WO2024207635A1 - 一种降低免疫原和增强抗肿瘤功效的asp-elp及其制备方法与应用 - Google Patents

一种降低免疫原和增强抗肿瘤功效的asp-elp及其制备方法与应用 Download PDF

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WO2024207635A1
WO2024207635A1 PCT/CN2023/105046 CN2023105046W WO2024207635A1 WO 2024207635 A1 WO2024207635 A1 WO 2024207635A1 CN 2023105046 W CN2023105046 W CN 2023105046W WO 2024207635 A1 WO2024207635 A1 WO 2024207635A1
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asp
elp
long
amino acid
elps
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高卫平
张三柯
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Peking University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6435—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a connective tissue peptide, e.g. collagen, fibronectin or gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6925—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a microcapsule, nanocapsule, microbubble or nanobubble
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/82—Asparaginase (3.5.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01001—Asparaginase (3.5.1.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)

Definitions

  • the present invention belongs to the field of biomedicine technology, and specifically relates to an ASP-ELP capable of reducing immunogens and enhancing anti-tumor efficacy, and a preparation method and application thereof.
  • Asparaginase is a bacterial enzyme that can selectively inhibit hematological malignant tumor cells, especially for pediatric acute lymphoblastic leukemia, and can significantly improve the complete remission rate (CR) and cure rate of hematological malignancies.
  • Most tumor cells in the hematological system cannot synthesize the amino acid asparagine necessary for growth by themselves, and must rely on the host for supply.
  • ASP can selectively induce tumor cell apoptosis by catalyzing the decomposition of asparagine necessary for tumor cell growth.
  • PEG-ASP liposomal asparaginase
  • FDA Food and Drug Administration
  • ALL acute lymphoblastic leukemia
  • NCCN National Comprehensive Cancer Network Clinical Practice Guidelines for Oncology
  • PCB polycarboxybetaine
  • EK glutamic acid and lysine
  • P(CB-EG3Glu) poly(L)-proline
  • PGP poly(L)-proline
  • the present invention provides an ASP-ELP capable of reducing immunogens and enhancing anti-tumor efficacy, and a preparation method and application thereof.
  • the present invention provides a long-acting sustained-release drug, which is obtained by artificial intelligence-assisted design; it includes a therapeutic agent and a drug carrier, the therapeutic agent is a protein drug; the drug carrier is ELP; the artificial intelligence-assisted design refers to: integrating the physical and biological knowledge of protein structure into the deep learning algorithm of AlphaFold2 using multiple sequence alignment, and the AlphaFold2 predicts the three-dimensional structure based on the amino acid sequence of the long-acting sustained-release drug, and finally determines the composition of the long-acting sustained-release drug.
  • the long-acting sustained-release drug provided by the present invention is a homotetramer complex composed of a core and an outer shell; wherein the therapeutic agent forms a tetramer core, and the ELP chain coils around and wraps the tetramer core to form a shell-like structure.
  • the amino acid sequence of the ELP is composed of a repeating unit of valine-proline-glycine-X amino acid-glycine;
  • the X amino acid is one or more of valine, alanine, glycine, leucine, isoleucine, lysine or histidine;
  • the amino acid sequence of the ELP consists of a pentapeptide repeating unit of valine-proline-glycine-X amino acid-glycine; the X amino acid is valine, alanine, glycine, leucine, isoleucine, lysine or histidine.
  • the pentapeptide repeating unit of ELP is valine-proline-glycine-valine- Glycine; the number of ELP repeats is 30-150;
  • the number of ELP repeats is 60-120.
  • the amino acid repeating unit of ELP is: 30 to 200 times, wherein X amino acid is one of valine, alanine, glycine, leucine, isoleucine, lysine, phenylalanine, and histidine. ELPs with different X amino acids can exhibit different biological functions.
  • the type of ELP is valine-proline-glycine-valine-glycine (VPGVG); based on the consideration of reducing immunogenicity and retaining activity, the number of ELP repeats is 30-150; preferably, the number of ELP repeats is 60-120.
  • an ELP protein is used to couple a protein with higher immunogenicity than the ELP protein; the low-immunogenic ELP acts as a physical barrier for the highly immunogenic protein, shielding the antigenic epitope of the highly immunogenic protein, thereby reducing the recognition of the immune system in the body and the clearance rate of the drug;
  • the therapeutic agent is asparaginase (ASP), interferon (IFN), growth hormone (GH) or tyrosine-rich protein.
  • the therapeutic agent is asparaginase.
  • amino acid sequence of the asparaginase is N-(2-aminoethyl)-2-aminoethyl
  • the asparaginase (ASP) applicable to the present invention may be a heterologous protein derived from Escherichia coli. ASP can stimulate the body to produce antibodies, causing allergic reactions, which are life-threatening in severe cases, and reduce the efficacy of ASP and the long-term disease-free survival of patients.
  • ASP Asparaginase
  • the present invention also provides a method for preparing a long-acting sustained-release drug, wherein the three-dimensional structure of the long-acting sustained-release drug is obtained by artificial intelligence-assisted design, the number of repetitions of asparaginase and ELP is determined, and elastin-like fusion asparaginase is obtained by biosynthesis, chemical coupling synthesis or solid phase synthesis; the biosynthesis refers to fusing the DNA fragment of asparaginase to the N-terminus of ELP through genetic engineering to construct an asparaginase-ELP plasmid.
  • the present invention provides an isolated polypeptide, the amino acid sequence of which includes the amino acid sequence of ELP and the amino acid sequence encoding the therapeutic agent; the amino acid sequence of ELP is composed of a pentapeptide repeating unit of valine-proline-glycine-X amino acid-glycine; the X amino acid is valine, alanine, glycine, leucine, isoleucine, lysine or histidine.
  • the present invention provides an isolated nucleic acid encoding the above-mentioned polypeptide.
  • the present invention provides a biomaterial, and the biomaterial provided by the present invention is used to generate the above-mentioned polypeptide or the biomaterial contains the above-mentioned nucleic acid; the biomaterial is a recombinant plasmid, a recombinant vector, or a recombinant cell.
  • the present invention also provides the use of the above-mentioned long-acting sustained-release drug or the above-mentioned polypeptide or the above-mentioned nucleic acid or the above-mentioned biological material in the preparation of a drug for treating malignant tumors of the blood system;
  • the hematological malignancy is acute lymphocytic leukemia, acute granulocytic leukemia, acute monocytic leukemia, malignant lymphoma or a hematological malignancy that is sensitive to asparaginase.
  • the present invention provides a drug for treating hematological malignancies, comprising the above-mentioned long-acting sustained-release drug.
  • the present invention provides an assessment of the in vivo safety of elastin-like fusion asparaginase (ASP-ELP) under single and multiple dosing modes, and the assessment of the in vivo safety under multiple dosing modes can comprehensively consider the effects of high-low concentration fluctuations and immunogenicity of the drug on in vivo safety.
  • ASP-ELP elastin-like fusion asparaginase
  • single administration is administered at the maximum tolerated dose (MTD) of the drug, and multiple administration is administered at the same dose once a week for 6 consecutive times, wherein the number of administrations in the multiple administrations is greater than 2.
  • MTD maximum tolerated dose
  • the present invention also carries out two methods: single administration of maximum tolerated dose (SA-MTD) and multiple administration of the same dose (MA-SD). Results of the evaluation of the effect of the dosing regimen.
  • SA-MTD maximum tolerated dose
  • MA-SD multiple administration of the same dose
  • ELP and asparaginase were fused through genetic engineering to form elastin-fused asparaginase (ASP-ELP), which not only maintained the biological activity of ASP but also reduced protein enzymatic degradation and renal excretion ( Figure 1).
  • ASP-ELP elastin-fused asparaginase
  • Figure 1 The ELP chain in ASP-ELP blocks the ASP antigen epitope, thereby effectively reducing the recognition of the human immune system.
  • thermosensitive property of ASP-ELP forms a sustained-release drug reservoir after intraperitoneal injection, thereby improving pharmacokinetics, reducing systemic toxicity, and enhancing anti-tumor efficacy (Figure 2).
  • the present invention tested and verified the performance of ASP-ELP through in vitro activity retention and stability studies and in vivo MTD, pharmacokinetics, immunogenicity, toxicity and anti-tumor effect studies.
  • FIG1 is a schematic diagram of the biosynthesis of ASP-ELP provided by the present invention.
  • FIG. 2 is a diagram showing the mechanism of ASP-ELP of the present invention in treating hematological malignancies.
  • Figure 3 is a structural diagram of ASP and ASP-ELPS predicted by AlphaFold2 of the present invention.
  • FIG. 4 is a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purification process of ASP-ELPs of the present invention.
  • FIG5 is a graph showing the purity of ASP and ASP-ELPs confirmed by SDS-PAGE.
  • FIG6 is a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis.
  • MALDI-TOF-MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry
  • FIG7 is a circular dichroism (CD) curve analysis, and the CD curve of ASP-ELPs completely overlaps with that of ASP, indicating that the secondary structure does not change after ASP fusion.
  • CD circular dichroism
  • FIG. 8 shows the hydrated diameters of ASP and ASP-ELPs measured by dynamic light scattering (DLS).
  • FIG9 shows the phase transition temperature of ASP-ELPs.
  • FIG. 10 shows the enzymatic activities of ASP, PEG-ASP and ASP-ELPs.
  • FIG. 11 shows the in vitro cytotoxicity of ASP, PEG-ASP and ASP-ELPs.
  • FIG. 12 shows the half maximal inhibitory concentration (IC 50 ) of ASP, PEG-ASP and ASP-ELPs on hematological tumors.
  • FIG. 13 shows the long-term storage stability of ASP, PEG-ASP and ASP-ELPs.
  • FIG. 14 is a representative image of the fluorescence intensity changes of Cy5-labeled ASP, PEG-ASP, and ASP-ELPs at the injection site.
  • FIG. 15 shows the in vivo fluorescence retention rates of Cy5-labeled ASP, PEG-ASP and ASP-ELPs at different times.
  • FIG. 16 is a graph showing the maximum tolerated dose (MTD) of ASP, PEG-ASP and ASP-ELPs in mice determined by a dose escalation experiment.
  • FIG. 17 is a graph showing the relationship between serum ASP activity level and time after intraperitoneal injection of the drug.
  • FIG. 18 shows the peak plasma concentrations (C max ) of ASP, PEG-ASP, and ASP-ELPs.
  • FIG. 19 shows the in vivo circulation half-life (t 1/2 ) of ASP, PEG-ASP and ASP-ELPs.
  • FIG. 20 is the area under the blood concentration-time curve (AUC) of ASP, PEG-ASP and ASP-ELPs.
  • FIG. 21 is the relationship between the plasma ASP activity level and time before and after immunization of mice.
  • FIG. 22 shows the in vivo circulation half-life (t 1/2 ) of ASP, PEG-ASP and ASP-ELPs before and after immunization of mice.
  • FIG. 23 shows the titers of anti-protein and anti-polymer immunoglobulin G (IgG) in mice after immunization.
  • FIG. 24 shows the titers of anti-protein and anti-polymer immunoglobulin M (IgM) in mice after immunization.
  • FIG. 25 shows representative images of H&E-stained pathological sections of the main organs of mice after administration of a single maximum tolerated dose (SA-MTD).
  • FIG. 26 shows representative images of H&E-stained pathological sections of the main organs of mice after multiple administration of the same dose (MA-SD).
  • FIG. 27 is an analysis of liver function indicators after administration of SA-MTD and MA-SD.
  • FIG. 28 is an analysis of renal function indicators after administration of SA-MTD and MA-SD.
  • FIG. 29 is an analysis of routine blood indexes after administration of SA-MTD and MA-SD.
  • FIG30 shows the anticancer effect of drug SA-MTD treatment on the leukemia (CCRF-CEM-Luc) CDX mouse model.
  • FIG31 shows the anticancer effect of drug SA-MTD treatment on the lymphoma (Raji-Luc) CDX mouse model.
  • FIG. 32 shows the anticancer effect of drug MA-SD treatment on the leukemia (CCRF-CEM-Luc) CDX mouse model.
  • FIG. 33 shows the anticancer effect of drug MA-SD treatment on the lymphoma (Raji-Luc) CDX mouse model.
  • ASP-ELPs structure of ASP-ELPs with different chain lengths was predicted by the multimer model of Alphafold2, and two promising candidates (ASP-ELPS) were selected for further study.
  • ASP-ELPS form a core-shell homotetrameric complex, in which their ASP molecules form a tetrameric core, similar to the tetrameric structure of ASP, while the flexible, randomly coiled ELP chains wrap around the core of their ASP to form a shell-like structure (Figure 3).
  • the predicted local distance difference test (plDDT) scores of the ELP chains are generally low, which confirms the disorder of the structure.
  • this example conducted a series of in vitro and in vivo experiments to more accurately evaluate the effect of ASP-ELPS on improving the performance of ASP.
  • the gene encoding ASP (UniProtKB/Swiss-Prot: P00805.2) was amplified by polymerase chain reaction (PCR) and inserted into the pET-25b (+) vector. Plasmids encoding ELP 60 and ELP 90 genes were constructed using the recursive directional ligation method (PRe-RDL) for plasmid reconstruction. The sequence of ELP consists of 60 or 90 copies of the pentapeptide Val-Pro-Gly-Val-Gly. The ASP fragment amplified by PCR was inserted into the plasmid containing the ELP 60 or ELP 90 gene to construct a recombinant plasmid encoding the ASP-ELPS gene.
  • PRe-RDL recursive directional ligation method
  • the plasmids carrying the ASP, ASP-ELP 60 or ASP-ELP 90 genes were transformed into Escherichia coli Rosetta-gami (DE3) pLys.
  • the expressed bacteria were transferred into 1L TB medium with 50 ⁇ g/mL antibiotics and cultured at 37°C, 220 rpm until the optical density (OD) at 600 nm was 0.8. After the temperature was adjusted to 20°C, protein expression was induced with 0.5 mM isopropyl- ⁇ -d-thiogalactopyranoside (IPTG) and allowed to continue overnight.
  • IPTG isopropyl- ⁇ -d-thiogalactopyranoside
  • ASP-ELPs fusion protein (ASP-ELPS) was purified by inverse phase cycling (ITC). The phase transition of ASP-ELPS was triggered by adding NaCl to a final concentration of 2 M and incubating in a water bath at 37°C for 15 min.
  • ASP-ELPs Aggregated ASP-ELPs were separated from the solution by centrifugation at 14000 rpm for 10 minutes at 37°C. ASP-ELPs were dissolved in pre-cooled PBS and centrifuged at 4°C to remove insoluble particles. Purified ASP-ELPs were obtained by repeating three consecutive rounds of ITC. ASP was purified by nickel column affinity chromatography. The supernatant containing ASP was filtered through a 0.45 ⁇ m pore size filter (Millipore, USA) and applied to a pre-equilibrated HisTrap HP 5 ml column (GE Healthcare, USA) installed on an AKTA Purifier system (GE Healthcare, USA) at a flow rate of 1 ml/min.
  • the column was washed with 20 mM HEPES, 500 mM NaCl, 5 mM imidazole, pH 7.4 (buffer A) and eluted with 20 mM HEPES, 500 mM NaCl, 500 mM imidazole, pH 7.4 (buffer B).
  • the His6-tagged ASP was finally eluted with 20% buffer A and 80% buffer B.
  • the desalting step was then performed on an AKTA system using a HiPrep 26/10 desalting column (GE Healthcare, USA).
  • the purified protein was then passed through an endotoxin removal gel (Thermo Scientific, USA) to remove bacterial endotoxins, lyophilized using an Alpha1-2LD plus freeze dryer (Christ, Germany), and stored at -80°C until use.
  • This example uses Coomassie Brilliant Blue stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to monitor the overexpression of ASP-ELPs in Escherichia coli and the purification by (ITC) ( Figure 4) and confirm the purity of ASP and ASP-ELPs ( Figure 5).
  • Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry MALDI-TOF-MS
  • the secondary structures of ASP and ASP-ELPs detected by circular dichroism (CD) are exactly the same, indicating that the ELP chain does not affect the structure of ASP ( Figure 7), as predicted by AlphaFold2.
  • the hydrodynamic diameters of ASP-ELPS measured by dynamic light scattering (DLS) are 18.6 and 22.5 nm, respectively, for ASP (8.4 nm) and 18.6 and 22.5 nm, respectively. 8), which means that the half-life of ASP-ELPs will be longer than that of ASP due to their larger size, resulting in reduced renal clearance.
  • phase transition behavior of ASP-ELPS was evaluated by monitoring the optical density (OD 350 ) of the solution in 10mM PBS at 350nm as a function of temperature on a SpectraMax Paradigm microplate reader (Molecular Devices, America).
  • OD 350 optical density
  • a series of diluted protein solutions (0.05, 0.2, 1, 5, 10, 50, 50, 100 and 200 ⁇ M) were added to a transparent 96-well plate (200 ⁇ L/well) and studied at a heating rate of 2°C/min over a temperature range of 10°C to 60°C to obtain OD350.
  • the transition temperature (Tt) is defined as the inflection point of the turbidity vs. temperature curve and is calculated as the maximum value of the first derivative.
  • ASP-ELPs have thermal expansion and will transition sharply from a soluble state to a co-osmotic state when the physical temperature exceeds its phase transition temperature (Tt).
  • Tt is an inflection point in the turbidity versus temperature curve, which decreases with increasing ASP-ELP concentration and ELP chain length (Figure 9).
  • the ASP activity of freshly prepared enzymes was detected using Nessler reagent (Merck, Germany). 50 mM Tris-HCl, 50 mM L-asparagine (L-Asn) and 10 nM enzyme were mixed in an Eppendorf microtube and incubated at 37°C for 10 minutes. Before adding the Nessler reagent, 1.5 M trichloroacetic acid (TCA) was added to terminate the reaction. The OD value at 410 nm was monitored on a SpectraMax Paradigm microplate reader (Molecular Devices, USA).
  • a standard curve for estimating enzyme activity was created by correlating the absorbance of ammonium sulfate at 410 nanometers with known standard concentrations.
  • One unit of ASP activity is defined as the amount of enzyme that produces 1.0 ⁇ M urea per minute at 37°C.
  • the activities of ASP, PEG-ASP, and ASP-ELPS measured by Nessler reagent in this example were 3.8 U/nM, 0.7 U/nM, 3.4 U/Nm, and 3.1 U/nM, respectively.
  • ASP-ELPS obtained a high level of activity retention of 89% and 81%, which is 4.7 and 4.3 times that of PEG-ASP.
  • ASP-ELP 90 The high activity retention of ASP-ELPs compared to PEG-ASP may be due to the simple biosynthesis procedure and precise structure.
  • the activity of ASP-ELP 90 is slightly lower than that of ASP-ELP 60 , which may be due to the higher coverage level of ELP chain on ASP in ASP-ELP 90 , as predicted by AlphaFold2 ( Figure 10).
  • CCRF-CEM, Jurkat, Raji and Ramos cells were seeded into 96-well plates (5000 cells/100 ⁇ l/well), and 10 ⁇ l of ASP, PEG-ASP, and ASP-ELPS were added to a final concentration of 0.5, 1, 2.5, 5, 10, 25, 50, 75, 100, 250, 500, and 1000 nM.
  • 10 ⁇ L of CCK-8 solution was added to each well and cultured in a 5% carbon dioxide incubator at 37°C for 1.5 hours.
  • the optical density (OD) of each well was measured at 450 nm using a SpectraMax Paradigm microplate reader (Molecular Devices, America).
  • the inhibition rate (%) was calculated as follows.
  • IC 50 half maximal inhibitory concentration
  • ASP, PEG-ASP, and ASP-ELPs were stored in 10 mM PBS at 37°C for 1, 3, 5, 7, 10, 14, 17, 21, and 25 days.
  • the enzyme activity was detected using the above-mentioned Nessler reagent ( Figure 13).
  • ASP-ELP 90 showed the best storage stability and the slowest loss of activity over time, and both ASP-ELPs conjugates performed better than ASP and PEG-ASP.
  • the remaining activity of ASP-ELP 90 was 93% and that of ASP-ELP 60 was 81%, which was significantly higher than 14% of ASP and 44% of PEG-ASP.
  • ASP-ELPs after 25 days of storage, ASP-ELPs still retained 54.5% and 25.6% of the initial activity, respectively.
  • the higher storage stability of ASP-ELPs is attributed to the protective effect of the ELP chain on ASP.
  • ASP-ELP 90 is more stable than ASP-ELP 60 , probably because the longer ELP chain provides more adequate packaging and protection.
  • the MTD of ASP, PEG-ASP, and ASP-ELPS was determined by a dose escalation study on healthy BALB/c mice ( Figure 14).
  • BALB/c mice were intraperitoneally injected with ASP at a dose of 500, 700, 900, and 1100 U/kg body weight;
  • PEG-ASP at a dose of 300, 500, 700, and 900 U/kg body weight;
  • ASP-ELP 60 at a dose of 1600, 1800, 2000, and 2200 U/kg body weight;
  • the survival and body weight changes of mice were monitored daily for two weeks.
  • the MTD was defined as the active dose below the dose that caused the death of any animal in the three animal groups or a weight loss of more than 10%.
  • the MTD of ASP-ELP 90 is 2400 U/kg body weight, which is 4.8, 3.4 and 1.3 times that of 500 U/kg body weight of ASP, 700 U/kg body weight of ASP-PEG and 1800 U/kg body weight of ASP-ELP 60 , respectively.
  • the MTD of ASP-ELPs is significantly higher than that of ASP and PEG-ASP.
  • concentration-dependent thermosensitivity of ASP-ELPs will help to form a reservoir for sustained release and avoid side effects associated with burst release caused by high concentrations;
  • the core-shell structure tetramer of ASP-ELPs will endow ASP-ELPs with low immunogenicity, resulting in insignificant immune-related side effects.
  • mice were anesthetized with 1.25% 2,2,2-tribromoethanol at a dose of 20 ⁇ L/g BW, the sedated mice were imaged at each time point (0, 1, 5, 10, 20 and 25 days) using an in vivo imaging system (IVIS) SPECTRUM/200 imaging system (Caliper Life Sciences, USA). The fluorescence intensity at the injection site was measured using Living Image 4.5 software (Caliper Life Sciences, USA).
  • IVIS in vivo imaging system
  • Cy5-ASP-ELPs formed fluorescent deposits after injection, and their fluorescence signals gradually decreased over time, while the fluorescence signals of Cy5-ASP and Cy5-PEG-ASP disappeared rapidly from the injection site ( Figure 15). Specifically, after 1 day, the fluorescence retention (ratio of remaining fluorescence to initial fluorescence) of Cy5-ASP-ELP 90 (91%) and Cy5-ASP-ELP 60 (78%) at the injection site was still high, while the fluorescence retention of PEG-ASP was significantly reduced to 17%, and the fluorescence of ASP almost disappeared (4%) ( Figure 16). Interestingly, on the 10th day, the fluorescence retention of Cy5-ASP-ELP 90 was 4.1 times higher than that of Cy5-ASP-ELP 60.
  • ASP-ELPs Due to the body temperature being higher than Tt and the large local concentration, ASP-ELPs can form a depression of sustained release after injection.
  • ASP-ELP 90 has a lower Tt than ASP-ELP 60 due to the longer length of its ELP chain, forming a more stable reservoir with slower fluorescence decay.
  • the time-dependent decrease in fluorescence at the injection site is due to the gradual dispersion of soluble ASP-ELP out of the depot, which provides a subtle strategy to avoid burst release of the drug and increase the circulation half-time.
  • the area under the curve (AUC) of ASP-ELP 90 at 360 hours and ASP-ELP 60 at 192 hours was linearly correlated with time ( FIG. 20 ), indicating zero-order release kinetics.
  • the AUCs of ASP-PEG and ASP were logarithmically correlated with time, indicating abrupt release kinetics, which may induce severe high-concentration-related side effects.
  • ASP-ELP 90 was more prominent than ASP-ELP 60 in improving pharmacokinetics, due to the longer zero-order release time caused by the lower Tt.
  • Blood samples were collected at specific time points (0.1, 0.5, 1, 2, 6, 12, 24, 48, 96, 144, 192 and 240 hours) after the first and third injections.
  • Serum ASP activity levels were estimated using an asparaginase activity assay kit (Biovision, USA). Mice were euthanized on day 42, and blood samples were collected and tested for immunogenicity.
  • ELP60 , ELP90 , and PEG-IFN were used as antigens in the coating process.
  • the expression and purification procedures of ELP60 and ELP90 were similar to those discussed above for ASP-ELPS. The same. According to the structures of ASP and ASP-ELPS predicted by AlphaFold2, it is speculated that the ASP-ELP conjugate reduces the immunogenicity of ASP by encapsulating the immunogenic sites on the surface of ASP, and the immunogenicity of ASP-ELP 90 will be lower than that of ASP-ELP 60 because the ELP 90 chain more fully encapsulates the surface of ASP ( Figure 3).
  • the anti-protein immunoglobulin G (IgG)/immunoglobulin M (IgM) titers in the serum after injection of ASP-ELP 60 , PEG-ASP and ASP were 10-/23-, 11-/28- and 90-/137-fold higher than those of ASP-ELP 90 , respectively, confirming that the anti-ASP immunogenicity of ASP-ELP 90 was significantly reduced.
  • the present example also measured the anti-polymer IgG/IgM titers to further clarify the above findings ( Figures 23 and 24).
  • the IgG/IgM titers of PEG-ASP against PEG polymer were 21-/31- and 13-/22-fold higher than those of ASP-ELP 60 against ELP 60 and ASP-ELP 90 against ELP 90 , respectively, indicating that the immunogenicity of ELP is much lower than that of PEG.
  • SA-MTD maximum tolerated dose
  • MA-SD same dose
  • SA-MTD is a single dose
  • ASP 500U/kg body weight
  • PEG-ASP is 700U/kg body weight
  • ASP-ELP 60 is 1800U/kg body weight
  • ASP-ELP 90 is 2400U/kg body weight
  • MA-SD is six doses per week, 400U/kg body weight.
  • major organs herein, liver, spleen, lung and kidney
  • blood samples were obtained on the 42nd day for histopathological and hematological analysis.
  • Celltac ⁇ MEK-6450 automatic hematology analyzer (Nihon Kohden, Japan) was used to obtain blood counts, including red blood cells (RBCs), white blood cells (WBCs), platelets (PLTs) and hemoglobin (HGB).
  • Serum biochemical parameters including liver function indicators such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and renal function indicators such as creatinine (CREA) and blood urea nitrogen (UREA), were measured using a Hitachi 7600 automatic analyzer (Hitachi, Japan).
  • liver function indicators such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT)
  • renal function indicators such as creatinine (CREA) and blood urea nitrogen (UREA)
  • Hitachi 7600 automatic analyzer Hitachi 7600 automatic analyzer
  • Paraffin sections were stained with hematoxylin and eosin (H&E) and Automated quantitative pathology imaging system (PerkinElmer, Hopkinton, MA, USA) imaging.
  • H&E hematoxylin and eosin
  • PerkinElmer Hopkinton, MA, USA
  • the experimental group received intraperitoneal injections at a dose of 400U/kg BW once a week for six weeks.
  • the total dose (2400U/kg BW) was equivalent to the MTD of ASP-ELP 90
  • the single dose (400U/kg BW) did not exceed the MTD of ASP (500U/kg BW). All other steps were as described above.
  • lipid vacuoles due to hepatocyte degeneration were observed in liver tissue, indicating liver damage; swollen, degenerated and necrotic splenocytes (black arrows) in spleen tissue, showing severe spleen damage; uniform red-stained protein deposition and sloughed necrotic renal tubular epithelial cells (black arrows) in kidney tissue, suggesting acute tubulointerstitial injury. Consistent with the histopathological results, the hematological parameters of the ASP and PEG-ASP groups showed statistically significant differences compared with the PBS group.
  • ASP-ELP 90 has a higher in vivo safety profile relative to ASP-ELP 60 due to its lower immunogenicity and peak concentration.
  • ASP-ELP The excellent performance of 90 in reducing the incidence of toxic side effects supports its status as a potential clinical candidate drug to replace ASP and PEG-ASP.
  • CCRF-CEM-Luc Fluorescein-expressing CCRF-CEM cells were injected into NOD/SCID mice via the tail vein to establish a mouse leukemia model; fluorescein-expressing Raji (Raji-Luc) cells were injected subcutaneously into the dorsal flank of NOD/SCID mice (2 ⁇ 10 6 cells/mouse) to establish a mouse lymphoma model.
  • mice carrying CCRF-CEM-Luc cells were randomly divided into four experimental groups and one control group.
  • the experimental groups were intraperitoneally injected with MTD of ASP, PEG-ASP, ASP-ELP 60 , or ASP-ELP 90 , and the control group received an equal volume of PBS.
  • Tumor burden in vivo was monitored by bioluminescence imaging (BLI) using an IVIS SPECTRUM/200 imaging system (Caliper Life Sciences, USA). Bioluminescence imaging was performed on days 0, 7, 14, and 21 after injection.
  • mice Prior to bioluminescence imaging, mice were anesthetized with 1.25% 2,2,2-tribromoethanol (20 ⁇ L/g BW) and injected with diphenylterazine (0.3 ⁇ M/mouse). The bioluminescence intensity of leukemia cells was measured using Living Image 4.5 software (Caliper Life Sciences, USA). For the study of SA-MTD in the mouse lymphoma model, bioluminescence imaging was performed on days 0, 7, 14, and 28 after injection, and all other steps were as described above.
  • the experimental groups received intraperitoneal injections of ASP, PEG-ASP, and ASP-ELPS once a week for six weeks, and bioluminescence imaging was performed on days 0, 14, 28, and 42 after injection. All other steps were as described above.
  • the in vivo leukemia proliferation in the ASP-ELP 90 group was significantly slower than that in the ASP, PEG-ASP, and ASP-ELP 60 groups ( Figure 30).
  • mice in the ASP-ELP 90 group were 1.25-, 1.79-, 2.12-, and 2.6-fold longer than that in the ASP-ELP 60 , PEG-ASP, ASP, and PBS groups, respectively ( Figure 30). Similar results were also observed in the SA-MTD lymphoma mouse model ( Figure 31). These results indicate that despite a single dose, ASP-ELP 90 significantly prolonged cancer remission more than ASP, PEG-ASP, and ASP-ELP 60 .
  • This example further evaluated the anti-leukemia and anti-lymphoma efficacy of MA-SD, and the ASP-ELP 90 group also showed stronger anti-tumor efficacy and significantly longer survival time than the ASP-ELP 60 , ASP-PEG and ASP groups ( Figures 32 and 33).
  • the above results show that under the same dosing regimen, the effect of ASP-ELP 90 in treating hematological cancer is significantly better than ASP, PEG-ASP and ASP-ELP 60.
  • ASP-ELP 90 is a more effective therapeutic agent and is promising as an alternative to ASP and PEG-ASP for the treatment of hematological malignancies.
  • the present invention provides an ASP-ELP that reduces immunogens and enhances anti-tumor efficacy, and a preparation method and application thereof.
  • the present invention uses the artificial intelligence tool AlphaFold2 for the first time to fuse ELPs of different lengths and compositions with ASP.
  • the designed ASP-ELPs have multiple advantages over the original drug asparaginase ASP and the long-acting asparaginase PEG-ASP.
  • the results of in vivo and in vitro experiments show that ASP-ELPs are superior to long-acting asparaginase in terms of enzyme activity retention rate, storage stability, drug metabolism dynamics, immunogenicity, biosafety, and anti-cancer efficacy.
  • the present invention not only provides a new design method for drug research and development based on artificial intelligence-assisted protein-polymer design, but also illustrates that ASP-ELPs is a new effective therapy for the treatment of hematological malignancies, with good economic value and application prospects.

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Abstract

本发明提供一种降低免疫原和增强抗肿瘤功效的ASP-ELP及其制备方法与应用。本发明首次采用人工智能工具AlphaFold2将不同长度和构成的ELP与ASP融合，所设计的ASP-ELPs与原药门冬酰胺酶ASP和长效门冬酰胺酶PEG-ASP相比，具有多种优势。体内外的实验结果显示，ASP-ELPs在酶活性保留率、储存稳定性、药物代谢动力学、免疫原性、生物安全性、抗癌疗效等方面均优于长效门冬酰胺酶。本发明不仅为基于人工智能辅助设计蛋白质-高分子聚合物的药物研发提供新的设计方法，同时也说明了ASP-ELPs是一种新的治疗血液恶性肿瘤有效疗法。

Description

一种降低免疫原和增强抗肿瘤功效的ASP-ELP及其制备方法与应用
交叉引用
本申请要求2023年4月6日提交的专利名称为“一种降低免疫原和增强抗肿瘤功效的ASP-ELP及其制备方法与应用”的第202310360892.5号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明属于生物医药技术领域，具体涉及一种降低免疫原和增强抗肿瘤功效的ASP-ELP及其制备方法与应用。
背景技术
门冬酰胺酶(Asparaginase,ASP)是一种能够选择性抑制血液系统恶性肿瘤细胞的细菌酶，尤其对小儿急性淋巴细胞白血病的疗效最好，能够显著提高血液恶性肿瘤的完全缓解率(CR)和治愈率。血液系统的肿瘤细胞大多不能自已合成生长必要的氨基酸门冬酰胺，必须依赖宿主供给，ASP可以通过催化分解肿瘤细胞生长所必需的门冬酰胺来选择性诱导肿瘤细胞凋亡。然而，由于ASP的免疫原性高、毒性强、半衰期短、稳定性差，通常需要多次大剂量给药才能达到足够的疗效，这就不可避免地诱发了严重的副作用，如超敏反应、胰腺炎、肝脂肪变性、肝硬化和凝血功能障碍等。
为了克服ASP的这些问题，人们把大肠杆菌来源的ASP与聚乙二醇及磷脂双分子层连接成为了脂质体门冬酰胺酶(PEG-ASP)。该药不但维持了门冬酰胺酶的生物活性，而且药物在到达肿瘤组织前保持脂质体形态，避免被蛋白质酶分解，使PEG-ASP的半衰期较ASP明显延长，成为长效门冬酰胺酶制剂。PEG-ASP(商品名为)于2006年被美国食品和药物管理局(FDA)批准为急性淋巴细胞白血病(ALL)的一线治疗方法，随后美国国家综合癌症网络肿瘤学临床实践指南(NCCN)2021版进一步推荐其取代ASP用于治疗小儿ALL。
然而，最近的研究发现，由于PEG在食品、化妆品和药品中的过度应用，人体中广泛存在着抗PEG抗体，这可能诱发加速血液清除(ABC)现象和严重的超敏反应，并降低其临床疗效。因此，研究人员试图用聚羧基甜菜碱(PCB)、谷氨酸和赖氨酸(EK)肽、P(CB-EG3Glu)或聚(L)-脯氨酸(PLP)代PEG与ASP偶联。然而，化学合成的固有缺点，包括复杂的合成过程、缺乏可重复性、产量低和成本高，使得这些ASP-高分子聚合物的临床转化极为困难。
发明内容
为了解决上述问题，本发明提供一种降低免疫原和增强抗肿瘤功效的ASP-ELP及其制备方法与应用。
第一方面，本发明提供一种长效缓释药物，所述长效缓释药物是由人工智能辅助设计得到；包括治疗剂和药物载体，所述治疗剂为蛋白类药物；所述药物载体为ELP；所述人工智能辅助设计是指：将蛋白质结构的物理和生物学知识，利用多序列比对，整合到AlphaFold2的深度学习算法中，所述AlphaFold2根据长效缓释药物的氨基酸序列预测出三维结构，最终确定长效缓释药物的组成。
本发明提供的长效缓释药物是由核心-外壳构成的同源四聚体复合物；其中，治疗剂形成四聚体核心，ELP链盘绕包裹所述四聚体核心，形成一个壳状结构。
本发明提供的长效缓释药物中，所述ELP的氨基酸序列由重复单元缬氨酸-脯氨酸-甘氨酸-X氨基酸-甘氨酸构成；所述X氨基酸是缬氨酸、丙氨酸、甘氨酸、亮氨酸、异亮氨酸、赖氨酸或组氨酸中的一种或多种；
优选地，所述ELP的氨基酸序列由五肽重复单元缬氨酸-脯氨酸-甘氨酸-X氨基酸-甘氨酸构成；所述X氨基酸是缬氨酸、丙氨酸、甘氨酸、亮氨酸、异亮氨酸、赖氨酸或组氨酸。
本发明提供的长效缓释药物中，ELP的五肽重复单元种类为缬氨酸-脯氨酸-甘氨酸-缬氨酸- 甘氨酸；ELP重复的数目为30-150；
优选地，ELP重复的数目为60-120。
在使用所述AlphaFold2预测ASP-ELP中，ELP的氨基酸重复单元为:30～200次,其中X氨酸是缬氨酸、丙氨酸、甘氨酸、亮氨酸、异亮氨酸、赖氨酸、苯丙氨酸、组氨酸其中一种。X氨基酸不同的ELP可表现不同的生物功能。
基于载体性能的考虑，ELP的种类为缬氨酸-脯氨酸-甘氨酸-缬氨酸-甘氨酸(VPGVG)；基于降低免疫原性和保留活性的考虑，ELP重复的数目为30-150；优选地，ELP重复的数目为60-120。
本发明提供的长效缓释药物中，使用ELP蛋白偶联比其免疫原性更高的蛋白质；低免疫原性的ELP作为高免疫原性蛋白质的物理屏障，遮挡所述高免疫原性蛋白质的抗原表位，降低体内免疫系统的识别和药物的清除速率；
本发明提供的长效缓释药物中，所述治疗剂为门冬酰胺酶(ASP)、干扰素(IFN)、生长激素(GH)或富含酪氨酸的蛋白质。
优选地，所述治疗剂为门冬酰胺酶。
所述门冬酰胺酶的氨基酸序列为
本发明所适用的门冬酰胺酶(ASP)可以是来源于大肠杆菌的一种异源性蛋白，ASP可刺激机体产生抗体，发生过敏反应，严重时危及生命，并降低ASP疗效和患者无病长期生存，
门冬酰胺酶(ASP)在治疗急性淋巴细胞白血病(ALL)方面发挥了重要作用，然而ASP的内在缺点，即高免疫原性、严重的毒性、短半衰期和差的稳定性，限制了它的临床应用。
第二方面，本发明还提供制备长效缓释药物的方法，由人工智能辅助设计得到长效缓释药物的三维结构，确定门冬酰胺酶和ELP的重复数目，采用生物合成、化学偶联合成或固相合成得到类弹性蛋白融合门冬酰胺酶；所述生物合成是指将门冬酰胺酶的DNA片段通过基因工程与ELP的N端融合，构建门冬酰胺酶-ELP质粒。
第三方面，本发明提供一种分离的多肽，所述多肽的氨基酸序列包括ELP的氨基酸序列和编码所述治疗剂的氨基酸序列；所述ELP的氨基酸序列由五肽重复单元缬氨酸-脯氨酸-甘氨酸-X氨基酸-甘氨酸构成；所述X氨基酸是缬氨酸、丙氨酸、甘氨酸、亮氨酸、异亮氨酸、赖氨酸或组氨酸。
第四方面，本发明提供一种分离的核酸，所述核酸编码上述的多肽。
第五方面，本发明提供一种生物材料，本发明所提供的生物材料用于生成上述的多肽或所述生物材料含有上述的核酸；所述生物材料为重组质粒、重组载体、重组细胞。
本发明还提供了上述长效缓释药物或上述的多肽或上述的核酸或上述的生物材料在制备血液系统恶性肿瘤治疗药物中的应用；
所述血液系统恶性肿瘤为急性淋巴细胞白血病、急性粒细胞白血病、急性单核细胞白血病、恶性淋巴瘤或对门冬酰胺酶敏感的血液系统恶性肿瘤。
第六方面，本发明提供一种血液系统恶性肿瘤治疗药物，含有上述的长效缓释药物。
更具体地，本发明提供了类弹性蛋白融合门冬酰胺酶(ASP-ELP)单次和多次给药模式下体内安全性的评估，多次给药模式下体内安全性的评估能够综合考虑体内药物高-低浓度波动和免疫原性对体内的安全性。
在本发明的ASP-ELP单次和多次给药模式中，单次给药是以药物的最大耐受剂量(MTD)给药的，多次给药是以相同剂量，每周一次，连续给药6次，其中，所述多次给药中，给药次数大于2。
本发明还进行了单次给药的最大耐受剂量(SA-MTD)和多次相同剂量给药(MA-SD)两种 给药方案的效果评估结果。
本发明的有益效果在于：
(1)将ELP和门冬酰胺酶通过基因工程融合成为弹性蛋白融合门冬酰胺酶(ASP-ELP)，不但维持了ASP的生物活性，而且减少蛋白质酶解及肾脏排泄(图1)。ASP-ELP中的ELP链遮挡ASP抗原表位，从而能够有效减低人体免疫系统的识别。
(2)本发明进一步发现，ASP-ELP独特的热敏性能在腹腔注射后形成一个持续释放的药物库，从而改善药代动力学，减少全身毒性，提高抗肿瘤疗效(图2)。为了证实ASP-ELP是否能替代PEG-ASP和ASP成为一种更有前途的治疗血液系统恶性肿瘤的药物，本发明通过体外的活性保持和稳定性研究以及体内的MTD、药代动力学、免疫原性、毒性和抗肿瘤效果研究来测试并验证了ASP-ELP的性能。
附图说明
为了更清楚地说明本发明或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是本发明提供的ASP-ELP生物合成示意图。
图2是本发明ASP-ELP治疗血液恶性肿瘤的机制图。
图3是本发明AlphaFold2预测的ASP、ASP-ELPS结构图。
图4是本发明十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析ASP-ELPs的纯化过程。
图5是SDS-PAGE确认ASP和ASP-ELPs的纯度。
图6是基质辅助激光解吸电离飞行时间质谱法(MALDI-TOF-MS)分析，括号内是根据氨基酸组成计算的理论分子量，检测结果确认了ASP和ASP-ELPs的实际分子量与理论值一致。
图7是圆二色谱(CD)曲线分析，ASP-ELPs的CD曲线与ASP的完全重叠，表明ASP融合后二级结构没有改变。
图8是动态光散射(DLS)测定ASP和ASP-ELPs的水合直径。
图9是ASP-ELPs的相转变温度。
图10是ASP、PEG-ASP和ASP-ELPs的酶活性。
图11是ASP、PEG-ASP和ASP-ELPs的体外细胞毒性。
图12是ASP、PEG-ASP和ASP-ELPs对血液肿瘤的半数最大抑制浓度(IC50)。
图13是ASP、PEG-ASP和ASP-ELPs的长期储存稳定性。
图14是Cy5标记的ASP、PEG-ASP和ASP-ELPs在注射部位的荧光强度变化的代表图像。
图15是Cy5标记的ASP、PEG-ASP和ASP-ELPs在不同时间的体内荧光保留率。
图16是剂量递增实验测定ASP、PEG-ASP和ASP-ELPs的小鼠最大耐受剂量(MTD)。
图17是腹腔注射药物后血清ASP活性水平与时间的关系。
图18是ASP、PEG-ASP和ASP-ELPs的血浆峰值浓度(Cmax)。
图19是ASP、PEG-ASP和ASP-ELPs的体内循环半衰期(t1/2)。
图20是ASP、PEG-ASP和ASP-ELPs的血药浓度-时间曲线下面积(AUC)。
图21是小鼠免疫前后血浆ASP活性水平与时间的关系。
图22是小鼠免疫前后ASP、PEG-ASP和ASP-ELPs的体内循环半衰期(t1/2)。
图23是小鼠免疫后的抗蛋白和抗聚合物免疫球蛋白G(IgG)的滴度。
图24是小鼠免疫后的抗蛋白和抗聚合物免疫球蛋白M(IgM)的滴度。
图25是单次最大耐受剂量给药(SA-MTD)后的小鼠主要脏器H&E染色的病理切片的代表图像。
图26是相同剂量多次给药(MA-SD)后的小鼠主要脏器H&E染色的病理切片的代表图像。
图27是SA-MTD和MA-SD给药后肝功能指标分析。
图28是SA-MTD和MA-SD给药后肾功能指标分析。
图29是SA-MTD和MA-SD给药后血常规指标分析。
图30是药物SA-MTD治疗对白血病(CCRF-CEM-Luc)CDX小鼠模型抗癌效果。
图31是药物SA-MTD治疗对淋巴瘤(Raji-Luc)CDX小鼠模型抗癌效果。
图32是药物MA-SD治疗对白血病(CCRF-CEM-Luc)CDX小鼠模型抗癌效果。
图33是药物MA-SD治疗对淋巴瘤(Raji-Luc)CDX小鼠模型抗癌效果。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合本发明中的附图，对本发明中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
本实施例中通过Alphafold2的多聚体模型对不同链长的ASP-ELPs进行了结构预测，并选择了两个有希望的候选者(ASP-ELPS)进行进一步研究。ASP-ELPS形成一个核心-外壳的同源四聚体复合物，它们的ASP分子形成一个四聚体核心，类似于ASP的四聚体结构，而灵活的、随机盘绕的ELP链则包裹着它们的ASP的核心，形成一个壳状结构(图3)。ELP链的预测局部距离差异测试(plDDT)得分普遍较低，这证实了结构的紊乱。考虑到ELP对ASP的双面影响，即既阻断ASP的免疫原性位点又降低其生物活性，本实施例进行了一系列的体外和体内实验，以更准确地评估ASP-ELPS对提高ASP性能的影响。
实施例2
本实施例中，编码ASP的基因(UniProtKB/Swiss-Prot：P00805.2)通过聚合酶链式反应(PCR)扩增，并插入到pET-25b(+)载体中。编码ELP60和ELP90基因的质粒是用质粒重建的递归定向连接法(PRe-RDL)构建的。ELP的序列是由60或90个复制的五肽Val-Pro-Gly-Val-Gly组成。将PCR扩增的ASP片段分别插入含有ELP60或ELP90基因的质粒中，构建编码ASP-ELPS基因的重组质粒。通过DNA测序验证基因后，将携带ASP、ASP-ELP60或ASP-ELP90基因的质粒分别转化到大肠杆菌Rosetta-gami(DE3)pLys中。表达的细菌被转移到1L TB培养基中，抗生素为50μg/mL，在37℃，220rpm下培养，直到600nm处的光密度(OD)为0.8。温度调整到20℃后，用0.5mM的异丙基-β-d-硫代半乳糖苷(IPTG)诱导蛋白表达，并让其继续过夜。在12,000rpm下离心15分钟，收获细胞，悬浮在10mM预冷PBS中，并使用scientz-IID超声波处理器(中国宁波新芝)裂解。10％(w/v)的聚乙烯亚胺(PEI)被用来沉淀裂解液中的核酸。ASP-ELPs融合蛋白(ASP-ELPS)通过反相循环(ITC)进行纯化。ASP-ELPS的相变是通过加入NaCl到最终浓度为2M并在37℃的水浴中孵育15分钟来触发。通过在37℃下以14000rpm离心10分钟，将聚集的ASP-ELPs从溶液中分离出来。将ASP-ELPs溶于预冷的PBS中，并在4℃下离心以去除不溶性颗粒。通过重复三轮连续的ITC，得到纯化的ASP-ELPs。通过镍柱亲和色谱法纯化ASP。含有ASP的上清液通过0.45微米孔径的过滤器(Millipore，美国)过滤，并以1毫升/分钟的流速应用于安装在AKTA Purifier系统(GE Healthcare，美国)上的预平衡的HisTrap HP 5毫升柱(GE Healthcare，美国)。柱子用20mM HEPES,500mM NaCl,5mM咪唑，pH 7.4(缓冲液A)清洗，并用20mM HEPES,500mM NaCl,500mM咪唑，pH 7.4(缓冲液B)洗脱。带His6标签的ASP最后用20％的缓冲液A和80％的缓冲液B洗脱。然后在AKTA系统上用HiPrep 26/10脱盐柱(GE Healthcare,USA)进行脱盐步骤。纯化的蛋白质随后通过内毒素去除凝胶(Thermo Scientific，美国)以去除细菌内毒素，使用Alpha1-2LD plus冻干机(Christ，德国)进行冻干，并储存在-80℃直至使用。
本实施例利用考马斯亮蓝染色的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)来监测ASP-ELPs在大肠杆菌中的过表达和通过(ITC)的纯化(图4)并确认ASP和ASP-ELPs的纯度(图5)。基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS)进一步证实，纯化的ASP和ASP-ELPs的分子质量与理论值大致相等(图6)。通过圆二色谱(CD)检测的ASP和ASP-ELPs的二级结构完全相同，表明ELP链并不影响ASP的结构(图7)，正如AlphaFold2所预测的。通过动态光散射(DLS)测量的ASP-ELPS的流体动力学直径为18.6和22.5纳米，分别是ASP(8.4纳米) 的2.2和2.7倍(图8)，这意味着ASP-ELPs的半衰期将比ASP长，因为它们的尺寸较大，导致肾脏清除率降低。
本实施例中，ASP-ELPS的相变行为是通过在SpectraMax Paradigm微板阅读器(Molecular Devices,America)上监测10mM PBS中的溶液在350nm处的光密度(OD350)作为温度的函数来评估的。为了研究相变，将一系列稀释的蛋白质溶液(0.05、0.2、1、5、10、50、50、100和200μM)加入到透明的96孔板中(200μL/孔)，并在10℃至60℃的温度范围内以2℃/分钟的加热速率进行研究，以获得OD350。过渡温度(Tt)被定义为浊度与温度曲线的拐点，并以第一导数的最大值计算。正如预期的那样，ASP-ELPs具有热膨胀性，当物理温度超过其相变温度(Tt)时，会从可溶状态急剧过渡到共渗状态。这里的Tt是浊度与温度曲线的一个拐点，它随着ASP-ELP浓度和ELP链长度的增加而减少(图9)。
实施例3
本实施例中，用Nessler试剂(德国Merck公司)检测新鲜制备的酶(ASP、PEG-ASP、ASP-ELPS)的ASP活性。将50mM Tris-HCl、50mM L-天冬酰胺(L-Asn)和10nM酶在Eppendorf微型试管中混合，并在37℃孵育10分钟。在加入Nessler试剂之前，加入1.5M三氯乙酸(TCA)以终止反应。在SpectraMax Paradigm微板阅读器(Molecular Devices,美国)上监测410nm处的OD值。通过将硫酸铵在410纳米处的吸光度与已知的标准浓度相关联，创建了一条用于估计酶活性的标准曲线。一个单位的ASP活性被定义为在37℃下每分钟产生1.0μM尿素的酶量。本实施例用Nessler试剂测定的ASP、PEG-ASP、ASP-ELPS的活性，分别为3.8U/nM、0.7U/nM、3.4U/Nm和3.1U/nM。与未修饰的ASP相比，ASP-ELPS获得了89％和81％的高水平活性保持率，是PEG-ASP的4.7和4.3倍。ASP-ELPs比PEG-ASP的高活性保持率可能是由于生物合成的程序简单和结构精确。ASP-ELP90的活性略低于ASP-ELP60，这可能是由于ASP-ELP90中ELP链对ASP的覆盖水平较高，正如AlphaFold2所预测的那样(图10)。
本实施例中，CCRF-CEM、Jurkat、Raji和Ramos细胞接种到96孔板中(5000个细胞/100微升/孔)，加入10微升ASP、PEG-ASP、ASP-ELPS，终浓度为0.5、1、2.5、5、10、25、50、75、100、250、500、1000nM。48小时后，在每个孔中加入10μL的CCK-8溶液，并在37℃的5％二氧化碳培养箱中培养1.5小时。使用SpectraMax Paradigm微板阅读器(Molecular Devices,America)在450nm处测量每个孔的光密度(OD)。抑制率(％)的计算方法如下。(OD样品–OD背景)/(OD对照–OD背景)×100％。半数最大抑制浓度(IC50)用Origin Pro 2021中的非线性拟合计算并获得了每种ASP类似物在每种细胞系上IC50(图11)。正如预期的那样，ASP-ELPs对每个细胞系的IC50s明显低于PEG-ASP，表明ASP-ELPs具有更强的细胞毒性，因为其具有更高的活性保留(图12)。
实施例4
本实施例中，将ASP、PEG-ASP、ASP-ELPs在37℃的10mM PBS中储存1、3、5、7、10、14、17、21和25天。使用上述的Nessler试剂检测酶的活性(图13)。ASP-ELP90显示出最好的储存稳定性，随着时间的推移活性损失最慢，两种ASP-ELPs共轭物的表现都优于ASP和PEG-ASP。例如，储存10天后，ASP-ELP90的剩余活性为93％，ASP-ELP60为81％，明显高于ASP的14％和PEG-ASP的44％。值得注意的是，在储存25天后，ASP-ELPs仍然分别保留了初始活性的54.5％和25.6％。将ASP-ELPs较高的储存稳定性归功于ELP链对ASP的保护作用。ASP-ELP90比ASP-ELP60更稳定，可能是由于更长的ELP链提供了更充分的包装和保护。
实施例5
本实施例中通过对健康BALB/c小鼠的剂量递增试验确定了ASP、PEG-ASP、ASP-ELPS的MTD(图14)。BALB/c小鼠腹腔注射ASP，剂量为500、700、900和1100U/kg体重；PEG-ASP，剂量为300、500、700和900U/kg体重；ASP-ELP60，剂量为1600、1800、2000和2200U/kg体重；ASP-ELP90，剂量为2200、2400、2600和2800U/kg体重(每次注射n＝3)。两周内每天监测小鼠的生存和体重变化。MTD被定义为低于导致三只动物组中任何动物死亡或体重下降超过10％的剂量的活性剂量。ASP-ELP90的MTD为2400U/kg体重，分别是ASP的500U/kg体重、ASP-PEG的700U/kg体重和ASP-ELP60的1800U/kg体重的4.8、3.4和1.3倍。
ASP-ELPs的MTD比ASP和PEG-ASP的MTD明显增加，的可能原因如下：ASP-ELPs的浓度依赖性热敏性将有助于形成持续释放的储库，避免高浓度引起的与突发释放有关的副作用；ASP-ELPs的核壳结构四聚体将赋予ASP-ELPs低免疫原性，导致不明显的免疫相关副作用。
实施例6
本实施例中，为了阐明体内的释放过程，用体内成像系统(IVIS)监测了Cy5标记的ASP、PEG-ASP、ASP-ELP60或ASP-ELP90在其MTD的荧光强度变化。BALB/c小鼠被随机分成五组(每组n＝3)。将Cy5标记的ASP、PEG-ASP、ASP-ELPs按其平均剂量腹腔注射到BALB/c小鼠体内，并按等量的PBS注射给BALB/c小鼠。用1.25％的2,2,2-三溴乙醇以20μL/g BW的剂量对小鼠进行麻醉后，使用体内成像系统(IVIS)SPECTRUM/200成像系统(Caliper Life Sciences,美国)在各时间点(0、1、5、10、20和25天)对镇静的小鼠进行成像。使用Living Image 4.5软件(Caliper Life Sciences,美国)测量注射部位的荧光强度。
Cy5-ASP-ELPs在注射后形成了荧光沉积，其荧光信号随着时间的推移逐渐减少，而Cy5-ASP和Cy5-PEG-ASP的荧光信号迅速从注射部位消失(图15)。具体来说，1天后，Cy5-ASP-ELP90(91％)和Cy5-ASP-ELP60(78％)在注射部位的荧光保留(剩余荧光与初始荧光之比)仍然很高，而PEG-ASP的荧光保留明显减少到17％，ASP的荧光几乎消失(4％)(图16)。有趣的是，在第10天，Cy5-ASP-ELP90的荧光保留比Cy5-ASP-ELP60高4.1倍。由于体温高于Tt和局部浓度大，ASP-ELPs可以在注射后形成持续释放的凹陷。此外，ASP-ELP90由于其ELP链的长度较长，与ASP-ELP60相比，Tt较低，形成了一个更稳定的储存库，荧光衰减较慢。注射部位的荧光随时间下降是由于可溶性ASP-ELP逐渐分散出储存库，这提供了一个微妙的策略来避免药物的爆发式释放，并增加循环半时间。
实施例7
本实施例中，ASP、PEG-ASP或ASP-ELPs以其MTD的剂量腹腔注射到BALB/c小鼠体内(每组n＝6)。在0.1、0.2、0.5、1、2、6、12、24、48、96、144、192、240、360、480和600小时从尾静脉采集血样，用门冬酰胺酶活性检测试剂盒(Biovision,美国)测量血清ASP活性水平(图17)。药代动力学参数由药代动力学模拟软件Drug and Statistics 2.0使用单室模型计算。ASP-ELP90(404U/L)和ASP-ELP60(661U/L)的峰值浓度(Cmax)明显低于PEG-ASP(1444U/L)和ASP(1766U/L)，这意味着高浓度诱发的副作用减少(图18)。ASP-ELP90(353.2小时)的终末半衰期(t1/2)比ASP-ELP60(191.0小时)、ASP-PEG(29.8小时)或ASP(3.0小时)长1.8倍、11.9倍和116.8倍(图19)，表明ASP-ELP结合物的治疗期更持久，尤其是ASP-ELP90。值得注意的是，ASP-ELP90在360小时内和ASP-ELP60在192小时内的曲线下面积(AUC)与时间呈线性相关(图20)，表明零阶释放动力学。相反，ASP-PEG和ASP的AUCs与时间呈对数关系，显示出突然的释放动力学，可能诱发严重的高浓度相关副作用。此外，ASP-ELP90比ASP-ELP60在改善药代动力学方面更为突出，这是因为较低的Tt引起的零阶释放时间较长。
实施例8
本实施例中，将BALB/c小鼠随机分为4组(n＝9)，腹腔注射ASP、PEG-ASP、ASP-ELP60或ASP-ELP90，剂量为400U/kg BW，每两周一次，持续六周。在第一次和第三次注射后的特定时间点(0.1、0.5、1、2、6、12、24、48、96、144、192和240小时)采集血样。使用门冬酰胺酶活性检测试剂盒(Biovision，美国)估计血清ASP活性水平。小鼠在第42天被安乐死，收集血样并进行免疫原性测试。将100μL的酶(1.0μg/mL/孔)加入到高结合力透明96孔板(Tymora，日本)中，并在4℃下孵育过夜，进行抗原包被。所有抗原包被孔用PBS清洗，并使用缓冲液(1％脱脂牛奶溶液在0.1M Tris缓冲液中，pH8.0)阻断2小时。丢弃阻断垫后，所有的孔用PBS洗五次，并注入100μL用PBS稀释的血清样品，在37℃下孵育2小时。用PBS洗五次，并加入100μLHRP结合的IgM和IgG第二抗体，在37℃下孵育1小时，使抗体与抗原结合。用PBS清洗5次后，所有孔都用100μL 3,3′,5,5′-四甲基联苯胺孵化，进行显色反应。15分钟后向每个孔中加入100μL的0.2M H2SO4以终止该反应。用SpectraMax M3微板阅读器(Molecular Devices,America)在450纳米处测量每个孔的光密度(OD)。为了分析抗ELP60、抗ELP90和抗PEG抗体，在包被过程中使用ELP60、ELP90和PEG-IFN作为抗原。ELP60和ELP90的表达和纯化过程与上面讨论的ASP-ELPS相 同。根据使用AlphaFold2预测的ASP、ASP-ELPS的结构，推测ASP-ELP共轭物通过包裹ASP表面的免疫原性部位来降低ASP的免疫原性，而ASP-ELP90的免疫原性会比ASP-ELP60低，因为ELP90链更充分地包裹了ASP的表面(图3)。为了验证本实施例的假设，通过对小鼠进行多次注射特定的ASP共轭物进行了免疫原性试验(图21)。发现ASP、PEG-ASP和ASP-ELP60组的血清ASP活性水平和t1/2在第三次注射后比第一次注射后低。相反，两次注射后，ASP-ELP90组的血清ASP活性水平和t1/2几乎相等(图22)。总之，这些药代动力学数据显示，与ASP、PEG-ASP和ASP-ELP60相比，ASP-ELP90的加速血液清除(ABC)现象并不明显。注射ASP-ELP60、PEG-ASP和ASP后血清中的抗蛋白免疫球蛋白G(IgG)/免疫球蛋白M(IgM)滴度分别比ASP-ELP90高10-/23-、11-/28-和90-/137-倍，证实ASP-ELP90的抗ASP免疫原性明显降低。本实施例还测量了抗聚合物的IgG/IgM滴度以进一步澄清上述发现(图23和图24)。PEG-ASP抗PEG聚合物的IgG/IgM滴度分别是ASP-ELP60抗ELP60和ASP-ELP90抗ELP90的21-/31-和13-/22-倍，表明ELP的免疫原性远远低于PEG的。此外，针对ELP的IgG/IgM滴度在ASP-ELP90组和ASP-ELP60组之间没有明显差异，表明ELP的免疫原性与ELP链长无关。
实施例9
本实施例中，在体内毒性研究中评估了两种给药方案：单次给药的最大耐受剂量(SA-MTD)和多次给药的相同剂量(MA-SD)。更具体地说，SA-MTD是单次给药，ASP为500U/kg体重，PEG-ASP为700U/kg体重，ASP-ELP60为1800U/kg体重，ASP-ELP90为2400U/kg体重；MA-SD是每周六次给药，400U/kg体重。在每个给药方案中，在第42天获得主要器官(心、肝、脾、肺和肾)和血液样本进行组织病理学和血液学分析。使用CelltacαMEK-6450自动血液分析仪(Nihon Kohden，日本)获得血液计数，包括红细胞(RBCs)、白细胞(WBCs)、血小板(PLTs)和血红蛋白(HGB)。血清生化指标，包括肝功能指标如天冬氨酸氨基转移酶(AST)和丙氨酸氨基转移酶(ALT)，以及肾功能指标如肌酐(CREA)和血尿素氮(UREA)，使用日立7600自动分析仪(日立，日本)测量。为了进行组织病理学评估，将动物新鲜组织块(厚度不超过0.5厘米)用4％中性多聚甲醛固定过夜，并嵌入石蜡中。石蜡切片用苏木精和伊红(H&E)染色，并用自动定量病理成像系统(PerkinElmer,Hopkinton,MA,USA)成像。对于MA-SD的研究，实验组接受腹腔注射，剂量为400U/kg BW，每周一次，持续六周。总剂量(2400U/kg BW)相当于ASP-ELP90的MTD，单剂量(400U/kg BW)不超过ASP的MTD(500U/kg BW)。所有其他步骤如上所述。对于SA-MTD的毒性评估，与PBS组相比，ASP、PEG-ASP、ASP-ELPs组没有观察到明显的组织病理学改变，血液学指标也没有统计学意义(图25)。尽管注射剂量较高，但ASP-ELP90与ASP、PEG-ASP和ASP-ELP60表现出同等的体内安全水平。然而，对于MA-SD的毒性评估，在ASP和PEG-ASP组的肝脏、脾脏和肾脏的苏木精和伊红(H&E)病理切片中观察到明显的组织学病变(图26)。特别是，观察到肝脏组织中由于肝细胞变性而产生的脂质空泡(黑色箭头)，表明肝脏损伤；脾脏组织中肿胀、变性和坏死的脾细胞(黑色箭头)，显示出严重的脾脏损伤；肾脏组织中均匀的红染蛋白沉积和脱落坏死的肾小管上皮细胞(黑色箭头)，提示急性肾小管间质损伤。与组织病理学结果一致，ASP和PEG-ASP组的血液学参数与PBS组相比显示出统计学上的显著差异。(i)天冬氨酸氨基转移酶(AST)和丙氨酸氨基转移酶(ALT)明显升高，表明ASP引起的肝损伤或肝功能异常(图27)。(肌酐(CREA)和血尿素氮(BUN)明显升高，表明ASP诱导的肾功能衰竭或肾功能异常(图28)；(iii)红细胞(RBC)、白细胞(WBC)、血小板(PLT)和血红蛋白(HGB)下降，标志着ASP诱导的造血功能抑制(图29)。将ASP和PEG-ASP组的毒副作用归因于高免疫原性ASP和PEG-ASP在六次注射后诱发的免疫相关毒性，以及每次注射后高活性峰值浓度诱发的剂量相关毒性。正如预期的那样，相对于PBS组，ASP-ELP60组和ASP-ELP90组没有明显的组织学异常，血液学指标也没有统计学上的显著差异，只是ASP-ELP60组的ALT和AST明显升高。相对于ASP-ELP60，ASP-ELP90的体内安全性更高，这是因为其免疫原性和峰值浓度较低。总之，ASP-ELP90在降低毒副作用发生率方面的出色表现支持其成为替代ASP和PEG-ASP的潜在临床候选药物。
实施例10
本实施例中，为了比较ASP、PEG-ASP和ASP-ELPs的抗肿瘤效果，在两个异种移植小鼠模 型(白血病小鼠模型和淋巴瘤小鼠模型)中评估了SA-MTD和MA-SD的治疗策略。将荧光素表达的CCRF-CEM(CCRF-CEM-Luc)细胞通过尾静脉注射到NOD/SCID小鼠体内，构建小鼠白血病模型；将荧光素表达的Raji(Raji-Luc)细胞皮下注射到NOD/SCID小鼠的背侧(2×106个细胞/小鼠)，构建小鼠淋巴瘤模型。为了研究SA-MTD在小鼠白血病模型中的作用，将携带CCRF-CEM-Luc细胞的小鼠随机分为四个实验组和一个对照组。实验组腹腔注射ASP、PEG-ASP、ASP-ELP60或ASP-ELP90的MTD，对照组接受同等体积的PBS。使用IVIS SPECTRUM/200成像系统(Caliper Life Sciences,USA)通过生物发光成像(BLI)监测体内的肿瘤负担。生物发光成像在注射后的第0、7、14和21天进行。在生物发光成像之前，用1.25％的2,2,2-三溴乙醇(20μL/g BW)麻醉小鼠，并注射二苯基特拉嗪(0.3μM/鼠)。使用Living Image 4.5软件(Caliper Life Sciences,USA)测量白血病细胞的生物发光强度。对于SA-MTD在小鼠淋巴瘤模型中的研究，生物发光成像在注射后的第0、7、14和28天进行，所有其他步骤如上所述。对于MA-SD在白血病小鼠模型和淋巴瘤小鼠模型中的研究，实验组接受腹腔注射ASP、PEG-ASP、ASP-ELPS，每周一次，持续六周，并在注射后第0、14、28和42天进行生物发光成像。所有其他步骤如上所述。对于SA-MTD的白血病小鼠模型，ASP-ELP90组的体内白血病增殖明显慢于ASP、PEG-ASP和ASP-ELP60组(图30)。同时，ASP-ELP90组小鼠的中位生存时间比ASP-ELP60、PEG-ASP、ASP和PBS组分别长1.25-、1.79-、2.12-和2.6倍(图30)。在SA-MTD的淋巴瘤小鼠模型中也观察到类似的结果(图31)。这些结果表明，尽管是单次给药，ASP-ELP90比ASP、PEG-ASP和ASP-ELP60更明显地延长了癌症缓解期。
本实施例进一步评估了MA-SD的抗白血病和抗淋巴瘤的疗效，ASP-ELP90组也比ASP-ELP60、ASP-PEG和ASP组表现出更强的抗肿瘤疗效和明显更长的生存时间(图32和图33)。上述结果表明，在相同的给药方案下，ASP-ELP90治疗血液病癌症的效果明显优于ASP、PEG-ASP和ASP-ELP60。总之，ASP-ELP90是一种更有效的治疗剂，作为ASP和PEG-ASP的替代品治疗血液系统恶性肿瘤是很有前途的。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
工业实用性
本发明提供一种降低免疫原和增强抗肿瘤功效的ASP-ELP及其制备方法与应用。本发明首次采用人工智能工具AlphaFold2将不同长度和构成的ELP与ASP融合，所设计的ASP-ELPs与原药门冬酰胺酶ASP和长效门冬酰胺酶PEG-ASP相比，具有多种优势。体内外的实验结果显示，ASP-ELPs在酶活性保留率、储存稳定性、药物代谢动力学、免疫原性、生物安全性、抗癌疗效等方面均优于长效门冬酰胺酶。本发明不仅为基于人工智能辅助设计蛋白质-高分子聚合物的药物研发提供新的设计方法，同时也说明了ASP-ELPs是一种新的治疗血液恶性肿瘤有效疗法，具有较好的经济价值和应用前景。

Claims (11)

  1. 一种长效缓释药物，其特征在于，所述长效缓释药物是由人工智能辅助设计得到，包括治疗剂和药物载体，所述治疗剂为蛋白类药物；所述药物载体为ELP；
    所述人工智能辅助设计是指：将蛋白质结构的物理和生物学知识，利用多序列比对，整合到AlphaFold2的深度学习算法中，所述AlphaFold2根据长效缓释药物的氨基酸序列预测出三维结构，最终确定长效缓释药物的组成。
  2. 根据权利要求1所述的长效缓释药物，其特征在于，所述长效缓释药物是由核心-外壳构成的同源四聚体复合物；其中，治疗剂形成四聚体核心，ELP链盘绕包裹所述四聚体核心，形成一个壳状结构。
  3. 根据权利要求2所述的长效缓释药物，其特征在于，所述ELP的氨基酸序列由重复单元缬氨酸-脯氨酸-甘氨酸-X氨基酸-甘氨酸构成；所述X氨基酸是缬氨酸、丙氨酸、甘氨酸、亮氨酸、异亮氨酸、赖氨酸或组氨酸中的一种或多种；
    优选地，所述ELP的氨基酸序列由五肽重复单元缬氨酸-脯氨酸-甘氨酸-X氨基酸-甘氨酸构成；所述X氨基酸是缬氨酸、丙氨酸、甘氨酸、亮氨酸、异亮氨酸、赖氨酸或组氨酸。
  4. 根据权利要求3所述的长效缓释药物，其特征在于，ELP的五肽重复单元种类为缬氨酸-脯氨酸-甘氨酸-缬氨酸-甘氨酸；ELP重复的数目为30-150；
    优选地，ELP重复的数目为60-120。
  5. 根据权利要求4所述的长效缓释药物，其特征在于，使用ELP偶联比其免疫原性更高的治疗剂；低免疫原性的ELP作为高免疫原性治疗剂的物理屏障，遮挡所述高免疫原性治疗剂的抗原表位，降低体内免疫系统的识别和药物的清除速率；
    优选地，所述治疗剂为门冬酰胺酶。
  6. 制备权利要求5所述长效缓释药物的方法，其特征在于，由人工智能辅助设计得到长效缓释药物的三维结构，确定门冬酰胺酶和ELP的重复数目，采用生物合成、化学偶联合成或固相合成得到类弹性蛋白融合门冬酰胺酶；所述生物合成是指将门冬酰胺酶的DNA片段通过基因工程与ELP的N端融合，构建门冬酰胺酶-ELP质粒。
  7. 一种分离的多肽，其特征在于，所述多肽的氨基酸序列包括ELP的氨基酸序列和编码权利要求1-5任一项所述治疗剂的氨基酸序列；所述ELP的氨基酸序列由五肽重复单元缬氨酸-脯氨酸-甘氨酸-X氨基酸-甘氨酸构成；所述X氨基酸是缬氨酸、丙氨酸、甘氨酸、亮氨酸、异亮氨酸、赖氨酸或组氨酸。
  8. 一种分离的核酸，其特征在于，所述核酸编码权利要求7所述的多肽。
  9. 一种生物材料，其特征在于，生成权利要求7所述的多肽或含有权利要求8所述的核酸；所述生物材料为重组质粒、重组载体、重组细胞。
  10. 权利要求1-5任一项所述长效缓释药物或权利要求7所述的多肽或权利要求8所述的核酸或权利要求9所述的生物材料在制备血液系统恶性肿瘤治疗药物中的应用；
    所述血液系统恶性肿瘤为急性淋巴细胞白血病、急性粒细胞白血病、急性单核细胞白血病、恶性淋巴瘤或对门冬酰胺酶敏感的血液系统恶性肿瘤。
  11. 一种血液系统恶性肿瘤治疗药物，其特征在于，含有权利要求1-5任一项所述的长效缓释药物。
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