Long-acting mono-modified pegylated double amino acid degradation activity of erwinia asparaginase conjugate and its preparation method and application

Abstract

The application discloses a long-acting single-modified polyethylene glycol double-amino acid degradation activity Escherichia asparaginease conjugate and a preparation method and application thereof, and belongs to the technical field of biological medicines. A protein skeleton of the conjugate is obtained by subjecting wild-type Escherichia L-asparaginease to S316C site-directed amino acid substitution mutation; a gene sequence of the mutant protein is shown as SEQ ID NO:1, and a corresponding amino acid sequence is shown as SEQ ID NO:2; and the mutant protein is coupled with a single molecule 40 kDa polyethylene glycol at the S316C mutation site. The conjugate of the application not only prolongs the half-life of the conjugate in the body, but also can maintain high activity in the consumption of L-glutamine, and can be applied to the preparation of a medicine for treating various types of solid tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a conjugate of a site-directed mutant of Erwinia chrysanthemi asparaginase and a single 40 kDa polyethylene glycol polymer, its preparation method and application. Background Technology

[0002] Erwinia L-asparaginase possesses dual catalytic activity as both L-asparaginase and L-glutaminase, degrading circulating L-asparagine and L-glutamine. It was approved by the FDA in 2011 for second-line treatment of acute lymphoblastic leukemia (ALL), particularly suitable for patients with allergic reactions to first-line E. coli-derived asparaginase (Pieters, R., Hunger, SP, Boos, J., Rizzari, C., Silverman, L., Baruchel, A., ... & Pui, CH (2011). L‐asparaginase treatment in acute lymphoblastic leukemia: a focus on Erwinia asparaginase. Cancer, 117(2), 238-249.).To prolong the half-life of Erwinia asparaginase in vivo, researchers randomly PEGylated it to obtain the derivative Pegcrisantaspase (PegC), which is used to treat non-L-asparagine auxotrophic cancers such as acute myeloid leukemia (AML) and pancreatic ductal adenocarcinoma (Liu, Y., Bollino, DR, Bah, OM, Strovel, ET, Le, TV, Zarrabi, J., ... & Emadi, A. (2025). A phase 1 study of the amino acid modulator pegcrisantaspase and venetoclax for relapsed or refractory acute myeloidleukemia. Blood, 145(5), 486-496. Liu, Y., Bollino, D., Bah, OM, Strovel, ET, Zarrabi, J., Philip, S., ... & Emadi, A. (2023). Overcoming Venetoclax (Ven) Resistance through glutamine (gln) depletion: final analysis of thephase 1 Trial of Ven and Pegcrisantaspase (PegC) combination in relapsed andrefractory (R / R) Acute myeloid leukemia (AML). Blood, 142, 60. Bollino, D.,Hameed, K., Bhat, A., Zarrabi, A., Casildo, A., Ma, X., ... & Emadi, A. (2024). Long-acting Erwinia chrysanthemi, Pegcrisantaspase, induces alternateamino acid biosynthetic pathways in a preclinical model of pancreatic ductaladenocarcinoma. Cancer & Metabolism, 12(1), 19.).After modification with 5 kDa PEGylation, the dosage of Erwinia asparaginase can be increased from the FDA-approved 25,000 U / m. 2 Reduced to 750 U / m 2A single dose can maintain its efficacy in vivo for about one week (Liu, Y., Bollino, DR, Bah, OM, Strovel, ET, Le, TV, Zarrabi, J., ... & Emadi, A. (2025). A phase 1 study of the amino acidmodulator pegcrisantaspase and venetoclax for relapsed or refractory acutemyeloid leukemia. Blood, 145(5), 486-496.). Current Phase I clinical data on PegC in combination with Venetoclax for the treatment of relapsed and refractory AML show that subjects achieving complete remission had the lowest plasma L-glutamine concentrations (Liu, Y., Bollino, D., Bah, OM, Strovel, ET, Zarrabi, J., Philip, S., ... & Emadi, A. (2023). Overcoming Venetoclax (Ven) Resistance through glutamine(gln) depletion: final analysis of the phase 1 Trial of Ven and Pegcrisantaspase (PegC) combination in relapsed and refractory (R / R) Acutemyeloid leukemia (AML). Blood, 142, 60.). Existing random PEGylation processes have significant drawbacks: random PEGylation of PegC not only reduces its efficiency in consuming cancer cell-dependent L-glutamine, but also makes it difficult to use in patients with acute myeloid leukemia (Liu, Y., Bollino, DR, Bah, OM, Strovel, ET, Le, TV, Zarrabi, J., ... & Emadi, A. (2025). A phase 1 study of the aminoacid modulator pegcrisantaspase and venetoclax for relapsed or refractoryacute myeloid leukemia. Blood, 145(5), 486-496. Liu, Y., Bollino, D., Bah, OM, Strovel, ET, Zarrabi, J., Philip, S., ... & Emadi, A. (2023). Overcoming Venetoclax (Ven) Resistance through glutamine (gln) depletion: final analysis of the phase 1 Trial of Ven and Pegcrisantaspase (PegC)combination in relapsed and refractory (R / R) Acute myeloid leukemia (AML). Blood, 142, 60.), mouse pancreatic xenograft tumor model (Bollino, D., Hameed, K., Bhat, A., Zarrabi, A., Casildo, A., Ma, X., ... & Emadi, A. (2024). Long-acting Erwiniachrysanthemi, Pegcrisantaspase, induces alternate amino acid biosyntheticpathways in a preclinical model of pancreatic ductal adenocarcinoma. Cancer & Metabolism, 12(1), 19.) and leukemia and lymphoma cells (Chien, WW, Allas, S., Rachinel, N., Sahakian, P., Julien, M., Le Beux, C., ... & Salles, G. (2014). Pharmacology, immunogenicity, and efficacy of a novel pegylated recombinant Erwinia chrysanthemi-derived L-asparaginase. Investigational new drugs, 32(5), 795-805.) maintain long-lasting yet low plasma L-glutamine concentrations. Furthermore, random polyethylene glycol modification can easily lead to batch-to-batch variations, increasing drug production costs. Summary of the Invention

[0003] To address the shortcomings of existing randomized polyethylene glycol-modified Erwinia asparaginases, such as decreased glutamine-consuming activity, significant batch-to-batch product variability, and limited drug efficacy, this invention aims to provide a highly efficient, low-immunogenic, and long-acting mono-PEGylated Erwinia asparaginase conjugate, its preparation process, and novel applications. This invention retains the enzyme's highly efficient amino acid degradation activity while improving in vivo drug stability and safety, thus expanding its application scenarios in tumor treatment.

[0004] This invention provides a long-acting, mono-modified polyethylene glycol-modified dual amino acid degradation activity Erwinia asparaginase (named NGAsparaginase40B and NGAsparaginase40L), belonging to the category of site-directed mono-polyethylene glycol-modified proteins. This modified asparaginase is based on wild-type Erwinia asparaginase, with a site-directed gene mutation at site S316C (serine at position 316 → cysteine). Its coding gene sequence is shown in SEQ ID NO:1, and the corresponding amino acid sequence is shown in SEQ ID NO:2.

[0005] This invention employs a site-specific PEGylation modification strategy, which, compared to traditional random PEG modification technology, can precisely achieve site-specific coupling of single-molecule PEG, completely avoiding the problems of product heterogeneity and large batch-to-batch variability caused by random modification. Simultaneously, the site-specific modification method can completely preserve the naturally high L-glutamine-consuming activity of Erwinia asparaginase, maintaining its dual amino acid consumption function; and through site-specific encapsulation modification with a single PEG molecule, it can effectively shield the enzyme protein antigen sites, reducing the recognition and attack of the body's own antibodies, significantly reducing drug immunogenicity, and greatly improving the drug's in vivo efficacy and long-lasting effect.

[0006] The polyethylene glycol (PEG) used in this invention has a molecular weight of 40 kDa and includes both linear and branched types, corresponding to the preparation of NGAsparaginase 40L (linear PEG modification) and NGAsparaginase 40B (branched PEG modification), respectively. In the PEGylation reaction system, the preferred molar ratio of modified protein to PEG molecules is 1:1.5, which can stably achieve single-molecule site-specific modification efficiency and avoid the problems of polymer modification or incomplete modification.

[0007] This invention also discloses a specific purification method for the above-mentioned polyethylene glycol-modified protein, which adopts a tandem purification process of anion and cation columns: preliminary purification is first performed through a Q column to remove impurities, nucleic acids and most impurities, followed by fine purification through an SP column, finally obtaining high-purity, uniform single-site PEG-modified Erwinia asparaginase, effectively ensuring batch consistency of the product.

[0008] The long-acting monomodified polyethylene glycol-modified Erwinia asparaginase conjugate prepared by this invention has the advantages of high amino acid consumption activity, low immunogenicity, and long-term stability. It can be applied to the treatment of solid tumors, especially suitable for the clinical treatment of lung cancer and colorectal cancer. It breaks through the traditional limitation of Erwinia asparaginase to be used only for hematologic malignancies and expands the anti-tumor application scenarios of this type of enzyme preparation.

[0009] The beneficial effects of this invention are:

[0010] This invention achieves specific site-directed binding to 40 kDa polyethylene glycol (PEG) through site-directed mutation of the S316C gene, yielding mono-PEGylated proteins NGAsparaginase40B and NGAsparaginase40L. This invention achieves single-site, uniform PEG modification through S316C site-directed mutation, effectively prolonging the drug's in vivo half-life while fully preserving the efficient L-glutamine-consuming activity of asparaginase. This overcomes the application limitations of existing technologies and can be effectively applied to the treatment of various solid tumors. The products of this invention achieve site-directed mono-PEG modification (distinct from random PEG modification), exhibiting extremely high reaction specificity. Attached Figure Description

[0011] Figure 1 SDS-PAGE analysis of NGAsparaginase expression and purification. Lane 1: Label; Lane 2: Total lysis buffer; Lane 3: Soluble fraction; Lane 4: Q column elution; Lane 5: Q column 100% elution buffer; Lane 6: SP column elution; Lane 7: SP column 5% elution buffer; Lane 8: SP column 10% elution buffer; Lane 9: SP column 100% elution buffer; Lane 10: NGAsparaginase after buffer exchange.

[0012] Figure 2 Molecular size of NGAsparaginase 40B / 40L before and after polyethylene glycolation.

[0013] Figure 3 : The change in plasma L-asparaginamide concentration in mice over time after a single intraperitoneal injection of NGAsparaginase 40B / 40L-.

[0014] Figure 4 The curve of plasma L-glutamine concentration in mice after a single intraperitoneal injection of NGAsparaginase 40B / 40L over time.

[0015] Figure 5 Inhibitory effects of different concentrations of NGAsaparaginas40B on A549 lung cancer cells.

[0016] Figure 6 The inhibitory effect of NGAsaparaginas40B at the same concentration on HCT116 colon cancer cells. Detailed Implementation

[0017] The reagents and instruments used in this invention are all commercially available products, and the experimental methods, unless otherwise specified, are all conventional laboratory methods.

[0018] This invention provides a site-directed conjugate formed by Erwinia asparaginase and polyethylene glycol, which achieves specific site-directed binding to 40 kDa polyethylene glycol through site-directed gene mutation of S316C, thereby producing polyethylene glycol-modified proteins NGAsparaginase40B and NGAsparaginase40L.

[0019] Natural Erwinia asparaginase is only used for the treatment of acute lymphoblastic leukemia (Pieters, R., Hunger, SP, Boos, J., Rizzari, C., Silverman, L., Baruchel, A., ... & Pui, CH (2011). L‐asparaginase treatment in acute lymphoblastic leukemia: a focus on Erwinia asparaginase. Cancer, 117(2), 238-249.), and has a half-life of only about 6 hours in the human body (Albertsen, B., Jakobsen, P., Schrøder, H., Schmiegelow, K., & Carlsen, NT (2001). Pharmacokinetics of Erwinia asparaginase after intravenous and intramuscular administration. Cancer chemotherapy and pharmacology, 48(1), 77-82.), and has a short duration of action.While existing random polyethylene glycol modification can prolong the in vivo half-life of enzymes, it significantly weakens their L-glutamine depletion activity, making it difficult to apply to the treatment of solid tumors (Liu, Y., Bollino, DR, Bah, OM, Strovel, ET, Le, TV, Zarrabi, J., ... & Emadi, A. (2025). A phase 1 study of the amino acid modulator pegcrisantaspase and venetoclax for relapsed orrefractory acute myeloid leukemia. Blood, 145(5), 486-496. Liu, Y., Bollino, D., Bah, OM, Strovel, ET, Zarrabi, J., Philip, S., ... & Emadi, A. (2023). Overcoming Venetoclax (Ven) Resistance through glutamine (gln) depletion: final analysis of the phase 1 Trial of Venetoclax and Pegcrisantaspase(PegC) combination in relapsed and refractory (R / R) Acute myeloid leukemia(AML). Blood, 142, 60. Bollino, D., Hameed, K., Bhat, A., Zarrabi, A., Casildo, A., Ma, X., ... & Emadi, A. (2024). Long-acting Erwiniachrysanthemi, Pegcrisantaspase, induces alternate amino acid biosynthetic pathways in a preclinical model of pancreatic ductal adenocarcinoma. Cancer &Metabolism, 12(1), 19.).

[0020] This invention provides a site-directed conjugate formed by Erwinia asparaginase and polyethylene glycol (PEG). Specific binding to 40 kDa PEG is achieved through site-directed mutation of the S316C gene. This specific binding originates from a thiol-maleimide coupling reaction, yielding PEG-modified proteins NGAsparaginase40B and NGAsparaginase40L. This single-site, uniform PEG modification achieved through S316C site-directed mutation effectively prolongs the drug's in vivo half-life while fully preserving the asparaginase's efficient L-glutamine-consuming activity. This overcomes the limitations of existing technologies and can be effectively applied to the treatment of various solid tumors.

[0021] The linear polyethylene glycol used in this invention was purchased from Xiamen Sinobond Biotechnology Co., Ltd., product model mPEG-MAL-40K, molecular weight 40 kDa, structural formula:

[0022]

[0023] The branched polyethylene glycol used in this invention was purchased from Xiamen Sinobond Biotechnology Co., Ltd., product model 2-arm PEG-MAL, molecular weight 40 kDa, structural formula:

[0024]

[0025] Example 1

[0026] Expression and purification of NGAsparaginase:

[0027] The gene encoding the mutant NGAsparaginase (S316C) in the pET9a vector was transformed into the BL21(DE3) strain for protein expression. The mutant protein was obtained by site-directed mutation of the wild-type Erwinia asparaginase gene into S316C. The gene sequence encoding this mutant protein is shown in SEQ ID NO:1, and the corresponding amino acid sequence is shown in SEQ ID NO:2. Single colonies were picked and inoculated into sterile LB medium containing 50 µg / mL kanamycin, and cultured overnight at 30°C and 250 rpm for activation. The overnight culture was then inoculated at a 1:100 ratio into 200 mL of sterile LB medium containing 50 µg / mL kanamycin and cultured further at 30°C and 250 rpm. The optical density (OD) of the culture was measured. 600When the eosinophil count reached 0.6-0.8, 1 mM IPTG was added to induce protein expression. After induction, the bacterial pellet was collected by centrifugation, and the cells were lysed using an ultrasonic homogenizer in resuspension buffer (20 mM Bis-Tris, 50 mM NaCl, pH 5.5). The supernatant was collected for later use. The supernatant was then loaded onto a HiTrap Q chromatography column for purification. The flow-through containing the target protein was collected, and the flow-through was loaded onto a HiTrap SP chromatography column for further gradient elution. The elution buffer consisted of 20 mM Bis-Tris, 1 M NaCl, and pH 5.5, with the target protein eluted in a range of 5%-10% elution buffer. The purity and expression levels of the samples at each stage were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0028] SDS-PAGE test results are as follows: Figure 1 As shown: Lanes 2 and 3 correspond to the total bacterial lysate and the soluble cell supernatant, respectively, both of which showed the presence of NGAsparaginase, proving that the protein is expressed in a soluble manner; Lane 4 is the HiTrap Q column flow-through component, in which the target protein is entirely present; After gradient elution of the flow-through using an SP column, lanes 7 and 8, corresponding to 5% and 10% elution ratios, respectively, were used to collect the target protein, which has a molecular weight of 36 kDa; Lane 10 is the purified protein sample after buffer replacement.

[0029] Example 2

[0030] Polyglycolation of NGAsparaginase:

[0031] The purified NGAsparaginase (S316C) obtained in Example 1 was reacted with 40 kDa linear polyethylene glycol and 40 kDa branched polyethylene glycol molecules at a molar ratio of 1:1.5. After the reaction, unreacted polyethylene glycol was removed using a 100 kDa molecular weight cutoff ultrafiltration device. NGAsparaginase 40L (linear PEG-modified product) and NGAsparaginase 40B (branched PEG-modified product) were prepared. NGAsparaginase 40B / 40L were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0032] The apparent molecular weight of NGArginase (S316C) after PEG modification increased significantly during electrophoresis. This is because the PEG chains can bind a large number of water molecules to form hydrated polymers, increasing the hydrodynamic size of the product. Figure 2As shown, 40 kDa linear PEG and branched PEG were coupled with NGArginase (S316C), resulting in NGArginase 40L with an apparent molecular weight of approximately 100 kDa and NGArginase 40B with an apparent molecular weight of approximately 140 kDa; however, their theoretical molecular weights are only about 76 kDa. The hydration effect of PEG is the core reason for the significantly higher apparent molecular weights on electrophoresis. The molecular weight of PEG is 40 kDa, and the molecular weight of the NGArginase (S316C) protein is approximately 36 kDa.

[0033] Example 3

[0034] Determination of the specific activity of NGAsparaginase 40B / 40L:

[0035] The concentration of ammonium ions released by substrate hydrolysis was detected using Nessler's reagent, and the specific enzyme activities against L-asparaginase and L-glutamine were determined separately. The simplified procedure is as follows: 5 μL of NGAsparaginase and NGAsparaginase 40B / 40L were mixed with 200 μL of L-asparaginase or L-glutamine solution (pH 7.4) and reacted at 37 °C. After the reaction was complete, 15 μL of 80% trichloroacetic acid was added to terminate the enzymatic reaction. Subsequently, the mixture was mixed with 800 μL of diluted Nessler's reagent and incubated at room temperature in the dark for 10 minutes. The absorbance of the reaction mixture at 430 nm was then measured and recorded using a UV-Vis spectrophotometer. The International Unit (IU) of enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of ammonia per minute from the substrate at 37 °C and pH 7.4.

[0036] Table 1 Comparison of specific activity of NGAsparaginase before and after modification with polyethylene glycol alone.

[0037]

[0038] Table 1 shows that the specific activities of NGAsparaginase for L-asparagine and L-glutamine remained basically the same before and after modification with a single polyethylene glycol.

[0039] Example 4

[0040] Pharmacodynamic studies of NGAsparaginase 40B / 40L:

[0041] To determine the pharmacodynamics of NGAsparaginase 40B / 40L in blood, plasma samples were collected from mice before and after injection of NGAsparaginase 40B / 40L to determine the concentrations of L-asparagine and L-glutamine. The specific method is as follows: On day 0, 6-week-old BALB / c mice were intraperitoneally injected (ip) with NGAsparaginase 40L (0.75 mg / kg) and NGAsparaginase 40B (0.75 mg / kg), respectively, with 3 mice in each group. Blood samples were collected from the lateral saphenous vein at different time points, and plasma was collected after centrifugation. An equal volume of plasma sample was mixed thoroughly with 10% sulfosalicylic acid solution and incubated overnight at 4°C to precipitate proteins. After precipitation, the sample was centrifuged at 13500 rpm for 10 minutes to remove the precipitated proteins. An equal volume of the supernatant was mixed with lithium citrate loading buffer. The processed samples and amino acid standards were loaded together into an amino acid analyzer (Hitachi L-8900) for the detection of serum L-asparagine and L-glutamine. Simultaneously, the weight changes of mice in each group were recorded.

[0042] like Figures 3-4 As shown, NGAsparaginase 40B / 40L completely consumed L-asparaginase to undetectable levels, with the effect lasting approximately 14 days. On the other hand, in mice injected with NGAsparaginase 40L, L-glutamine levels recovered to approximately 170 µM on day 7, while in mice injected with NGAsparaginase 40B, L-glutamine levels only began to recover to approximately 20 µM on day 10. Conversely, natural Erwinia chrysanthemi asparaginase, when injected into mice at a dose of 0.68 mg / kg, only consumed L-asparaginase for 72 hours and did not show any effect on L-glutamine consumption (Modi, T., & Gervais, D. (2022). Improved pharmacokinetic and pharmacodynamic profile of a novel PEGylated native Erwinia chrysanthemi L-Asparaginase. Investigational New Drugs, 40(1), 21-29.). Compared to natural Erwinia asparaginases, NGAsparaginase 40B / 40L exhibited a longer duration of action in consuming L-asparaginase and L-glutamine, demonstrating a significantly prolonged half-life. Furthermore, NGAsparaginase 40B was more effective than NGAsparaginase 40L in eliminating L-glutamine.

[0043] Example 5

[0044] Inhibition test of NGAsparaginase40B on A549 lung cancer cells and HCT116 colon cancer cells:

[0045] A549 or HCT116 cancer cells were seeded at a density of 3000 cells per well in 96-well plates and cultured overnight in RPMI 1640 medium (containing 10% fetal bovine serum and penicillin-streptomycin) at 37°C with 5% CO2. After 24 hours, different concentrations of NGAsparaginase 40B were added, and the cells were cultured for another 72 hours. 10 µL of 5 mg / mL MTT reagent was added to each well, and the cells were incubated at 37°C for 4 hours. After incubation, the culture medium was removed, and 100 µL of DMSO was added to dissolve formazan crystals. Cell viability was determined by absorbance at 540 nm, and IC50 was calculated using Prism software. 50 Value. n=6.

[0046] like Figures 5-6 As shown, NGAsparaginase 40B can effectively inhibit the proliferation of A549 lung cancer cells and HCT116 colon cancer cells, with an IC50 value of 1.5%. 50 The values ​​were 0.101 ± 0.006 µg / mL and 0.020 ± 0.004 µg / mL, respectively.

Claims

1. A long-lasting, mono-modified polyethylene glycol-modified Erwinia asparaginase conjugate with dual amino acid degradation activity, characterized in that, The protein backbone of the conjugate is obtained by site-directed amino acid substitution mutation of wild-type Erwinia L-asparaginase at S316C; the gene sequence encoding the mutant protein is shown in SEQ ID NO:1, and the corresponding amino acid sequence is shown in SEQ ID NO:2; the mutant protein is site-directedly coupled to a single molecule of 40 kDa polyethylene glycol at the S316C mutation site.

2. The Erwinia asparaginase conjugate with long-lasting mono-modified polyethylene glycol-modified dual amino acid degradation activity according to claim 1, characterized in that, The 40 kDa polyethylene glycol is either linear polyethylene glycol or branched polyethylene glycol; the linear 40 kDa polyethylene glycol modified product is named NGAsparaginase40L, and the branched 40 kDa polyethylene glycol modified product is named NGAsparaginase40B.

3. The Erwinia asparaginase conjugate with long-acting monomodified polyethylene glycol-modified dual amino acid degradation activity according to claim 2, wherein the conjugate simultaneously and efficiently degrades L-asparagine and L-glutamine, and the specific enzyme activity before and after modification does not decrease significantly.

4. A method for preparing the long-acting mono-modified polyethylene glycol-modified Erwinia asparaginase conjugate with dual amino acid degradation activity as described in claim 2, characterized in that, Includes the following steps: The pET9a vector containing the gene encoded by SEQ ID NO:1 was transformed into BL21 (DE3) competent strain, and the S316C mutant Erwinia L-asparaginase was obtained by IPTG induction. The bacterial cell lysis supernatant was sequentially passed through a HiTrap Q anion exchange chromatography column to collect the flow-through fraction, and then eluted with a HiTrap SP cation exchange chromatography column to obtain high-purity mutant naked protein. The purified mutant naked protein was coupled to 40 kDa polyethylene glycol at a protein:PEG molar ratio of 1:1.

5. Unreacted free polyethylene glycol was removed using an ultrafiltration device with a molecular weight cutoff of 100 kDa to obtain the target monoPEG-modified conjugate.

5. The preparation method according to claim 4, characterized in that, The HiTrap SP cation exchange column was used for linear gradient elution with 20 mM Bis-Tris, 1 M NaCl, and pH 5.5 elution buffer. The target protein was collected by elution within the range of 5%-10% elution buffer ratio.

6. The use of the long-acting monomodified polyethylene glycol-modified Erwinia asparaginase conjugate with dual amino acid degradation activity as described in any one of claims 1-3 in the preparation of antitumor drugs.

7. The application according to claim 6, characterized in that, The tumors include lung cancer and colorectal cancer.

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