLM-13 (CD33 + (p53 wild), SUP-B15 (CD33) - Cell nuclei were stained with DAPI (p53 wild), and cell cycle changes were quantified by flow cytometry.

[0088] Example

[0089] The embodiments provided by the present invention will be described in detail with reference to specific examples, but the present invention is not limited to the following scope.

[0090] The following embodiments are included to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the embodiments representing the inventors' discoveries are well-suited to implementing the invention and can thus be considered preferred modes of implementation. However, those skilled in the art will understand from this disclosure that many variations can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, while still obtaining the same or similar results.

[0091] Example 1: Preparation and characterization of p53 agonist peptide and humanized CD33 antibody

[0092] 1. Synthesis and characterization of p53 agonist peptide

[0093] PMI peptides (PMI-N8A: TSFAEYWALLSP, PMI-M3: LTFLEYWAQLMQ, D-PMI-ω: efwyvef(p-Cl)ekllr; uppercase letters represent L-configured amino acids, and lowercase letters represent D-configured amino acids) were synthesized using a solid-phase peptide synthesis method.

[0094] Specific method: Using the Boc solid-phase peptide synthesis method, amino acids (Jier Biochemical) were sequentially added to PAM-Boc resin (Xi'an Chuangjing Biotechnology Co., Ltd.) according to their sequence. HBTU (Sigma-Aldrich) / DIEA (Jier Biochemical) was used as the condensing agent, and trifluoroacetic acid (TFA) (Shanghai Renran Trading Co., Ltd.) was used as the deprotecting agent. After the reaction, the resin was cleaved with hydrogen fluoride containing P-cresol (Sigma-Aldrich), and the reaction was stirred in an ice bath for 1 hour. After the reaction, the hydrogen fluoride in the tube was removed under reduced pressure, and the precipitate was precipitated with ice-cold diethyl ether (Sinopharm Chemical Reagent Co., Ltd.) and washed three times. The precipitate was redissolved in 20% acetonitrile (Sinopharm Chemical Reagent Co., Ltd.), and the filtrate was collected and rotary evaporated to obtain a crude peptide solution. The crude peptide was purified using an acetonitrile / water (containing 0.1% TFA) system.

[0095] The purity and molecular weight (Mw) of PMI-N8A, PMI-M3, and D-PMI-ω were characterized by HPLC (Agilent 1260) and ESI-MS (AB Sciex, AB 4000Q TRAP). The HPLC and mass spectra of PMI-N8A, PMI-M3, and D-PMI-ω are attached. Figure 1 , Figure 2 , Figure 3 .

[0096] Figure 1 HPLC and ESI-MS chromatograms of PMI-N8A are shown. Chromatographic methods: Column (YMC, C18): 150 × 4.6 mm; Mobile phase A: water (containing 0.1% TFA), mobile phase B: acetonitrile (containing 0.1% TFA); Elution program: 2-32 min 5% B-65% B; Flow rate: 0.7 mL / min; Column temperature: 40℃; Detection: UV 280 nm, retention time: 22.415 min. ESI-MS: 1383.6, consistent with the theoretical molecular weight.

[0097] Figure 2 The HPLC and ESI-MS chromatograms of PMI-M3 are shown. The chromatographic method was the same as above, with a retention time of 25.651 min. The ESI-MS value was 1542, consistent with the theoretical molecular weight.

[0098] Figure 3 The HPLC and ESI-MS chromatograms of D-PMI-ω are shown. The chromatographic method was the same as above, with a retention time of 23.704 min. The ESI-MS value was 1692.4, consistent with the theoretical molecular weight.

[0099] 2. Preparation and characterization of humanized CD33 antibodies

[0100] Based on experimental requirements, a humanization modification scheme for the anti-CD33 antibody was established. Using molecular cloning techniques, pCDNA3.1+ eukaryotic expression vectors carrying the coding sequences for the humanized anti-CD33 antibody light and heavy chains were successfully constructed. The expression vectors were then amplified using competent DH5α cells to obtain a large number of endotoxin-free plasmid vectors, which were then introduced into a eukaryotic cell expression system using transfection reagents.

[0101] The specific steps are as follows:

[0102] 1) Enzyme digestion reaction

[0103] The synthesized antibody heavy chain DNA vector, light chain vector, and pCDNA3.1 vector all require double digestion with XbaI and HindIII (New England Biolabs) to obtain the desired fragments. For the digestion reaction, approximately 2 μg of DNA was added, along with 1 μL each of restriction endonucleases XbaI and HindIII, and 10X NEB2.1 digestion buffer (New England Biolabs). Finally, an appropriate amount of double-distilled water was added to bring the total volume to 20 μL. The mixture was incubated at 37°C for 1.5 h.

[0104] 2) Agarose gel electrophoresis

[0105] Agarose gel electrophoresis is used to separate DNA molecules based on fragment size. To prepare the agarose gel, first prepare a 1% agarose solution (Biowest) using approximately 30 mL of 1x TAE buffer. Shake well and heat in a microwave until completely dissolved. After cooling to approximately 60°C or below, add 30 μL of 0.5 mg / mL ethidium bromide (Solarbio), mix well, pour into a gel casting plate, insert an appropriate comb, and allow to stand at room temperature until completely solidified. For electrophoresis, place the gel in the electrophoresis tank, add 1x TAE buffer, and remove the comb. Use a pipette to pipette the DNA sample, which has been pre-mixed with the loading buffer, into the sample wells. Adjust the voltage to 80V, and the electrophoresis separation time is approximately 40 minutes. After electrophoresis, remove the gel and place it in a UV transilluminator to observe the fluorescent DNA bands.

[0106] 50x TAE buffer: 242g Tris, 57.1mL glacial acetic acid, 0.5mol / L EDTA, pH 8.0; 6x loading buffer: 0.25% bromophenol blue, 0.25% xylene cyanide, 30% glycerol.

[0107] 3) DNA agarose gel recovery

[0108] This experiment used a DNA gel extraction kit (Sangon Biotech Co., Ltd.). First, an agarose gel containing the target fragment was excised using a UV transilluminator and placed in a 1.5 mL centrifuge tube. After weighing, QG buffer equivalent to three times the gel volume was added, and the mixture was heated to 50°C until the gel was completely dissolved. The resulting solution containing the target DNA fragment was then added to an adsorption column, centrifuged at 13000 rpm for 1 minute, and the liquid was discarded. After two steps of washing with PE buffer, the target DNA fragment was eluted with approximately 50 μL of elution buffer (10 mM Tris.HCl, pH 8.0) and centrifuged for 1 minute.

[0109] 4) DNA fragment ligation reaction

[0110] The antibody DNA fragment obtained by enzyme digestion and purification was mixed with the pCDNA3.1 vector at a molar ratio of 7:1. Then, 1 μL of T4 ligase and 2 μL of 10x T4 ligase buffer (New England Biolabs) were added. Finally, an appropriate volume of double-distilled water was added to a final volume of 20 μL. The mixture was then incubated at 16°C for approximately 8 hours.

[0111] 5) Preparation of DH5α competent cells

[0112] In principle, the preparation of competent cells utilizes CaCl2 solution to enhance the permeability of the DH5α strain, allowing macromolecules such as DNA to enter the bacterial interior. First, the DH5α strain is streaked onto an LB agar plate and incubated overnight at 37°C. Then, single bacterial colonies are picked and transferred to 2 mL of LB liquid medium, incubated at 37°C and 220 rpm for approximately 8 hours. The bacterial suspension is then diluted with LB medium at approximately a 1:100 ratio, incubated at 37°C and 220 rpm for approximately 2-3 hours, and the absorbance (OD) at 600 nm is monitored. 600 The bacterial growth rate was approximately 0.4-0.6 (in the logarithmic growth phase). The obtained bacterial suspension was placed on ice for about 20 minutes, then centrifuged at 300 rpm at 4°C for 10 minutes. The supernatant was discarded, and the bacterial pellet was retained. The bacterial cells were resuspended in 200 mL of pre-chilled 0.1 M CaCl2 solution and incubated on ice for about 30 minutes. Then, it was centrifuged again at 3000 rpm at 4°C for 10 minutes. The supernatant was discarded, and the bacterial cells were retained. Finally, the bacteria were resuspended in 50 mL of pre-chilled 0.1 M CaCl2, 10% glycerol solution. After aliquoting, the solution was stored at -80°C.

[0113] 6) Transformation of DH5α competent states

[0114] Transformation of DH5α competent cells involves introducing a DNA vector into *E. coli* via heat shock to amplify the DNA vector. First, approximately 200 μL of aliquoted DH5α competent cells are thawed on ice, and about 50 ng of DNA is added. The mixture is then incubated on ice for 30 minutes. Next, the mixture of competent cells and DNA is heat-shocked in a 42°C water bath for 60 seconds, followed by a rapid reset on ice for approximately 2 minutes. Approximately 800 μL of LB medium is added to the heat-shocked competent cells, and the mixture is incubated at 37°C and 220 rpm for approximately 1 hour. This step is to allow the bacteria to fully recover. Finally, the recovered bacteria are plated onto LB agar plates containing an antibiotic corresponding to the DNA vector's resistance. The plates are then inverted and incubated at 37°C for approximately 12 hours.

[0115] 7) DNA plasmid preparation

[0116] The methods for extracting DNA plasmids from bacteria mainly include three basic steps: bacterial culture to amplify the plasmid; collection and lysis of bacterial cells; and isolation and purification of plasmid DNA.

[0117] Mini-scale extraction of DNA plasmid molecules was performed using a plasmid mini-preparation kit (Sangon Biotech Co., Ltd.). First, 5-10 mL of bacterial culture was incubated overnight at 37°C. The bacterial culture was then centrifuged at 8000 rpm for 10 minutes, and the supernatant was discarded, retaining the bacterial cells. 250 μL of Solution I (5 mM glucose, 25 mM Tris pH 8.0, 10 mM EDTA) containing RNase A was added to the bacterial cells to fully resuspend the bacteria. Then, 250 μL of Solution II (0.2 mM NaOH, 10 g / L SDS) was added, and the mixture was gently inverted 6-8 times. Next, 350 μL of Solution II (5 M sodium acetate, 60 mL glacial acetic acid, 28.5 mL double-distilled water) was added to lyse the bacteria. After gently inverting the mixture 6-8 times, a white precipitate appeared in the centrifuge tube. The obtained bacterial lysate was centrifuged at 12000 rpm for 10 minutes, and the supernatant was transferred to an adsorption column. Centrifuge the adsorption column at 12,000 rpm for 1 minute, discard the liquid, and wash the adsorption column twice with 600 μL of wash buffer, centrifuging at 12,000 rpm for 1 minute each time and discarding the liquid. Finally, place the adsorption column into a new collection tube and elute the DNA plasmid with approximately 50-100 μL of elution buffer.

[0118] DNA plasmids used for transient transfection of cells expressing proteins require stringent requirements in terms of concentration and purity; therefore, an endotoxin-free plasmid mass production kit (Qiagen) is necessary. The principle is the same as for small-scale plasmid production. First, 100-250 mL of bacterial culture needs to be cultured overnight. After centrifuging at 4°C to obtain bacterial cells, the bacteria are resuspended in 10 mL of Solution I, Solution II is added, the centrifuge tube is inverted 6-8 times, and the mixture is incubated at room temperature for 5 minutes. Then, the bacterial lysate is filtered and transferred to a new centrifuge tube, along with 2.5 mL of endotoxin remover. The mixture is thoroughly mixed and incubated on ice for 30 minutes. Next, the liquid is placed into an adsorption column, allowing gravity to allow the liquid to pass through the resin and exit the column. The adsorption column is washed with 2 x 30 mL washing buffer, and then the DNA is eluted with 15 mL of elution buffer. Add 10.5 mL of isopropanol (Sinopharm Reagent Co., Ltd.) to the elution buffer and mix thoroughly to precipitate the DNA. Centrifuge at a speed exceeding 15000 x g for 30 minutes. After centrifugation, carefully discard the supernatant and allow the DNA precipitate to stand at room temperature until the liquid has completely evaporated. Finally, reconstitute the DNA plasmid with an appropriate volume (approximately 2 mL) of endotoxin-free TE buffer.

[0119] The plasmid concentration was determined by measuring the absorbance of the sample at a wavelength of 260 nm using a micro-ultraviolet spectrophotometer (Implen). An OD260 of 1 corresponds to a double-stranded DNA concentration of 50 μg / ml. The OD260 / OD280 ratio should be close to 1.8. A ratio less than 1.6 usually indicates protein contamination in the sample, while a ratio greater than 1.9 indicates RNA contamination.

[0120] 8) Site-directed mutagenesis at the ends of antibody heavy chain expression vectors

[0121] We employed site-directed mutagenesis (PCM) to introduce an additional cysteine ​​residue at the C-terminus of the antibody heavy chain. The first step in PCM involved designing PCR primers (Sangon Biotech Co., Ltd.) capable of introducing the mutated sequence. Specifically, 10-20 base pairs were taken from before and after the desired mutation site in the pCDNA3.1 vector containing the inserted antibody heavy chain DNA sequence, and a cysteine ​​codon was added in the middle. The resulting sequence served as the forward primer, and its reverse complementary sequence served as the reverse primer. Then, using the synthesized PCR primers and the vector containing the antibody heavy chain DNA sequence as a template, a 50 μL reaction system was prepared (5 μL 10x reaction buffer, 2 μL approximately 50 ng template, 1 μL each of 10 nM forward and reverse primers, 2.5 mM each of 2 μL dNTP (New England Biolabs) mixture, 1 μL pfu DNA polymerase (New England Biolabs), and 39 μL H2O). PCR reaction conditions: denaturation temperature 95℃ for 30 s, annealing temperature 65℃ for 1 min, extension temperature 72℃ for 6 min, 20 cycles. Then, 1 μL of DpnI enzyme (New England Biolabs) was added to the obtained PCR product to remove the non-mutated DNA template, as the template plasmid DNA is methylated and can be cleaved by DpnI. Finally, the reaction product was used to transform DH5α competent cells, single clones were picked, and sequencing was used to verify whether the cysteine ​​residue DNA sequence was successfully inserted.

[0122] 9) Cell Culture

[0123] Expi293FTM cells (Thermo Fisher) should be cultured in suspension. The culture medium is Expi293TM Expression Medium (Thermo Fisher). Place the cells in an Erlenmeyer flask and incubate on a horizontal rotating shaker within a CO2 incubator at 37°C, 8% CO2, and 125 rpm. The cell density should be maintained at 0.3 x 10⁻⁶ cells / year. 6 -3x10 6 Cells / mL, and the percentage of live cells must be maintained above 95%.

[0124] 10) Cell transfection

[0125] Transient transfection of Expi293FTM suspension cells was used for high-dose expression of anti-CD33 antibody. Before transfection, Expi293FTM cells should be cultured to a density greater than 3 x 102. 7 Cells / mL, cell viability not less than 95%. Before transfection, dilute cells to 2.9 x 10⁻⁶ cells / mL. 7Cells / mL, volume 25.5 mL. In centrifuge tube I, 15 μg each of the antibody heavy chain and light chain expression vector were mixed with 1.5 mL of opti-MEM medium (Gibco). In centrifuge tube II, 81 μL of ExpiFectamine™ transfection reagent (Thermo Fisher) was diluted with 1.5 mL of opti-MEM medium and carefully mixed with a pipette. After standing at room temperature for 5 minutes, the mixture from centrifuge tube I was added to centrifuge tube II, carefully mixed, and incubated at room temperature for 20 minutes. Finally, the transfection mixture was added dropwise to the cell culture system and cultured under appropriate conditions. 16 to 24 hours after transfection, 150 μL of ExpiFectamine™ 293 Transfection Enhancer I and 1.5 mL of ExpiFectamine™ 293 Transfection Enhancer II were added. After 7 days of continuous culture, the culture supernatant was collected by centrifugation for detection and purification of anti-CD33 antibody.

[0126] 10) Antibody purification

[0127] The isolation and purification of anti-CD33 antibodies were performed using a Protein A antibody affinity chromatography column (Saifen Technology). Proteins A and G are derived from the bacterial surface, and both can bind to the Fc region of human immunoglobulin IgG. During antibody purification, the column was first equilibrated with 10 column volumes of sample buffer (PBS). Simultaneously, the antibody-expressing cell supernatant was diluted 1:1 with sample buffer. The cell solution was then passed through the affinity column at a rate of 1 column volume per minute to allow the antibody to load onto the column. The column was then washed with 20 column volumes of sample buffer. Finally, the antibody was eluted with acidic elution buffer (100 mM pH = 3.0 Gly-HCl) and collected. The elution profile of the anti-CD33 antibody is attached. Figure 4 .

[0128] Figure 4 The elution profile of humanized CD33 antibody on a Protein A antibody affinity chromatography column is shown. After the eukaryotic cell protein expression system produced the antibody, the cell culture medium was collected and loaded onto a Protein A antibody affinity chromatography column (loading buffer: PBS), followed by elution with 100 mM pH=3.0 Gly-HCl. The antibody eluted in approximately 37 min.

[0129] 11) SDS-PAGE detection of antibody expression

[0130] Polyacrylamide gel electrophoresis (SDS-PAGE) is a commonly used protein analysis technique. It allows proteins to be separated into different bands according to their molecular weight under denaturing conditions and an electric field. The first step in SDS-PAGE detection is sample preparation. Take 2 x 10 μL of each of the liquid fractions from antibody purification (eluent and elution), and add 2.5 μL of 5x SDS-PAGE loading buffer (Yisheng Biotechnology) with or without β-mercaptoethanol. Mix well and heat at 100°C for 10 minutes. Prepare a 10% polyacrylamide separating gel containing SDS and a 3% compression gel. Add the prepared samples to the wells, compress at 80V for approximately 15 minutes, then adjust the voltage to 120V and continue separation for 35 minutes. After electrophoresis, stain the gel with Coomassie Brilliant Blue for approximately two hours, then destain with destaining solution until the protein bands are clear. See the attached image for the SDS-PAGE bands of the Anti-CD33 antibody. Figure 5 .

[0131] Figure 5 The results show SDS-PAGE characterization of antibody purity and molecular weight. After reduction with loading buffer (containing β-mercaptoethanol), SDS-PAGE showed heavy chain bands at 55–70 kDa and light chain bands at approximately 25 kDa, consistent with the theoretical molecular weight, and no other significant contaminant bands. When the antibody was loaded onto SDS-PAGE with native loading buffer (without β-mercaptoethanol) (Sangon Biotech Co., Ltd.), only one protein band was observed, significantly higher than either the heavy or light chain. The efflux buffer did not contain the corresponding antibody band, indicating that the purification method was appropriate.

[0132] 12) HPLC-MS determination of antibody molecular weight

[0133] The prepared antibody was dialyzed with pure water, lyophilized, and then dissolved in a certain amount of pure water. Its molecular weight was detected by HPLC-MS (Agilent 6230LC / TOF). A certain amount of DTT (Sigma) was added to reduce the antibody. The heavy and light chains of the reduced antibody were then injected into HPLC-MS to confirm its molecular weight.

[0134] Figure 6 The figure shows the molecular weight characterized by HPLC-MS. As can be seen from the figure, the antibody molecular weight is 148,128, the antibody heavy chain molecular weight is 50,190, and the antibody light chain molecular weight is 23,759.

[0135] Chromatographic method: Column (Agilent, C4): 150 × 2.1 mm; Mobile phase A: water (containing 0.1% trifluoroacetic acid), mobile phase B: 70% isopropanol + 20% acetonitrile + 10% water (containing 0.1% trifluoroacetic acid); Elution program: 2-10 min 100% B; Flow rate: 0.7 mL / min; Column temperature: 25℃; Detection: UV 280 nm, consistent with theoretical molecular weight.

[0136] Example 2: Construction and optimization of antibody-liposome coupling reaction

[0137] 1. Screening for the maximum amount of antibody conjugated to liposomes

[0138] 1) Detection of unconjugated residual antibodies

[0139] Ab-Lip 1-3 was designed with low (1 / 92.24), medium (1 / 36.89), and high (1 / 18.48) antibody / liposome dosage ratios, and unconjugated free antibodies were detected using various methods.

[0140] Unconjugated residual antibodies in the reaction solution exhibited stronger translocation ability on Native-PAGE than antibodies conjugated to liposomes, and this was characterized using Native-PAGE. 20 μL of each reaction solution was added to 5 μL of 5x loading buffer without β-mercaptoethanol. Using a 10% isocratic gel, the prepared samples were added to the wells with the same total antibody amount. The voltage was adjusted to 120V, and the gel was separated for 40 minutes. After electrophoresis, the gel was stained with Coomassie Brilliant Blue for approximately two hours, followed by destaining with destaining solution until the protein bands were clear. The Native-PAGE bands of the reaction solution are shown in the attached image. Figure 7 .

[0141] Figure 7 The image shows the Native-PAGE characterization of the antibody-liposome coupling reaction. As can be seen from the figure, the amount of uncoupled residual antibody in Ab-Lip1-2 is significantly lower than that in Ab-Lip3, indicating that the antibody / liposome dosage ratio of Ab-Lip2 (1 / 36.89) represents the maximum antibody modification ratio.

[0142] Liposomes and unconjugated residual antibodies were separated using Sepharose CL-4B size exclusion gel chromatography (Shanghai Sepharose Biotechnology Co., Ltd.). The reaction solution was loaded onto a Sepharose CL-4B column, and PBS was used as the mobile phase. The detection wavelength was UV: 214 nm. The eluents of liposomes and unconjugated residual antibodies were observed and collected. The elution signal curves are shown in the attached figure. Figure 8 .

[0143] Figure 8The results show that the antibody-liposome coupling reaction was characterized by Sepharose CL-4B size exclusion gel chromatography. Liposomes eluted in Sepharose CL-4B gel chromatography at 6–8 min, while uncoupled free antibody eluted in Sepharose CL-4B at 10–25 min. The results indicate that Ab-Lip 1–2 showed virtually no uncoupled residual antibody elution, while Ab-Lip 3 showed significant free antibody elution, suggesting that the antibody / liposome dosage ratio of 1 / 36.89 for Ab-Lip 2 represents the maximum antibody modification ratio.

[0144] 2) Quantitative analysis of the amount of antibody already coupled to liposomes

[0145] After removing unconjugated residual antibody using Sepharose CL-4B, the antibody-conjugated amount was quantified using a BCA kit (ThermoFisher). A standard curve was prepared using bovine serum albumin (Sinopharm Chemical Reagent Co., Ltd.), and the antibody-conjugated amount of Ab-Lip 1-3 was determined by measuring the UV absorbance at 562 nm. The results are shown in the appendix. Figure 9 .

[0146] Figure 9 The results show the antibody-conjugated amount on liposomes as determined by the BCA method. After conjugation of liposomes with antibodies, unconjugated free antibodies were removed using Sepharose CL-4B. The amount of antibody conjugated on the liposomes was then determined using the BCA method. Ab-Lip 2-3 showed the highest antibody-conjugated amount, more than twice that of Ab-Lip 1, indicating that the antibody / liposome dosage ratio of Ab-Lip 2 (1 / 36.89) represents the maximum antibody modification ratio.

[0147] Use Goat Anti-Mouse IgG (Alexa) 488)(Abcam) was used to detect the amount of antibody conjugated on liposomes. 150 μL of liposomes were mixed with 3 μL of 20 mg / mL goat anti-mouse IgG (Alexa). 488) was incubated at room temperature for 1 hour, followed by removal of excess goat anti-mouse IgG (Alexa) using Sepharose CL-4B. 488), the fluorescence intensity of the collected liposome solution under excitation light of 488 nm was measured, and the results are shown in the appendix. Figure 10 .

[0148] Figure 10 It showed Goat Anti-Mouse IgG (Alexa) 488) Detection of antibody-conjugated liposomes. After conjugation of liposomes with antibodies, unconjugated free antibodies were removed using Sepharose CL-4B, and then the antibody levels were measured using goat anti-mouse IgG (Alexa). 488) The antibody conjugated on the liposomes was identified and bound, and the amount of antibody conjugation was determined based on the fluorescence intensity. The antibody conjugation amount of Ab-Lip 2 was the highest, and the antibody conjugation amount of Ab-Lip 3 was basically the same, both greater than that of Ab-Lip 1, indicating that the antibody / liposome dosage ratio of Ab-Lip 2 was 1 / 36.89, which is the maximum proportion of antibody modification.

[0149] 3) Particle size characterization

[0150] After removing unconjugated residual antibody using Sepharose CL-4B, the particle size distribution of the collected Ab-Lip 1-3 was determined using a laser particle size analyzer (Malvern Zeta-sizer Nano ZS). The results are shown in the appendix. Figure 11 .

[0151] Figure 11 The particle size characterization of liposomes with different antibody-conjugated amounts is shown. The particle size of Ab-Lip1-3 with low (1 / 92.24), medium (1 / 36.89), and high (1 / 18.48) antibody / liposome dosage ratios was determined using a laser particle size analyzer. The particle size of unconjugated liposomes (Mal-Lip) was 122.53±0.68 nm, with a PDI of 0.098±0.026; Ab-Lip 1 had a particle size of 133.97±1.44 nm, with a PDI of 0.126±0.016; Ab-Lip 2 had a particle size of 149.43±0.76 nm, with a PDI of 0.117±0.005; and Ab-Lip 3 had a particle size of 142.50±1.35 nm, with a PDI of 0.093±0.030. The antibody conjugation amount of Ab-Lip 2 was significantly greater than that of Ab-Lip 1, and the increase in liposome size was significantly greater for Ab-Lip 2 than for Ab-Lip 1. The antibody conjugation amount of Ab-Lip 2 was basically the same as that of Ab-Lip 3, and the increase in liposome size for both was also similar.

[0152] 4) SDS-PAGE characterizes the conjugation mechanism between antibody and liposome.

[0153] Conjugation was performed using different concentrations of reducing agent and different treatment methods at the Ab-Lip 2 antibody / liposome dosage ratio. Specifically, Ab-Lip 2a indicates that Tcep (Sigma) is not removed after reduction at an antibody / Tcep molar ratio of 7.5:1; Ab-Lip 2b indicates that Tcep is removed by ultrafiltration centrifugation after reduction at an antibody / Tcep molar ratio of 7.5:1; Ab-Lip 2c indicates that Tcep is not removed after reduction at an antibody / Tcep molar ratio of 10:1; and Ab-Lip 2d indicates that Tcep is removed by ultrafiltration centrifugation after reduction at an antibody / Tcep molar ratio of 10:1. The liposomes after reaction were passed through a Sepharose CL-4B column, and the liposome eluent was collected for SDS-PAGE characterization using the same method as above. The results are shown in the appendix. Figure 12 .

[0154] Figure 12 The results show SDS-PAGE characterization of antibody-liposome conjugation. Mal-Lip represents liposomes without antibody conjugation, Antibody represents free antibody, and Ab-Lip 2a-d represent conjugation using different concentrations of reducing agent and different treatment methods at the Ab-Lip 2 antibody / liposome dosage ratio. The SDS-PAGE results show that the light chain band shift remains unchanged, while the heavy chain band shifts upwards. Except for cases where one heavy chain in the conjugated antibody did not react, the uppermost band in the Ab-Lip 2b group is the lightest, indicating that the optimal reduction method is a 7.5:1 antibody / Tcep molar ratio followed by ultrafiltration centrifugation to remove Tcep.

[0155] 2. Optimization of the optimal amount of antibody conjugated to liposomes

[0156] Liposomes Ab-Lip@DiD①-③, loaded with the red fluorescent probe DiD, were designed with antibody / liposome dosage ratios of low (1 / 590.24), medium (1 / 147.56), and high (1 / 36.89), respectively. After diluting each group of liposomes to 1.5 μM in cell culture medium, they were applied to AML cells HL-60 (CD33). + MOLM-13 (CD33) + SUP-B15 (CD33) - After incubating at 37°C for 2 hours, the cells were centrifuged at 1000 rpm for 5 minutes, collected, washed once with PBS, and the fluorescence signal of the cells was quantified by flow cytometry (BD Biosciences, FACSCanto™). The results are shown in the appendix. Figure 13 .

[0157] Figure 13The antibody / liposome dosage ratio was optimized based on antibody targeting efficiency. Liposomes Ab-Lip@DiD①-③, loaded with the red fluorescent probe DiD, were designed with low (1 / 590.24), medium (1 / 147.56), and high (1 / 36.89) antibody / liposome dosage ratios, respectively. Lip@DiD represents DiD-loaded liposomes without antibody conjugation. Each group of liposomes was applied to AML cells HL-60 (CD33) + MOLM-13 (CD33) + SUP-B15 (CD33) - Figure A shows the quantitative analysis of DiD red fluorescence by flow cytometry; Figure B shows the average fluorescence intensity during uptake; Figure C shows the median fluorescence intensity during uptake; and Figure D shows the percentage of positive cells. HL-60 and MOLM-13 showed the highest uptake of Ab-Lip@DiD② and exhibited strong cell selectivity, while SUP-B15 showed uptake of Ab-Lip@DiD①-③ comparable to Lip@DiD. These results indicate that an antibody / liposome dosage ratio of 1 / 147.56 is the optimal ratio for antibody modification, and antibody modification to liposomes maximizes targeting efficiency.

[0158] Example 3: Preparation and characterization of liposomes conjugated with humanized CD33 antibody and loaded with p53 agonist peptide

[0159] 1. Preparation of liposomes conjugated with humanized CD33 antibody carrying p53 agonist peptide

[0160] Hydrogenated soybean phosphatidylcholine (Avanti), cholesterol (Sigma), polyethylene glycol-distearate phosphatidylethanolamine (Laysan Bio), and maleimide-polyethylene glycol-distearate phosphatidylethanolamine (Laysan Bio) were weighed in a molar ratio of 50:38.5:3:2 as lipid excipients for liposome preparation. The lipid excipients were dissolved in chloroform (Sinopharm Chemical Reagent Co., Ltd.), and p53 agonist peptide was dissolved in methanol (Sinopharm Chemical Reagent Co., Ltd.). The two mixtures were thoroughly mixed, with a lipid excipient to peptide mass ratio of 15.3:1. This ratio balances peptide encapsulation efficiency and peptide concentration. Increasing the peptide dosage ratio increases the peptide concentration but decreases the encapsulation efficiency; conversely, decreasing the peptide dosage ratio increases the encapsulation efficiency but decreases the peptide concentration. The volume ratio of chloroform to methanol was 4.5:1, as chloroform and methanol are miscible and have an azeotropic point. When the volume ratio of chloroform to methanol is 4.5 / 1, heating to the azeotropic point allows chloroform and methanol to evaporate simultaneously, preventing the precipitation of lipid excipients or peptides due to changes in solubility caused by the evaporation of one solvent first. Then, 10 mM phosphate buffer (pH 6.5) is added. Because the lipid excipient contains maleimide-polyethylene glycol-distearate phosphatidylethanolamine, the maleimide group exhibits the highest stability within the pH range of 6.0-6.5. At pH 6.5, the addition reaction activity between the thiol group and maleimide is higher than at pH 6.0; therefore, 10 mM phosphate buffer (pH 6.5) is chosen as the aqueous phase medium. This results in a 9:1 volume ratio of organic to aqueous phase. This 9:1 volume ratio ensures that the aqueous suspension formed after rotary evaporation has a small and uniform particle size. If there is too much organic phase, the aqueous suspension formed after rotary evaporation will have excessively large particle sizes; if there is too much aqueous phase, an oil-in-water emulsion cannot be formed by sonication. The aqueous and organic phases are thoroughly mixed and sonicated until a homogeneous oil-in-water emulsion is formed. The resulting emulsion is then rotary evaporated under vacuum for half an hour, followed by replenishment with 10 mM phosphate buffer (pH 6.5). Vacuum evaporation continues until a homogeneous liposome suspension is formed. The suspension is then incubated in a 50°C water bath shaker for 1 hour. The liposomes are then sequentially extruded through 400 nm, 200 nm, and 100 nm polyester membranes (Whatman, track-etch membrane). Humanized CD33 antibody and tris(2-carboxyethyl)phosphine (Tcep) are incubated at room temperature at a molar ratio of 1:7.5 for 1 hour, followed by removal of Tcep by ultrafiltration centrifugation. The reduced antibody was mixed with the extruded liposomes at a molar ratio of antibody / maleimide-polyethylene glycol-distearate phosphatidylethanolamine of 1:147, and stirred at low speed at 4°C for 3 hours. The free peptides and uncoupled antibody were then separated by Sephadex CL-4B size exclusion gel chromatography (see Appendix). Figure 14 ).

[0161] Figure 14The results show that liposomes conjugated with humanized CD33 antibody and loaded with p53 agonist peptide were eluted using Sepharose CL-4B. Liposomes loaded with p53 agonist peptide were prepared by reverse evaporation and then conjugated with antibody at an antibody / liposome ratio of 1 / 147.56. Finally, unloaded peptides and unconjugated antibodies were removed using Sepharose CL-4B, and the dispersion medium was replaced with PBS. The results show that Ab-Lip@PMI-N8A, Ab-Lip@D-PMI-ω, and Ab-Lip@PMI-M3 all eluted within approximately 10 minutes.

[0162] The particle size distribution of the collected liposome eluent was determined by a laser particle size analyzer, and the results are shown in the appendix. Figure 15 . Figure 15 Particle size characterization of liposomes conjugated with humanized CD33 antibody and loaded with p53 agonist peptide is shown. The particle size of Ab-Lip@PMI-N8A is 115.1 nm with a PDI of 0.151, the particle size of Ab-Lip@D-PMI-ω is 166.6 nm with a PDI of 0.210, and the particle size of Ab-Lip@PMI-M3 is 123.1 nm with a PDI of 0.157.

[0163] After the liposomes were diluted with twice their volume of acetonitrile for demulsification, they were centrifuged at 10,000 rpm for 5 min. The supernatant was then injected into RP-HPLC to determine the drug content. The results are shown in the appendix. Figure 16 . Figure 16 This study demonstrates the quantitative analysis of peptide drug content in liposomes using RP-HPLC. After demulsification and dilution with acetonitrile, the supernatant was injected into RP-HPLC (Agilent, 1260) to determine the drug content. The drug concentration ranged from approximately 280 to 425 μg / mL, meeting experimental requirements. The encapsulation efficiency of the PMI peptide reached 40-52%, which is a satisfactory encapsulation effect for a large molecule drug. Chromatographic methods: Column (YMC, C18): 150 × 4.6 mm; Mobile phase A: water (containing 0.1% trifluoroacetic acid); Mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid); Elution program: 2-32 min 15% B-90% B; Flow rate: 0.7 mL / min; Column temperature: 40℃; Detection: UV 214 nm.

[0164] 2. Particle size stability of humanized CD33 antibody-conjugated p53 agonist peptide

[0165] The prepared liposomes were placed in a shaker at 37°C, and their particle size distribution was measured by a laser particle size analyzer at a specified time. The results are shown in the appendix. Figure 17 . Figure 17The particle size stability of liposomes conjugated with humanized CD33 antibody and loaded with p53 agonist peptide was demonstrated. The prepared liposomes were incubated at 37°C, and particle size changes were detected at different time points. The results showed that Ab-Lip@PMI-N8A, Ab-Lip@D-PMI-ω, and Ab-Lip@PMI-M3 all exhibited good particle size stability, with only a 10% change in particle size within 72 hours. The particle size index (PDI) increased slightly after 24 hours, and increased by approximately 10–40% at 72 hours.

[0166] Each prepared liposome was mixed with an equal volume of ICR mouse serum and placed in a shaker at 37°C. The particle size distribution was measured using a laser particle size analyzer at a specified time. The results are shown in the appendix. Figure 18 . Figure 18 The particle size stability of liposomes conjugated with humanized CD33 antibody and loaded with p53 agonist peptide was demonstrated in mouse serum. Each prepared liposome was mixed with an equal volume of mouse serum, and particle size changes were detected at different time points. The results showed that Ab-Lip@PMI-N8A, Ab-Lip@D-PMI-ω, and Ab-Lip@PMI-M3 all exhibited good particle size stability, with particle size changes of only 10–35% within 48 h, and a sudden increase in PDI of 20–40% at 24 h.

[0167] Example 4: In vitro targeting of the formulation

[0168] 1. Cell surface binding

[0169] AML cells HL-60(CD33) + MOLM-13 (CD33) + SUP-B15 (CD33) - Incubate with 1% BSA / PBS at room temperature for 30 min, centrifuge at 1000 rpm for 5 min, and remove the liquid. Dilute the antibody and antibody-conjugated liposomes in cell culture medium to an antibody concentration of 10 μg / mL, add the antibody and incubate at 4°C for 1 h, centrifuge at 1000 rpm for 5 min, collect the cells, wash three times with PBS, and add 2 μg / mL of goat anti-mouse IgG (Alexa). 488) Incubate at room temperature for 1 hour, centrifuge at 1000 rpm for 5 minutes, wash three times with PBS, and quantify the fluorescence signal of cells using flow cytometry. Results are shown in the appendix. Figure 19 .

[0170] Figure 19 The binding of antibody-conjugated liposomes to the surface of AML cells was demonstrated. The antibody and antibody-conjugated liposomes were administered to AML cells HL-60 (CD33). + MOLM-13 (CD33) +SUP-B15 (CD33) - Incubation at 4°C inhibited energy entry into cells, allowing the antibody to continue binding to the cell surface. The figure shows that the antibody and antibody-conjugated liposomes exhibited excellent recognition ability on the HL-60 and MOLM-13 surfaces. The Ab-Lip positive signal was higher than the Ab signal, possibly due to the amplification effect of multiple antibodies conjugated to a single liposome. Furthermore, the antibody and antibody-conjugated liposomes showed no significant binding to SUP-B15, demonstrating good targeting performance.

[0171] 2. Cellular uptake

[0172] The antibody and antibody-conjugated liposomes were diluted from cell culture medium to an antibody concentration of 50 μg / mL and added to AML cells HL-60 (CD33). + MOLM-13 (CD33) + SUP-B15 (CD33) - After incubation at 37°C for 2 hours, centrifuge at 1000 rpm for 5 minutes to remove the drug solution, wash twice with PBS, fix with paraformaldehyde for 15 minutes, wash once with PBS, incubate with 0.2% Triton X-100 for 5 minutes, and wash once with PBS. Add 1% BSA / PBS and incubate at room temperature for 30 minutes, then add 2 μg / mL goat anti-mouse IgG (Alexa). 488) Incubate at room temperature for 1 hour, wash twice with PBS, stain with 10 ng / mL DAPI (Dalian Meilun Biotechnology Co., Ltd.) for 10 minutes, wash twice with PBS, add to a glass slide, cover with a coverslip, and observe under a laser confocal microscope (ZEISS, LSM 710). The results are shown in the appendix. Figure 20 .

[0173] Figure 20 The uptake of antibody-coupled liposomes by AML cells was demonstrated. Antibodies and antibody-coupled liposomes were administered to AML cells HL-60 (CD33). + MOLM-13 (CD33) + SUP-B15 (CD33) - After incubation at 37°C to disrupt the cell membrane, goat anti-mouse IgG (Alexa) was used. 488) Detection of intracellular antibody distribution. As shown in the figure, the humanized anti-CD33 antibody can be well taken up by HL-60 and MOLM-13 cells, and modifying it onto the surface of liposomes also endows the liposomes with CD33 targeting capabilities. + The formulation enables cell entry and maintains the cell selectivity of the humanized anti-CD33 antibody; SUP-B15 shows no visible fluorescence distribution within cells.

[0174] 3. Competitive inhibition

[0175] The humanized CD33 antibody was diluted from the cell culture medium to an antibody concentration of 8000 μg / mL and added to MOLM-13 (CD33) + After incubation at 4°C for 1 hour, a certain volume of Ab-Lip@DiD and Lip@DiD was added to bring the final DiD drug concentration to 1.5 μM. Incubation was continued at 4°C for 2 hours, followed by centrifugation at 1000 rpm for 5 minutes. Cells were collected, washed once with PBS, and their fluorescence signal was quantified using flow cytometry. A control group was used, consisting of cells that had not been pre-incubated with CD33 antibody but underwent the same procedures. Results are shown in the appendix. Figure 21 .

[0176] Figure 21 The competitive inhibitors are shown. Figure A shows MOLM-13 (CD33). + The fluorescence signals of Ab-Lip@DiD and Lip@DiD uptake before and after pre-incubation with CD33 antibody are shown in Figure B. Figure B shows the average fluorescence uptake by MOLM-13, and Figure C shows the median fluorescence uptake by MOLM-13. The results indicate that MOLM-13 uptake of Ab-Lip@DiD can be competitively inhibited by CD33 antibody, suggesting that the cellular entry pathway of Ab-Lip@DiD is mediated by CD33 protein.

[0177] 4. Lysosomal characterization

[0178] HPTS (Sigma) was coated using a film-forming hydration method, similar to the antibody conjugation method described above. Uncoated free HPTS was removed using G50 size exclusion gel chromatography. Liposomes conjugated with CD33 and loaded with HPTS were diluted in cell culture medium to an antibody concentration of 8 μM, incubated at 37°C, centrifuged at 1000 rpm for 5 min at the specified time, and the cells were collected. The cells were washed three times with PBS, and the fluorescence intensity was measured under excitation light at 413 nm and 454 nm (emission wavelength 510 nm). The dispersion medium was then replaced with a 5 mM NH4Cl PBS solution, and the fluorescence intensity was measured again under the same conditions. Ig was calculated. 413 / I 454 Values ​​and results are attached. Figure 22 .

[0179] Figure 22 The results demonstrate that the acidic fluorescent probe HPTS characterizes the acidic environment of lysosomes. The fluorescence intensity of HPTS decreased with decreasing pH upon excitation at 454 nm, while it remained unchanged with pH upon excitation at 413 nm. Therefore, I... 454 / I 413This can characterize pH changes in the environment in which HPTS reside. Antibody-conjugated liposomes loaded with HPTS were administered to MOLM-13 (CD33). + I was measured at each time point. 454 / I 413 Value. The results show that within 0.25 h to 4 h after drug administration to cells, I... 454 / I 413 The value continued to decrease, reaching its lowest point after 4 hours, indicating that the liposomes entered the cell and entered the acidic intracellular environment, i.e., the lysosomes. After the lysosomes were destroyed using NH4Cl, I 454 / I 413 The value rebounded.

[0180] The antibody and antibody-conjugated liposomes were diluted in cell culture medium to a DiD concentration of 1.5 μM and then applied to AML cells MOLM-13 (CD33). + After incubating at 37°C for 0.5 or 2 hours, add a certain volume of Lyso-sensor (Dalian Meilun Biotechnology Co., Ltd.) to bring the final concentration to 1 μM. Incubate at 37°C for 0.5 hours, centrifuge at 1000 rpm for 5 minutes, remove the drug solution, wash once with PBS, stain with 10 ng / mL DAPI for 10 minutes, wash twice with PBS, add to a glass slide, cover with a coverslip, and observe under a laser confocal microscope. The results are shown in the appendix. Figure 23 .

[0181] Figure 23 Lysosomal colocalization was demonstrated. Ab-Lip@DiD was administered to MOLM-13 (CD33) + After incubation at 37℃ for 0.5 h and 4 h, lysosomes were labeled with a Lyso-sensor, and lysosomal co-localization was observed using a laser confocal microscope. The figures show that Lip@DiD was not taken up by MOLM-13, while Ab-Lip@DiD at 0.5 h had mostly just crossed the membrane and entered the cell, showing poor co-localization with lysosomes. At 4 h, most of Ab-Lip@DiD entered the lysosome, showing good co-localization, indicating that Ab-Lip@DiD is degraded within the lysosome after entering the cell.

[0182] Example 5: In vitro pharmacodynamic evaluation of the formulation

[0183] 1. Cytotoxicity

[0184] Liposomes containing CD33 antibody-conjugated sPMI peptide were prepared, diluted to gradient concentrations using cell culture medium, and then administered to CD33 antibody-conjugated cells. + , p53 depleted), MOLM-13 (CD33 + (p53 wild), SUP-B15 (CD33) -(p53 wild). After incubation at 37℃ for 72 h, 20 μL of CCK-8 was added to each well, and incubation was continued at 37℃ for another 2 h. The absorbance was measured at 450 nm using a microplate reader, and cell viability was determined using CCK-8 (Dalian Meilun Biotechnology Co., Ltd.). The results are shown in the appendix. Figure 24 .

[0185] Figure 24 The results of the cytotoxicity assay are shown. Liposomes conjugated with CD33 antibody and loaded with sPMI peptide were prepared, diluted to gradient concentrations using cell culture medium, and then administered to CD33 antibody-dependent ... + , p53 depleted), MOLM-13 (CD33 + (p53wild), SUP-B15 (CD33) - (p53 wild). After 72 hours, Ab-Lip@sPMI showed the best killing effect on MOLM-13, followed by SUP-B15. Due to the p53 deficiency in HL-60, HL-60 was insensitive to Ab-Lip@sPMI. The killing effect of Ab-Lip@sPMI on MOLM-13 was better than that of free sPMI, better than Lip@sPMI, but less effective than the small molecule p53 activator Nutlin-3a. However, Nutlin-3a has lower cell selectivity than peptide drugs, and at high concentrations, it still exhibits strong toxicity to p53-deficient HL-60.

[0186] 2. Apoptosis

[0187] Liposomes conjugated with CD33 antibody and loaded with sPMI peptide were prepared, diluted to a drug concentration of 30 μM using cell culture medium, and then administered to CD33 antibody-conjugated liposomes. + , p53 depleted), MOLM-13 (CD33 + (p53 wild), SUP-B15 (CD33) - After incubating at 37℃ for 8 hours (p53 wild), centrifuge at 1000 rpm for 5 minutes, remove the drug solution, and replace with fresh culture medium to culture for 48 hours. Centrifuge at 1000 rpm for 5 minutes, collect cells, wash once with PBS, add 200 μL Binding Buffer (Dalian Meilun Biotechnology Co., Ltd.), mix with a pipette, add 5 μL Annexin V-FITC (Dalian Meilun Biotechnology Co., Ltd.), and 5 μL PI (Dalian Meilun Biotechnology Co., Ltd.). Quantify the fluorescence signal of the cells using flow cytometry. The results are shown in the appendix. Figure 25 .

[0188] Figure 25The results of apoptosis evaluation were shown. Liposomes conjugated with CD33 antibody and loaded with sPMI peptide were prepared, diluted to a drug concentration of 30 μM using cell culture medium, and administered to CD33 antibody-dependent cell culture media. + , p53depleted), MOLM-13 (CD33 + (p53wild), SUP-B15 (CD33) - After 8 hours, the drug solution was aspirated and replaced with fresh culture medium for 48 hours. As shown in the figure, due to the p53 deficiency in HL-60 cells, Ab-Lip@sPMI, Lip@sPMI, sPMI, and Nutlin-3a did not induce significant apoptosis. For MOLM-13 cells, due to antibody-mediated infiltration, the amount of Ab-Lip@sPMI absorbed into cells was greater than that of Lip@sPMI in a short time, resulting in a significantly better apoptosis effect. For SUP-B15 cells, neither Ab-Lip@sPMI nor Lip@sPMI was taken up, thus no significant apoptosis was observed. In conclusion, the liposome-encapsulated, antibody-conjugated delivery method, compared to free peptide drugs, exhibits better cell selectivity, which is beneficial for targeted therapy.

[0189] 3. Cell cycle

[0190] Liposomes conjugated with CD33 antibody and loaded with sPMI peptide were prepared, diluted to a drug concentration of 30 μM using cell culture medium, and then administered to CD33 antibody-conjugated liposomes. + , p53 depleted), MOLM-13 (CD33 + (p53 wild), SUP-B15 (CD33) - After incubating at 37℃ for 8 hours (p53 wild), the cells were centrifuged at 1000 rpm for 5 minutes, the drug solution was removed, and the cells were cultured in fresh medium for 48 hours. The cells were then centrifuged at 1000 rpm for 5 minutes, collected, washed once with PBS, and mixed with pre-chilled 70% ethanol (at -20℃) using a pipette. The cells were then incubated at 4℃ overnight. Finally, the cells were centrifuged at 1000 rpm for 5 minutes, collected, washed once with PBS, and stained with 1 μg / mL DAPI at room temperature for 10 minutes. The fluorescence signal of the cells was quantified using flow cytometry. The results are shown in the appendix. Figure 26 .

[0191] Figure 26The results of cell cycle evaluation are shown. The method was the same as above. The results show that, due to the p53 deficiency in HL-60, Ab-Lip@sPMI, Lip@sPMI, sPMI, and Nutlin-3a did not significantly affect the cell cycle. For MOLM-13 cells, the influx of Ab-Lip@sPMI into cells was greater than that of Lip@sPMI in a short period. Due to antibody mediation, the proportion of cells in the S+G2 phase was significantly lower than that of Lip@sPMI, and cell proliferation was inhibited. For SUP-B15, neither Ab-Lip@sPMI nor Lip@sPMI was taken up; therefore, the cell cycle did not change significantly compared to sPMI and Nutlin-3a.

[0192] Although the invention and its advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made to this document without departing from the spirit and scope of the invention as defined by the appended claims. Furthermore, the scope of this application is not intended to be limited to the specific embodiments of the forms, means, methods, and steps described herein, including processes, machines, manufactures, and compositions. Those skilled in the art will readily understand from the content of this invention that existing or subsequently developed forms, means, methods, or steps that perform substantially the same function or achieve substantially the same results as the corresponding embodiments described herein may be used. Therefore, the appended claims are intended to include the forms, means, methods, or steps, processes, machines, manufactures, and compositions within their scope. sequence list <110> Fudan University Shanghai Xuhui Shangyi Zhongshan Immunotherapy Technology Transformation Research Center <120> Liposome drug delivery system for p53 agonist peptide mediated by humanized CD33 antibody <141> 2021-07-12 <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 238 <212> PRT <213> Artificial sequence <400> 1 Met Glu Lys Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser 35 40 45 Val Asp Asn Tyr Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro 50 55 60 Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys 100 105 110 Gln Gln Ser Lys Glu Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 2 <211> 462 <212> PRT <213> Artificial Sequence <400> 2 Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn 65 70 75 80 Gln Lys Phe Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly Gln Gly 115 120 125 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 130 135 140 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 145 150 155 160 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 165 170 175 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 180 185 190 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 195 200 205 Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro 210 215 220 Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu 225 230 235 240 Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu 245 250 255 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 260 265 270 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 275 280 285 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 290 295 300 Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu 305 310 315 320 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 325 330 335 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 340 345 350 Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 355 360 365 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 370 375 380 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 385 390 395 400 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly 405 410 415 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 420 425 430 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 435 440 445 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Cys 450 455 460 <210> 3 <211> 12 <212> PRT <213> Artificial sequence <400> 3 Thr Ser Phe Ala Glu Tyr Trp Ala Leu Leu Ser Pro 1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial sequence <400> 4 Leu Thr Phe Leu Glu Tyr Trp Ala Gln Leu Met Gln 1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial sequence <223> D-configuration amino acids, Phe has (p-Cl) modification. <400> 5 Glu Phe Trp Tyr Val Glu Phe Glu Lys Leu Leu Arg 1 5 10

Claims

1. A liposome-based drug delivery system, comprising liposomes, a targeting molecule, and a polypeptide drug encapsulated within the liposomes, characterized in that, The liposomes were prepared using hydrogenated soybean phosphatidylcholine, cholesterol, polyethylene glycol-distearate phosphatidylethanolamine, and maleimide-polyethylene glycol-distearate phosphatidylethanolamine in a molar ratio of 50:38.5:3:

2. The targeting molecule is an antibody conjugated to the surface of a liposome, the antibody is a humanized CD33 antibody with targeting activity, the light chain sequence of the humanized CD33 antibody is SEQ ID NO: 1, and the heavy chain sequence of the humanized CD33 antibody is SEQ ID NO: 2; The polypeptide drug is a p53 agonist peptide. The liposome drug delivery system was prepared using the following method: a) A solution providing hydrogenated soybean phosphatidylcholine, cholesterol, polyethylene glycol-distearyl phosphatidylethanolamine and maleimide-polyethylene glycol-distearyl phosphatidylethanolamine as lipid excipients; b) A solution providing the polypeptide drug; c) Mix the solution from step a) with the solution from step b), wherein the weight ratio of lipid excipient to peptide is 10:1 to 20:1, and the volume ratio of the solution from step a) to the solution from step b) is 4:1 to 5:

1. d) Liposomes loaded with polypeptide drugs were prepared using the reverse evaporation method; e) The antibody is mixed with tris(2-carboxyethyl)phosphine to obtain a reduced antibody; f) The reduced antibody is mixed with the liposomes loaded with the peptide drug prepared in step d) to obtain an antibody-conjugated liposome drug delivery system. In step c), the weight ratio of lipid excipient to peptide is 15.3:1, and the volume ratio of the solution in step a) to the solution in step b) is 4.5:

1. Step d) includes: adding 10 mM phosphate buffer solution with pH=6.5 to make the volume ratio of organic phase to aqueous phase 9:

1.

2. The liposome drug delivery system as described in claim 1, characterized in that, The p53 agonist peptide is selected from: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:

5.

3. The liposome drug delivery system as described in claim 1, characterized in that, The molar ratio of the antibody to maleimide-polyethylene glycol-distearate phosphatidylethanolamine is 1:36-1:

200.

4. The liposome drug delivery system as described in claim 1, characterized in that, Step d) further includes: sequentially extruding the membrane through 400 nm, 200 nm and 100 nm.

5. The liposome drug delivery system as described in claim 1, characterized in that, In step e), the molar ratio of antibody to tris(2-carboxyethyl)phosphine is 1:5 to 1:

10.

6. The liposome drug delivery system as described in claim 1, characterized in that, In step f), the molar ratio of antibody to maleimide-polyethylene glycol-distearate phosphatidylethanolamine is 1:147.

Images