Highly active and immunogenicly attenuated human uricase mutants

Abstract

The application belongs to the technical field of biology and relates to a human uric acid oxidase mutant with high activity and weakened immunogenicity. The human uric acid oxidase mutant with high activity and weakened immunogenicity is obtained by mutation of an inactivated human uric acid oxidase with an amino acid sequence of SEQ ID NO. 1; the mutant has amino acid substitutions at positions 83, 84, 112, 119, 121, 151, 208, 219, 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 application belongs to the field of biotechnology, and relates to uric acid oxidase, in particular to a human uric acid oxidase mutant with high activity and weakened immunogenicity. BACKGROUND

[0002] The main clinical treatment drugs for hyperuricemia and gout are allopurinol, xanthine oxidase inhibitors, benzbromarone, probenecid, colchicine, etc. These drugs generally have the risk of multi-organ toxicity, and their application is severely limited. Uric acid oxidase is a key enzyme in the terminal stage of purine metabolism, which is responsible for catalyzing the conversion of uric acid into allantoin with higher solubility, showing a more ideal effect of lowering uric acid. Currently, the recombinant uric acid oxidase approved for the treatment of hyperuricemia in the clinic mainly comes from heterologous species such as Aspergillus flavus and pig-baboon chimeras, which often causes serious allergic reactions in patients due to heterogeneity. Although the half-life and stability of the uric acid oxidase modified by polyethylene glycol (PEG) are improved, patients may still produce anti-drug antibodies (ADAs) and infusion reactions, thereby limiting its wide application in the clinic, and it is mainly used for the treatment of acute, severe, life-threatening refractory hyperuricemia. The existence of immunogenicity has become the main limitation of the application of uric acid oxidase. The development of uric acid oxidase with low immunogenicity has become a hot research field of the current recombinant uric acid oxidase 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 are found in human sweat glands, there is no urate oxidase activity. JIANG et al. reported that a low immunogenicity therapeutic urate oxidase was obtained by reviving the human urate oxidase pseudogene (Document 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 document shows that by analyzing the multiple sequence alignment of the inactive human urate oxidase gene, mutations were made at 15 highly conserved sites, obtaining a “revived” urate oxidase rHU15 with a sequence consistency of 95.06% with the theoretical sequence of human urate oxidase amino acid. However, the specific enzyme activity was only 2.30 U / mg. Further mutation of the expanded conserved site obtained a mutant rHU19 with 19 sites (the sequence consistency with the theoretical sequence of human urate oxidase amino acid decreased to 93.75%), and the specific enzyme activity reached 8.29 U / mg. The document did not test the immunogenicity of the mutant. Another document reports that even the baboon urate oxidase with a sequence consistency of up to 93.75% with the theoretical sequence of human urate oxidase amino acid can cause significant immunogenicity (Document 2: XIONG R, Cloning and molecular modification of baboon urate oxidase gene [D]. Beijing University of Chemical Technology, 2012.). Compared with baboon urate oxidase, rHU19 has lower sequence consistency, so it can be inferred that rHU19 is more similar to heterologous urate oxidase than to human origin, and still has significant immunogenicity. Since the human urate oxidase pseudogene is revived by back-mutation of all highly conserved sites, it is essentially non-human, increasing the heterogeneity of the revived enzyme and thus increasing the immunogenicity.

[0004] As a protein that has never existed in the form of a complete protein molecule in the human body during development, there are two problems with the revival strategy of human urate oxidase: first, by analyzing the conservation of amino acid sequences of isozymes from different species to restore the activity of human urate oxidase, the protein amino acid sequence is actually non-humanized, and the restoration of conserved sites enhances the immunogenicity while restoring enzyme activity; second, when the humanization rate of amino acid sequence reaches a threshold (e.g., 95%), further increasing the humanization rate actually leads to a significant decrease in enzyme activity, and even loss of activity.

[0005] Therefore, the current strategy of functional recovery of human urate oxidase pseudogene by homologous alignment and conservative amino acid mutation is faced with challenges and difficulties, and it is necessary to find a new strategy to obtain a human urate oxidase mutant with high activity and weakened immunogenicity. SUMMARY

[0006] The primary object of the present application is to provide a human urate oxidase mutant with high activity and weakened immunogenicity, which has high homology with the theoretical amino acid sequence of human urate oxidase and has high enzyme activity while reducing immunogenicity.

[0007] The human urate oxidase mutant with high activity and weakened immunogenicity according to the present application is a human urate oxidase mutant obtained by mutation of the inactive human urate oxidase with the amino acid sequence of SEQ ID NO. 1; it has amino acid substitutions at positions 83, 84, 112, 119, 121, 151, 208, 219, 222, 232, 233, 240 and 252.

[0008] According to a further feature of the human urate oxidase mutant with high activity and weakened immunogenicity according to the present application, the amino acid substitution at position 83 is substitution of glutamic acid (E) with glycine (G), the amino acid substitution at position 112 is substitution of methionine (M) with valine (V), the amino acid substitution at position 84 is substitution of isoleucine (I) with cysteine (C) or histidine (H) or asparagine (N) or arginine (R), the amino acid substitution at position 119 is substitution of histidine (H) with arginine (R), the amino acid substitution at position 121 is substitution of glycine (G) with glutamic acid (E), the amino acid substitution at position 151 is substitution of glutamine (Q) with proline (P), the amino acid substitution at position 208 is substitution of lysine (K) with glutamic acid (E), the amino acid substitution at position 219 is substitution of methionine (M) with leucine (L), the amino acid substitution at position 222 is substitution of serine (S) with phenylalanine (F), the amino acid substitution at position 232 is substitution of leucine (L) with serine (S), the amino acid substitution at position 233 is substitution of threonine (T) with proline (P), the amino acid substitution at position 240 is substitution of cysteine (C) with tyrosine (Y), and the amino acid substitution at position 252 is substitution of alanine (A) with glutamic acid (E); the amino acid sequences of the site-directed mutant human urate oxidase mutants are SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

[0009] When the 84th amino acid substitution is substitution of isoleucine (I) with cysteine (C), the amino acid sequence of the site-directed mutagenesis engineered human-derived urate oxidase mutant is SEQ ID NO. 2.

[0010] When the 84th amino acid substitution is substitution of isoleucine (I) with histidine (H), the amino acid sequence of the site-directed mutagenesis engineered human-derived urate oxidase mutant is SEQ ID NO. 3.

[0011] When the 84th amino acid substitution is substitution of isoleucine (I) with asparagine (N), the amino acid sequence of the site-directed mutagenesis engineered human-derived urate oxidase mutant is SEQ ID NO. 4.

[0012] When the 84th amino acid substitution is substitution of isoleucine (I) with arginine (R), the amino acid sequence of the site-directed mutagenesis engineered human-derived urate oxidase mutant is SEQ ID NO. 5.

[0013] The second object of the present application is to provide a DNA molecule encoding the high-activity and weakly immunogenic human-derived urate oxidase mutant of the present application.

[0014] When the amino acid sequence of the human-derived urate oxidase mutant of the present application is SEQ ID NO. 2, the nucleotide sequence of the human-derived urate oxidase mutant is SEQ ID NO. 6.

[0015] When the amino acid sequence of the human-derived urate oxidase mutant of the present application is SEQ ID NO. 3, the nucleotide sequence of the human-derived urate oxidase mutant is SEQ ID NO. 7.

[0016] When the amino acid sequence of the human-derived urate oxidase mutant of the present application is SEQ ID NO. 4, the nucleotide sequence of the human-derived urate oxidase mutant is SEQ ID NO. 8.

[0017] When the amino acid sequence of the human-derived urate oxidase mutant of the present application is SEQ ID NO. 5, the nucleotide sequence of the human-derived urate oxidase mutant is SEQ ID NO. 9.

[0018] The third object of the present application is to provide a vector containing the DNA molecule of the present application.

[0019] The fourth object of the present application is to provide a host cell containing the DNA molecule of the present application, or containing the vector of the present application.

[0020] The above-mentioned vector and host cell can be prepared by techniques well known in the art.

[0021] A fifth object of the present application is to provide a method for producing a human urate oxidase mutant having high activity and weakened immunogenicity.

[0022] The method for producing a human urate oxidase mutant having high activity and weakened immunogenicity according to the present application comprises culturing the host cell according to the present application under conditions suitable for expression of the urate oxidase mutant, and isolating the human urate oxidase mutant having high activity and weakened immunogenicity from the culture medium.

[0023] When the DNA molecule according to the present application is inserted into the vector in the appropriate orientation and in the correct reading frame, or is transferred into the host cell, the DNA molecule can be expressed in any eukaryotic or prokaryotic expression system. Many host-vector systems can be used to express protein coding sequences. Host-vector systems include, but are not limited to, bacteria transformed with bacteriophage, plasmid or cosmid; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with a virus; insect cell systems infected with a virus; plant cell systems infected with bacteria. Preferred vectors according to the present application include viral vectors, plasmids, cosmids or oligonucleotides.

[0024] Preferred hosts according to the present application are prokaryotic systems such as E. coli, and preferred protein expression methods according to the present application are baculovirus expression.

[0025] A sixth object of the present application is to provide the use of the human urate oxidase mutant having high activity and weakened immunogenicity for the preparation of a medicament for the treatment of hyperuricemia and gout.

[0026] The present application is a site-directed mutation of the amino acid sequence of an inactive human urate oxidase from a human urate oxidase pseudogene (referred to as hUOX gene). The GENBANK accession number of the human urate oxidase pseudogene sequence is NR_003927.2. The stop codons at positions 3 and 187 in the sequence are changed to R, becoming the amino acid sequence of an inactive human urate oxidase (SEQ ID NO. 1).

[0027] The inventors have obtained a human urate oxidase mutant having high activity and weakened immunogenicity, rhUOX mut3 / I84C (SEQ ID NO. 2), rhUOX mut3 / I84H (SEQ ID NO. 3), rhUOX mut3 / I84N (SEQ ID NO. 4) and rhUOX mut3 / I84R(SEQ ID NO. 5), with a homology of 95.06% to the theoretical sequence of human uricase amino acids, and specific enzyme activities of 7.36 U / mg, 7.55 U / mg, 7.41 U / mg and 7.47 U / mg, respectively, indicating that the immunological weakening design can still maintain good activity and has a high degree of humanization, and the hotspot amino acids of the protein surface antigen peptide are further mutated so as to be unable to be recognized, thereby reducing immunogenicity, and thus the humanized uricase mutant of the application is more suitable for the clinical needs of low immunogenic uricase and is suitable for long-term use in the clinical treatment of hyperuricemia and gout, and can be used for preparing a drug for treating hyperuricemia and gout.

[0028] The inventors obtained the uricase mutant rhUOX mut3 / I84C (SEQ ID NO. 2), rhUOX mut3 / I84H (SEQ ID NO. 3), rhUOX mut3 / I84N (SEQ ID NO. 4) and rhUOX mut3 / I84R (SEQ ID NO. 5) based on SEQ ID NO. 1 through site-directed mutagenesis, which can still maintain good activity after immunological weakening design and has a high degree of humanization. They have a homology of 95.06% to the theoretical sequence of human uricase amino acids and specific enzyme activities of 7.36 U / mg, 7.55 U / mg, 7.41 U / mg and 7.47 U / mg, respectively, which breaks through the threshold of 95% homology reported in the literature and shows high enzyme activity. Experiments show that they have low immunogenicity and are similar to human serum albumin (HSA) in immunogenicity, because the hotspot amino acids of the protein surface antigen peptide are further mutated so as to be unable to be recognized, thereby reducing immunogenicity. Therefore, the humanized uricase mutant of the application with high activity and weakened immunogenicity is more suitable for the clinical needs of low immunogenic uricase and is suitable for long-term use in the clinical treatment of hyperuricemia and gout, and can be used for preparing a drug for treating hyperuricemia and gout. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 SDS-PAGE protein electropherogram of the humanized uricase mutant of the application, wherein 1: rhUOX wt ; 2: rhUOX mut3 / I84C mutant; 3: rhUOX mut3 / I84H mutant; 4: rhUOX mut3 / I84N mutant; 5: rhUOX mut3 / I84R mutant; M is Marker.

[0030] Figure 2 showing rhUOX wt , rhUOXmut3 / I84C , rhUOX mut3 / I84H , rhUOX mut3 / I84N , rhUOX mut3 / I84R , rhUOX

[0031] Figure 3 The rhUOX mut3 / I84C , rhUOX mut3 / I84H , rhUOX mut3 / I84N , rhUOX mut3 / I84R and rhUOX wt Af The results of human blood monocyte immunogenicity test of Aspergillus flavus-derived uric acid oxidase (UOX) and human serum albumin (HAS).

[0032] Figure 4 The results of in vivo efficacy of human-derived uric acid oxidase, wherein A: the results of in vivo efficacy in male mice; B: the results of in vivo efficacy in female mice. DETAILED DESCRIPTION

[0033] The terms used herein, unless otherwise stated, are the meanings commonly understood by those skilled in the art. The following provides definitions of some special terms used in the present application.

[0034] "rhUOX wt " means an inactive human-derived uric acid oxidase in which the missense (stopper) codons at positions 33 and 187 of the human-derived uric acid oxidase pseudogene are restored to arginine (R), and the gene is represented in italics "rhUOX rhuox wt " means. The amino acid sequence of rhUOX wt is SEQ ID NO. 1.

[0035] "rhUOX mut3 / I84C , rhUOX mut3 / I84H , rhUOX mut3 / I84N , and rhUOX mut3 / I84R " means an immunogenicity-weakened hUOX mutant, and the gene is represented in italics "rhUOX rhuox mut3 / I84C , rhuox mut3 / I84H , rhuox mut3 / I84N , rhuox mut3 / I84R " means. rhUOX mut3 / I84C , rhUOX mut3 / I84H , rhUOX mut3 / I84N , and rhUOX mut3 / I84RThe amino acid sequences of the mutants are SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5 respectively.

[0036] Example 1 rhuox wt rhuox mut3 / I84C rhuox mut3 / I84H rhuox mut3 / I84N rhuox mut3 / I84R Synthesis of genes

[0037] The present application adopts human uric acid oxidase pseudogene sequence rhuox wt (GenBank registration number NR_003927.2), which is modified to obtain human uric acid oxidase mutants rhuox mut3 / I84C rhuox mut3 / I84H rhuox mut3 / I84N rhuox mut3 / I84R The above-mentioned genes are synthesized by Huada Gene Company, and are cloned between NcoI and XhoI of pET28a plasmid (Invitrogen Company), and the recombinant plasmids are named as pET28a-huox wt , pET28a-huox mut3 / I84C , pET28a-huox mut3 / I84H , pET28a-huox mut3 / I84N , pET28a-huox mut3 / I84R .

[0038] Example 2 rhuox wt Figure 1 mut3 / I84C Figure 1 mut3 / I84H Figure 2 mut3 / I84N Af mut3 / I84R The genes are respectively transformed into Escherichia coli Rosetta (DE3), and the induction expression of recombinant proteins

[0039] The receptor bacteria of the experiment are Escherichia coli Rosetta (DE3), and pET28a-huox wt , pET28a-huox mut3 / I84C , pET28a-huox mut3 / I84H , pET28a-huox mut3 / I84N, pET28a-huox mut3 / I84R After transformation of the recombinant plasmid, Kanamycin (Kan) resistant plates were used for preliminary screening, and then single colonies on the Kan plates were picked and cultured in 5 mL of LB liquid medium (containing 50 mg / mL Kan) for 12-16 h, and plasmids were extracted for PCR verification, and positive recombinants were further screened.

[0040] The positive recombinants were inoculated in 5 mL of LB medium (50 mg / mL Kan) and cultured at 37°C, 200 rpm overnight. The recovered bacterial liquid was inoculated in 500 mL of LB medium (50 mg / mL Kan) at 1%, and cultured at 37°C, 200 rpm until the OD 600 was 0.6, 60 μM lactose was added, and expression was induced at 30°C, 200 rpm for 12 h. After induction, the bacterial cells were collected, resuspended in carbonate buffer at pH 10.0 (mass / volume ratio 1:20), and the bacterial cells were broken using an ultrasonic disrupter. The supernatant was collected by centrifugation at 6000 g for 10 min, and the recombinant protein was obtained.

[0041] Example 3: rhUOX wt , rhUOX mut3 / I84C , rhUOX mut3 / I84H , rhUOX mut3 / I84N , rhUOX mut3 / I84R SDS-PAGE electrophoresis detection of mutant recombinant proteins

[0042] The SDS-PAGE electrophoresis detection of this example includes the following steps:

[0043] (1) Prepare 10 mL of 10% separation gel, mix well, and pour the gel into the glass plate with a micropipette until the gel stops at 2-3 cm from the top of the short glass plate, then seal the gel surface with distilled water. Gently lift one end of the gel maker and then put it down to make the gel surface flat. After polymerization for 40 min, discard the distilled water and absorb the excess water with filter paper.

[0044] (2) Prepare 4 mL of 5% concentrated gel and pour it evenly on the separation gel. Insert the corresponding comb while avoiding air bubbles. Polymerize for 30 min until the gel solidifies.

[0045] (3) Assemble the electrophoresis tank, pour the electrophoresis solution into the tank, and the volume should be more than half of the tank volume. Move the prepared gel into the electrophoresis tank and carefully remove the comb.

[0046] (4) Point the sample in turn, and the sample volume should not be too much, 15 μL per well is appropriate.

[0047] (5) At the beginning of electrophoresis, set 90 V to run the gel, and change the voltage to 120 V for the indicator to the concentrated gel part. Stop electrophoresis when the target band reaches the middle position (the target band corresponds to the corresponding band of Maker, which can be known in advance).

[0048] (6) Carefully peel off the gel, and after staining with Coomassie Brilliant Blue R-250 for 30 min, decolorize it with decolorizing solution until the background is light and the protein bands are clear.

[0049] (7) Image the gel and observe the results. The results of SDS-PAGE protein electrophoresis are shown in Af .

[0050] The results of SDS-PAGE protein electrophoresis are shown in Af . Lane 1 is the denatured rhUOX wt protein sample, and lanes 2-5 are mutant rhUOX mut3 / I84C , rhUOX mut3 / I84H , rhUOX mut3 / I84N , and rhUOX mut3 / I84R protein samples. The target protein is about 34 kDa in size, and the results show that the target gene is successfully expressed in E. coli Rosetta and can be used for subsequent experiments.

[0051] Example 4: Activity characterization of mutants

[0052] At 37°C, take 20 μL of the purified protein (rhUOX mut3 / I84C , rhUOX mut3 / I84H , rhUOX mut3 / I84N , and rhUOX mut3 / I84R ) in 600 μL of a solution containing 0.1 M uric acid (pH 8.0), and after 10 min of reaction, add an equal volume of pure methanol to terminate the reaction. Calculate the enzyme activity by measuring the reduced ultraviolet absorption value at 293 nm by ultraviolet spectrophotometry. The control group is inactivated uric acid oxidase. The experimental results are shown in Figure 3 . The specific enzyme activities of rhUOX mut3 / I84C , rhUOX mut3 / I84H , rhUOX mut3 / I84N , and rhUOX mut3 / I84R are 7.36 U / mg, 7.55 U / mg, 7.41 U / mg, and 7.47 U / mg, respectively, and these mutants all have high activity.

[0053] Definition of enzyme activity unit: 1 unit is the amount of enzyme required to catalyze the oxidation of 1 µmol of uric acid per minute.

[0054] E (U / mg) = (△OD 293 × V1 × D) / (12.3 × V2 × T × C)

[0055] 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.

[0056] Example 5: Human PBMC cell immunogenicity analysis

[0057] Human serum albumin (HAS) and urate oxidase from Aspergillus flavus ( Figure 4 UOX) as the control group, rhUOX mut3 / I84C 、rhUOX mut3 / I84H 、rhUOX mut3 / I84N and rhUOX mut3 / I84R 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.

[0058] Indirect ELISA specific steps:

[0059] (1) Antibody coating: protein samples (HSA, ​ UOX、rhUOX mut3 / I84C 、rhUOX mut3 / I84H 、rhUOX mut3 / I84N and rhUOX mut3 / I84R 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.

[0060] (2) Blocking: Block with Elisa blocking solution for 1 h.

[0061] (3) Washing: Wash three times with PBST and pat dry.

[0062] (4) Incubation: Add 100 μL of the supernatant prepared from the above cell culture (HSA, ​ UOX、rhUOXmut3 / I84C , rhUOX mut3 / I84H , rhUOX mut3 / I84N , and rhUOX mut3 / I84R , the blank control is PBS, and incubated at 37 ℃ for 1.5 h.

[0063] (5) Washing: wash three times with washing solution. Pat dry.

[0064] (6) HRP-goat anti-human IgG incubation: add 100 μL of diluted HRP-goat anti-human IgG (1:3000, PBST, pH 7.4) per well, and incubate at 37 ℃ for 1 h.

[0065] (7) Washing: wash three times with washing solution. Pat dry.

[0066] (8) Reaction color development: add 100 μL of freshly prepared TMB working solution per well, and react at room temperature in the dark for 15 min. Add 50 μL of stop solution (2 M H2SO4) per well. Read the OD value of each well with an enzyme-labeled instrument. 450

[0067] Take the well A450 greater than or equal to 2.1 times the negative control well as positive: P / N = OD 450 Test serum / OD 450 The negative control serum (i.e., P / N ≥ 2.1 is positive).

[0068] The results are shown in ​ , mutant rhUOX mut3 / I84C , rhUOX mut3 / I84H , rhUOX mut3 / I84N , and rhUOX mut3 / I84R have lower immunogenicity, which is comparable to that of HSA.

[0069] Example 7: In vivo efficacy verification of uric acid oxidase gene KO mice

[0070] C57BL / 6J mice (UOX- / -) were used in the experiment, 48 in total, half male and half female, 5-8 weeks old, 25-30 g, SPF level, purchased from Saiye Biotechnology Co., Ltd., and raised in the SPF level experimental animal room of the Experimental Animal Management Center of Jinan University, with a room temperature of 20-25 ℃. The experimental animal use license is SYXK (Yue) 2022-0174. The C57BL / 6J mice were given clean drinking water before the experiment, and were allowed to eat freely for 3 days before being used in the experiment.

[0071] Normal control group (normal serum level), high uric acid control group, and experimental group (rhUOX mut3 / I84C , rhUOX mut3 / I84H ​, rhUOX mut3 / I84N and rhUOX mut3 / I84R ). The normal control group was wild-type C57BL / 6J mice, and the other groups used uricase knockout KO mice for experiments, 12 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, and the experimental group was given a dose of 2 U / kg of rilonacept. Intraperitoneal injection was given, and blood was taken from the orbital vein at 0, 0.5, 1, 3, and 6 hours after administration. The serum was treated as above, and the collected serum was detected by HPLC for uric acid content. The results are shown in ​ Under the experimental conditions, the immunogenicity weakened rhUOX mut3 / I84C , rhUOX mut3 / I84H , rhUOX mut3 / I84N and rhUOX mut3 / I84R can maintain the uric acid level of KO mice at a normal level for at least 3 hours, and still have a uric acid-lowering effect after 6 hours.

[0072] HPLC detection conditions

[0073] 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℃; injection volume: 20 uL; mobile phase: A 0.1% formic acid-water; B pure methanol.

[0074] Regarding the number of point mutations, position, homology, enzyme specific activity, immunogenicity, etc., the uricase mutants described in the present application are compared with literature 1 and 2 in Table 1 below.

[0075] Table 1:

[0076]

[0077] Literature 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.

[0078] Literature 2: Xiong Runsong. Chimpanzee uricase gene cloning and molecular modification research [D]. Beijing University of Chemical Technology, 2012.

[0079] As shown in the above table, the present application first discloses human urate oxidase mutants with high activity and weakened immunogenicity, rhUOX mut3 / I84C , rhUOX mut3 / I84H , rhUOX mut3 / I84N and rhUOX mut3 / I84R , which have a homology of 95.06% with the theoretical amino acid sequence of human urate oxidase (the same as rHU15 reported in document 1), and the specific activities are 7.36 U / mg, 7.55 U / mg, 7.41 U / mg and 7.47 U / mg respectively (significantly higher than rHU15 reported in document 1). The mutants have high activity while reducing the mutation sites, and maintain a high humanization rate. Experiments show that they have low immunogenicity, similar to that of HSA. The reason is that the hot amino acids of the protein surface antigen peptide are further mutated and thus cannot be recognized, thereby reducing the immunogenicity. Therefore, the human urate oxidase mutants with weakened immunogenicity and high activity disclosed in the present application are more in line with the clinical needs of low immunogenicity urate oxidase, and are suitable for application in long-term use in the clinical treatment of hyperuricemia and gout, and can be used for preparing drugs for treating hyperuricemia and gout.

Claims

1. A highly active and immunogenic human urate oxidase mutant, characterized by: The mutant is a human urate oxidase mutant obtained by mutating the inactive human urate oxidase with the amino acid sequence of SEQ ID NO.1; the amino acid sequences of the human urate oxidase mutants are SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5, respectively.

2. A DNA molecule, characterized in that: It encodes the highly active and immunogenic weakened human uricase mutant according to claim 1.

3. The DNA molecule according to claim 2, characterized in that: Its nucleotide sequence is SEQ ID NO. 6 or SEQ ID NO. 7 or SEQ ID NO. 8 or SEQ ID NO.

9.

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 highly active and immunogenic human urate oxidase mutant according to claim 1, characterized in that: The method comprises: culturing the host cell according to claim 5 under conditions suitable for the expression of uricase, and isolating the human uricase mutant from the culture medium.

7. Use of the highly active and immunogenic weakened human urate oxidase mutant according to claim 1 for preparing a drug for treating hyperuricemia and gout.

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