Human urate oxidase mutants with reduced immunogenicity and high activity

Abstract

The application belongs to the technical field of biology and relates to a human uric acid oxidase mutant with weakened immunogenicity and high activity. The human uric acid oxidase mutant with weakened immunogenicity and high activity is obtained by mutating an inactive human uric acid oxidase with an amino acid sequence of SEQ ID NO. 1, and has amino acid substitutions at positions 83, 85, 119, 121, 151, 222, 232, 233, 240 and 252. The human uric acid oxidase mutant has high homology with a theoretical amino acid sequence of human uric acid oxidase, has high activity while reducing immunogenicity, is suitable for long-term application in clinical treatment of hyperuricemia and gout, and can be used for preparing a drug for treating hyperuricemia and gout.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to urate oxidase, in particular to a human urate oxidase mutant with weakened immunogenicity and high activity. Background Art

[0002] The main clinical treatments for hyperuricemia and gout include allopurinol, xanthine oxidase inhibitors, benzbromarone, probenecid, and colchicine. These drugs are commonly associated with the risk of multi-organ toxicity, severely limiting their use. Urate oxidase, a key enzyme in the terminal link of purine metabolism, catalyzes the conversion of uric acid to the more soluble allantoin, exhibiting a more desirable urate-lowering effect. Currently, recombinant urate oxidases approved for the treatment of hyperuricemia are primarily derived from heterologous species, such as Aspergillus flavus and pig-baboon chimeras. Due to their heterologous nature, these agents often cause severe allergic reactions in patients. Although polyethylene glycol (PEG)-modified uricases have improved half-life and stability, patients may still develop anti-drug antibodies (ADAs) and infusion reactions, limiting their widespread clinical application. Consequently, they are primarily used to treat acute, severe, and life-threatening refractory hyperuricemia. Immunogenicity has become a major limitation to the application of uricases. Developing uricases with reduced immunogenicity has become a hot topic in the research of recombinant uricase modification.

[0003] The human urate oxidase gene has become a pseudogene due to complex missense mutations and accumulation during evolution. Although short peptides of urate oxidase have been found in human sweat glands, they lack urate oxidase activity. Jiang et al. reported resurrecting a human urate oxidase pseudogene to produce a low-immunogenic therapeutic urate oxidase (Reference 1: Jiang N, Xu C, Zhang L, et al. “Resurrected” human-source urate oxidase with high uricolytic activity and stability [J]. Enzyme and Microbial Technology, 2021, 149: 109852). The paper reports that, through multiple sequence alignment analysis of the inactive human uricase gene, mutations at 15 highly conserved sites resulted in a "revived" uricase, rHU15, with a theoretical amino acid sequence identity of 95.06% to human uricase. However, the specific enzyme activity was only 2.30 U / mg. Further mutations at more conserved sites resulted in a mutant, rHU19, with a 19-site identity (reducing the theoretical amino acid sequence identity to 93.75%) and a specific enzyme activity of 8.29 U / mg. The paper did not test the immunogenicity of these mutants. Other literature reports that even a baboon uricase with a theoretical amino acid sequence identity of 93.75% to human uricase can cause significant immunogenicity (Reference 2: Xiong Runsong. Cloning and Molecular Modification of the Baboon Urate Oxidase Gene [D]. Beijing University of Chemical Technology, 2012). Compared to baboon uricase, rHU19 has lower sequence identity, suggesting that it is more similar to a heterologous uricase than to a human one, and thus exhibits significant immunogenicity. Because the human uricase pseudogene was revived through back mutations at all highly conserved sites, it is inherently non-humanized, increasing the heterologous nature of the revived enzyme and, consequently, its immunogenicity.

[0004] Human urate oxidase, a protein that has never existed in the body as a complete protein molecule during human development, has two problems in its resurrection strategy: first, restoring the activity of human urate oxidase by mutation through conservation analysis of the amino acid sequences of isoenzymes from different species actually dehumanizes the protein amino acid sequence. Repair of conservative sites restores enzyme activity while enhancing immunogenicity; second, when the humanization rate of the amino acid sequence reaches a threshold (for example, 95%), continuing to increase the humanization rate will lead to a significant decrease in enzyme activity or even loss of activity.

[0005] Therefore, the current strategy of functional resurrection of the human uricase pseudogene through homology alignment and conservative amino acid mutation faces challenges and difficulties. Therefore, it is necessary to find new strategies to obtain human uricase mutants with weakened immunogenicity and high activity. Summary of the Invention

[0006] The primary purpose of the present invention is to provide a human uricase mutant with weakened immunogenicity and high activity. The human uricase mutant maintains a high homology with the theoretical amino acid sequence of human uricase, and has high enzyme activity while reducing immunogenicity.

[0007] The human uricase mutant with weakened immunogenicity and high activity described in the present invention is a human uricase mutant obtained by mutating an inactive human uricase with an amino acid sequence of SEQ ID NO. 1; it has amino acid substitutions at positions 83, 85, 119, 121, 151, 222, 232, 233, 240 and 252.

[0008] According to a further feature of the human uricase mutant with weakened immunogenicity and high activity of the present invention, the amino acid substitution at position 83 is a substitution of glutamic acid (E) with glycine (G), the amino acid substitution at position 85 is a substitution of lysine (K) with cysteine ​​(C), the amino acid substitution at position 119 is a substitution of histidine (H) with arginine (R), the amino acid substitution at position 121 is a substitution of glycine (G) with glutamic acid (E), the amino acid substitution at position 151 is a substitution of glutamine (Q) with proline (P), the amino acid substitution at position 222 is a substitution of serine (S) with phenylalanine (F), the amino acid substitution at position 232 is a substitution of leucine (L) with serine (S), the amino acid substitution at position 233 is a substitution of threonine (T) with proline (P), the amino acid substitution at position 240 is a substitution of cysteine ​​(C) with tyrosine (Y), and the amino acid substitution at position 252 is a substitution of alanine (A) with glutamic acid (E); the amino acid sequence of the human uricase mutant modified by site-directed mutagenesis is SEQ ID NO. 2.

[0009] The second object of the present invention is to provide a DNA molecule encoding the human uricase mutant with weakened immunogenicity and high activity according to the present invention.

[0010] The DNA molecule according to the present invention has a nucleotide sequence of SEQ ID NO. 3.

[0011] The third object of the present invention is to provide a vector containing the DNA molecule of the present invention.

[0012] The fourth object of the present invention is to provide a host cell containing the DNA molecule of the present invention, or containing the vector of the present invention.

[0013] The above-mentioned vectors and host cells can be prepared by techniques well known in the art.

[0014] The fifth object of the present invention is to provide a method for producing a human uricase mutant with weakened immunogenicity and high activity.

[0015] The method for producing the human uricase mutant with weakened immunogenicity and high activity described in the present invention comprises: culturing the host cell described in the present invention under conditions suitable for the expression of the uricase mutant, and isolating the human uricase mutant with weakened immunogenicity and high activity from the culture medium.

[0016] When the DNA molecules of the present invention are inserted into the vector or transformed into the host cell in the proper orientation and correct reading frame, the DNA molecules can be expressed in any eukaryotic or prokaryotic expression system. A variety of host-vector systems can be used to express protein coding sequences. Host-vector systems include, but are not limited to, bacteria transformed with bacteriophages, plasmids, or cosmids; microorganisms containing yeast vectors, such as yeast; mammalian cell systems infected with viruses; insect cell systems infected with viruses; and plant cell systems infected with bacteria. Preferred vectors of the present invention include viral vectors, plasmids, cosmids, or oligonucleotides.

[0017] The preferred host of the present invention is a prokaryotic system such as Escherichia coli; the preferred protein expression method of the present invention is expression in Bacillus.

[0018] The sixth object of the present invention is to provide the use of the human urate oxidase mutant with weakened immunogenicity and high activity for preparing a drug for treating hyperuricemia and gout.

[0019] The present invention involves site-directed mutagenesis of the amino acid sequence of an inactive human uricase pseudogene (hUOX). The GENBANK accession number for the human uricase pseudogene sequence is NR_003927.2. By replacing the terminators at positions 3 and 187 with R, the amino acid sequence of the inactive human uricase (SEQ ID NO. 1) was obtained.

[0020] Based on SEQ ID NO.1, the inventors obtained a urate oxidase mutant rhUOX through site-directed mutagenesis that can still maintain good activity and a high degree of humanization after immunoweakening design. mut1 / K85C(SEQ ID NO. 2). This mutant shares 96.05% homology with the theoretical amino acid sequence of human uricase, demonstrating a higher humanization rate than previously reported uricases. Its specific enzyme activity, 7.766 U / mg, surpasses the 95% homology threshold reported in the literature and demonstrates high enzymatic activity. Experiments have shown that this mutant has low immunogenicity, similar to that of human serum albumin (HSA). This is because the hotspot amino acids of its surface antigenic peptide have been further mutated, rendering it unrecognizable and thus reducing its immunogenicity. Therefore, the human uricase mutant with weakened immunogenicity and high activity described in the present invention better meets the clinical need for a less immunogenic uricase, is suitable for long-term use in the clinical treatment of hyperuricemia and gout, and can be used to prepare drugs for the treatment of hyperuricemia and gout. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The SDS-PAGE protein electrophoresis diagram of the human urate oxidase mutant of the present invention, wherein 1: rhUOX wt ;3:rhUOX mut1 / K85C ; M stands for Marker.

[0022] Figure 2 The rhUOX of the present invention is shown wt 、rhUOX mut1 / K85C uric acid degradation activity.

[0023] Figure 3 The rhUOX of the present invention is shown mut1 / K85C and urate oxidase from Aspergillus flavus ( Af Results of human blood monocyte immunogenicity test of human serum albumin (HSA) and human serum albumin (HUOX).

[0024] Figure 4 The in vivo efficacy results of human urate oxidase are shown, where A: efficacy results in male mice; B: efficacy results in female mice. DETAILED DESCRIPTION

[0025] The terms used herein, unless otherwise specified, have the meanings commonly understood by those skilled in the art. The following provides definitions of some special terms used in the present invention.

[0026] “rhUOX wt " indicates an inactive human uricase in which the missense (terminator) codons at positions 33 and 187 of the human uricase pseudogene have been restored to arginine (R). The gene is in italics. rhuox wt " said. wtThe amino acid sequence is SEQ ID NO.1.

[0027] “rhUOX mut1 / K85C " indicates the hUOX mutant of the present invention, whose gene is in italics" rhuox mut1 / K85C " said. mut1 / K85C The amino acid sequence of the mutant is SEQ ID NO.2.

[0028] Example 1: rhuox wt 、rhuox mut1 / K85C Gene synthesis

[0029] The present invention adopts human urate oxidase pseudogene sequence rhuox wt (GenBank accession number is NR_003927.2), and a human urate oxidase mutant was obtained by transformation rhuox mut1 / K85C The above genes were synthesized by BGI and cloned between NcoI and XhoI of pET28a plasmid (Invitrogen). The recombinant plasmids were named pET28a-huox wt and pET28a-huox mut1 / K85C .

[0030] Example 2: rhuox wt and rhuox mut1 / K85C Genes were transformed into Escherichia coli Rosetta (DE3) and recombinant protein was induced to express

[0031] The recipient bacteria of this experiment was Escherichia coli Rosetta (DE3), and pET28a-huox wt and pET28a-huox mut1 / K85C After transformation with the recombinant plasmid, perform preliminary screening using kanamycin (Kan)-resistant plates. Then, pick a single colony on the Kan plate and culture it in 5 mL of LB liquid medium (containing 50 mg / mL Kan) for 12-16 hours. Then, extract the plasmid for PCR verification and further screen for positive clone recombinants.

[0032] The positive recombinants were inoculated into 5 mL of LB medium (50 mg / mL Kan) and cultured at 37°C and 200 rpm overnight. The recovered bacterial suspension was inoculated into 500 mL of LB medium (50 mg / mL Kan) at a rate of 1% and cultured at 37°C and 200 rpm until the OD 600The expression of the protein was induced at 0.6 by adding 60 μM lactose and inducing expression for 12 h at 30°C and 200 rpm. After induction, the cells were collected and resuspended in carbonate buffer (pH 10.0) (mass to volume ratio of 1:20). The cells were disrupted using an ultrasonic disruptor and centrifuged at 6000 g for 10 min. The supernatant was collected to obtain the recombinant protein.

[0033] Example 3: rhUOX wt and rhUOX mut1 / K85C Detection of mutant recombinant proteins by SDS-PAGE electrophoresis

[0034] The SDS-PAGE electrophoresis detection of this embodiment comprises the following steps:

[0035] (1) Prepare 10 mL of 10% separation gel, mix well, and pour the gel into the glass plate with a micropipette until it stops 2 to 3 cm from the upper edge of the short glass plate. Then seal the gel surface with distilled water. Gently lift one end of the gel dispenser and then put it down to make the gel surface flat. After polymerization for 40 minutes, discard the distilled water and absorb excess water with filter paper.

[0036] (2) Prepare 4 mL of 5% concentrated gel and pour it evenly on the separation gel. Insert a comb of corresponding specifications while avoiding the generation of bubbles. Allow the gel to solidify for 30 minutes.

[0037] (3) Install the electrophoresis tank and fill it with electrophoresis liquid. The volume should be larger than half of the volume of the electrophoresis tank. Move the prepared gel into the electrophoresis tank and carefully remove the comb.

[0038] (4) Spot the samples one by one. The amount of sample should not be too much. 15 μL per well is appropriate.

[0039] (5) At the beginning of electrophoresis, set the voltage to 90 V. When the indicator reaches the concentrated gel, change the voltage to 120 V and continue electrophoresis. Stop electrophoresis when the target band reaches the middle position (the target band corresponds to the corresponding band of the Maker, which can be known in advance).

[0040] (6) Carefully peel off the gel and stain with Coomassie Brilliant Blue R-250 for 30 minutes. Then decolorize with destaining buffer until the background is light and the protein bands are clear.

[0041] (7) Gel imaging and observation of the results. The SDS-PAGE protein electrophoresis results are as follows Figure 1 shown.

[0042] The results of SDS-PAGE protein electrophoresis are as follows Figure 1 Lane 1 is denatured rhUOX wt Protein sample, lane 2 is mutant rhUOX mut1 / K85CTarget protein sample. The target protein is approximately 34 kDa in size. This result indicates that the target gene was successfully expressed in E. coli Rosetta and can be used for subsequent experiments.

[0043] Example 4: Characterization of the activity of mutants

[0044] At 37 ° C, take 20 μL of purified protein rhUOX mut1 / K85C In 600 μL of 0.1 M uric acid solution (pH 8.0), the reaction was allowed to proceed for 10 minutes, followed by the addition of an equal volume of pure methanol to terminate the reaction. The enzyme activity was calculated by measuring the decrease in UV absorbance at 293 nm using UV spectrophotometry. The control group consisted of inactivated urate oxidase. Figure 2 As shown. mut1 / K85C The specific enzyme activity was 7.766 U / mg, indicating that the mutant had high activity.

[0045] Enzyme activity unit definition: The amount of enzyme required to catalyze the oxidation of 1 μmol of uric acid per minute is 1 unit.

[0046] E (U / mg) = (△OD 293 × V1 × D) / (12.3 × V2 × T × C)

[0047] Wherein, V1 is the total reaction volume; D is the dilution factor; 12.3 is the molar extinction coefficient of uric acid; T is the reaction time; and C is the protein concentration.

[0048] Example 5: Human PBMC cell immunogenicity analysis

[0049] Human serum albumin (HAS) and urate oxidase from Aspergillus flavus ( Af UOX) as the control group, rhUOX mut1 / K85C The experimental group was prepared by adding 50 μg / mL of the above proteins and PWM (20 μg / mL) into a 24-well cell culture plate at a volume of 100 μL (5 μg) per well, and then adding PBMC cell suspension (1×10 6 The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, with a half-volume medium change after 2-3 days. After the incubation period, the cells were centrifuged at 3000 rpm for 15 minutes and harvested. The cells were added to the corresponding protein-coated cell culture plates, with 500 μL per well. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, with a half-volume medium change after 2-3 days. After the incubation period, the supernatant was collected. Anti-URIC oxidase IgG was detected by indirect ELISA to analyze immunogenicity.

[0050] Indirect ELISA specific steps:

[0051] (1) Antibody coating: protein samples (HSA, Af UOX and rhUOX mut1 / K85C 100 μL (5 μg) was added to each well of a 96-well plate. Each sample was prepared in duplicate. After overnight at 4°C, the plates were washed three times with PBST and patted dry.

[0052] (2) Blocking: Block with Elisa blocking solution for 1 h.

[0053] (3) Washing: Wash three times with PBST and pat dry.

[0054] (4) Incubation: Add 100 μL of the supernatant prepared from the above cell culture (HSA, Af UOX and rhUOX mut1 / K85C ), blank control was PBS, and the cells were incubated at 37°C for 1.5 h.

[0055] (5) Washing: Wash three times with detergent. Pat dry.

[0056] (6) Incubation with HRP-goat anti-human IgG: Add 100 μL of diluted HRP-goat anti-human IgG (1:3000, PBST, pH 7.4) to each well and incubate at 37 °C for 1 h.

[0057] (7) Washing: Wash three times with detergent. Pat dry.

[0058] (8) Reaction color development: Add 100 μL of freshly prepared TMB working solution to each well and react in the dark at room temperature for 15 minutes. Add 50 μL of stop solution (2 M H2SO4) to each well. Read the OD value using a microplate reader. 450 value.

[0059] A450 of the test well is greater than or equal to 2.1 times that of the negative control well, which is considered positive: P / N = OD 450 Serum to be tested / OD 450 Negative control serum (i.e., P / N ≥ 2.1 is positive).

[0060] The results are as follows Figure 3 As shown, the mutant rhUOX mut1 / K85C It has low immunogenicity, similar to that of HSA.

[0061] Example 7: In vivo efficacy verification in uricase gene KO mice

[0062] Forty-eight C57BL / 6J mice (UOX- / -), half male and half female, 5-8 weeks old, weighing 25-30 g, were purchased from Saiye Biotechnology Co., Ltd. and housed in the SPF-grade laboratory animal room of the Experimental Animal Management Center of Jinan University at a room temperature of 20-25°C. Laboratory Animal Use Permit: SYXK (Guangdong) 2022-0174. C57BL / 6J mice were provided with clean water and free access to food before the experiment and were used after 3 days of feeding.

[0063] A normal control group (normal serum level), a high uric acid control group, and an experimental group (rhUOX mut1 / K85C ). The normal control group consisted of wild-type C57BL / 6J mice, while the other groups used KO mice with urate oxidase knockout for the experiment, with 12 mice in each group (6 males and 6 females). The normal control group and the high uric acid control group were given an equal volume of normal saline, while the experimental group was given the reference dose of rasburicase (2U / kg). The drug was administered by intraperitoneal injection, and blood was collected from the orbital vein at 0, 0.5, 1, 3, and 6 hours after administration. The serum was processed as above, and the uric acid content of the collected serum was detected by HPLC. The results are shown in Figure 2. Figure 4 As shown in the pharmacodynamics experiment of hyperuricemia KO mice, under the experimental conditions, the immunogenicity of rhUOX was weakened. mut1 / K85C It can maintain the blood uric acid level of KO mice at normal levels for at least 3 hours and can still exert the uric acid-lowering effect after 6 hours.

[0064] HPLC detection conditions

[0065] Chromatographic column: C18 column; length 150 mm, inner diameter 3 mm (Part NO: 70105-154330); flow rate: 1 mL / min; UV detector: UV wavelength 225, 293 nm; column temperature: 30°C; injection volume: 20 uL; mobile phase: A 0.1% formic acid-water; B pure methanol.

[0066] Table 1 below shows a comparison of the uricase mutants described in the present invention with those in References 1 and 2 regarding the number and position of point mutations, homology, enzyme specific activity, and immunogenicity.

[0067] Table 1:

[0068]

[0069] Document 1: JIANG N, XU C, ZHANG L, et al. “Resurrected” human-source urateoxidase with high uricolytic activity and stability [J]. Enzyme and MicrobialTechnology, 2021, 149: 109852.

[0070] Reference 2: Xiong Runsong. Study on cloning and molecular modification of baboon urate oxidase gene[D]. Beijing University of Chemical Technology, 2012.

[0071] As can be seen from the above table, the present invention discloses for the first time a human urate oxidase mutant rhUOX with weakened immunogenicity and high activity. mut1 / K85C This mutant has a homology of 96.05% with the theoretical amino acid sequence of human urate oxidase and an enzyme specific activity of 7.766 U / mg. While the number of mutation sites has been reduced, high activity is retained, and a high humanization rate is maintained. This mutant has a higher humanization rate and activity than rHU15 (Reference 1). Experiments have shown that this mutant has low immunogenicity, similar to that of HSA. This is because the hotspot amino acids of its protein surface antigen peptide have been further mutated, making it unrecognizable, thereby reducing immunogenicity. Therefore, the human urate oxidase mutant with weakened immunogenicity and high activity described in the present invention better meets the clinical needs of low-immunogenic urate oxidase, is suitable for long-term use in the clinical treatment of hyperuricemia and gout, and can be used to prepare drugs for the treatment of hyperuricemia and gout.

Claims

1. A human urate oxidase mutant with weakened immunogenicity and high activity, characterized by: The mutant is a human uricase mutant obtained by mutating an inactive human uricase with an amino acid sequence of SEQ ID NO. 1; the amino acid sequence of the human uricase mutant is SEQ ID NO.

2.

2. A DNA molecule, characterized in that: It encodes the human uricase mutant with weakened immunogenicity and high activity as described in claim 1.

3. The DNA molecule according to claim 2, characterized in that: Its nucleotide sequence is SEQ ID NO.

3.

4. A carrier, characterized in that: It contains the DNA molecule according to claim 2 or 3.

5. A host cell, characterized in that: It contains the DNA molecule according to claim 2 or 3, or the vector according to claim 4.

6. A method for producing a human urate oxidase mutant with weakened immunogenicity and high activity according to claim 1, characterized in that: include: The host cell of claim 5 is cultured under conditions suitable for the expression of the uricase mutant, and the highly active and immunogenic human uricase mutant is isolated from the culture medium.

7. Use of the human urate oxidase mutant with weakened immunogenicity and high activity as claimed in claim 1 for preparing a medicament for treating hyperuricemia and gout.

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