Human urate oxidase mutant with high activity and immunogenicity weakening

Abstract

The invention belongs to the technical field of biology, and relates to a high-activity human urate oxidase mutant with immunogenicity weakening. The human urate oxidase mutant with high activity and immunogenicity weakening, disclosed by the invention, is a human urate oxidase mutant obtained by mutation of inactive human urate oxidase with an amino acid sequence of SEQ ID NO.1; the amino acid sequence of the human urate oxidase mutant is SEQ ID NO.2, and the human urate oxidase mutant has 83rd, 85th, 119th, 121st, 151st, 208th, 219th, 222nd, 232, 233rd, 240th and 252nd amino acid substitutions. The human uricase mutant keeps high homology with the theoretical amino acid sequence of human uricase, has high activity while reducing immunogenicity, is suitable for long-term use in clinical treatment of hyperuricemia and gout, and can be used for preparing drugs for treating hyperuricemia and gout.

Description

Technical Field

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

[0002] The main clinical treatment drugs for hyperuricemia and gout are allopurinol, xanthine oxidase inhibitors, benzbromarone, probenecid, and colchicine. These drugs generally have the risk of multi-organ toxicity, and their application is severely limited. Urate oxidase, as a key enzyme in the terminal link of purine metabolism, is responsible for catalyzing the conversion of uric acid into more soluble allantoin, showing a more ideal uric acid-lowering effect. Currently, the recombinant urate oxidase approved for the treatment of hyperuricemia in clinical practice is mainly derived from heterologous species, such as Aspergillus flavus and pig-baboon chimeras, which often cause severe allergic reactions in patients due to heterologousness. Although the half-life and stability of uricase modified with polyethylene glycol (PEG) have been improved, patients may still develop anti-drug antibodies (ADAs) and infusion reactions, which limits its wide clinical application. Therefore, it is mainly used to treat acute, severe, life-threatening and refractory hyperuricemia. The existence of immunogenicity has become the main limitation of uricase application. The development of uricase with low immunogenicity has become a hot area in the current 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 are found in human sweat glands, there is no urate oxidase activity. Jiang et al. reported that low-immunogenicity therapeutic urate oxidase was obtained by resurrecting the human urate oxidase pseudogene (Reference 1: Jiang N, Xuc, 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 performing multiple sequence alignment analysis on the inactive human uricase gene and mutating 15 highly conserved sites, a "resurrected" uricase rHU15 with a theoretical sequence identity of 95.06% with human uricase was obtained, but the enzyme activity was only 2.30U / mg. After further expanding the mutation of conservative sites, a mutant rHU19 with 19 sites was obtained (the theoretical sequence identity with human uricase was reduced to 93.75%), and the enzyme activity reached 8.29U / mg. The document did not test the immunogenicity of the mutant. Another document reported that even baboon uricase with a theoretical sequence identity of 93.75% with human uricase may cause significant immunogenicity (Document 2: Xiong Runsong. Study on cloning and molecular modification of baboon uricase gene [D]. Beijing University of Chemical Technology, 2012.). Compared with baboon uricase, rHU19 has lower sequence identity, so it can be inferred that rHU19 is more similar to heterologous uricase rather than human uricase, and still has significant immunogenicity. Since the human uricase pseudogene is revived by back mutation of all highly conserved sites, there is an essential non-humanization, which increases the heterologous nature of the revived enzyme and thus increases immunogenicity.

[0004] Human urate oxidase is a protein that has never existed in the body as a complete protein molecule during human development. There are two problems with its resurrection strategy: first, the activity of human urate oxidase is restored by mutation through conservation analysis of the amino acid sequences of isozymes from different species, which 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 mutations faces challenges and difficulties. Therefore, it is necessary to find new strategies to obtain highly active human uricase mutants with weakened immunogenicity. Summary of the invention

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

[0007] The highly active human uricase mutant with weakened immunogenicity 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, 208, 219, 222, 232, 233, 240 and 252.

[0008] According to the further characteristics of the highly active and immunogenic human uricase mutant of the present invention, the amino acid substitution at position 83 is to replace glutamic acid (E) with glycine (G), the amino acid substitution at position 85 is to replace lysine (K) with cysteine ​​(C), the amino acid substitution at position 119 is to replace histidine (H) with arginine (R), the amino acid substitution at position 121 is to replace glycine (G) with glutamic acid (E), the amino acid substitution at position 151 is to replace glutamine (Q) with proline (P), and the amino acid substitution at position 208 is to replace lysine with glutamic acid (E). (K), the 219th amino acid substitution is a substitution of methionine (M) with leucine (L), the 222nd amino acid substitution is a substitution of serine (S) with phenylalanine (F), the 232nd amino acid substitution is a substitution of leucine (L) with serine (S), the 233rd amino acid substitution is a substitution of threonine (T) with proline (P), the 240th amino acid substitution is a substitution of cysteine ​​(C) with tyrosine (Y), and the 252nd amino acid substitution is a substitution of alanine (A) with glutamic acid (E); the amino acid sequence of the human urate oxidase 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 urate oxidase mutant with weakened immunogenicity and high activity as described in the present invention. According to the DNA molecule of the present invention, its nucleotide sequence is SEQ ID NO. 3. The third object of the present invention is to provide a vector containing the DNA molecule of the present invention.

[0010] The fourth object of the present invention is to provide a host cell, which contains the DNA molecule of the present invention, or contains the vector of the present invention.

[0011] The above-mentioned vectors and host cells can be prepared by techniques well known in the art.

[0012] The fifth object of the present invention is to provide a method for producing a human uricase mutant with high activity and weakened immunogenicity.

[0013] The method for producing the highly active and immunogenic weakened human uricase mutant of the present invention comprises: culturing the host cell of the present invention under conditions suitable for the expression of the uricase mutant, and isolating the highly active and immunogenic weakened human uricase mutant from the culture medium.

[0014] When the DNA molecule of the present invention is inserted into the vector or transferred into the host cell in the appropriate orientation and correct reading frame, 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 bacteriophages, plasmids or cosmids; microorganisms containing yeast vectors, such as yeast; mammalian cell systems infected with viruses; insect cell systems infected with viruses; plant cell systems infected with bacteria. Preferred vectors of the present invention include viral vectors, plasmids, cosmids or oligonucleotides.

[0015] 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.

[0016] The sixth object of the present invention is to provide the use of the highly active and immunogenic weakened human urate oxidase mutant for preparing a drug for treating hyperuricemia and gout.

[0017] The present invention is to perform site-directed mutagenesis on the amino acid sequence of the inactive human uricase pseudogene (called hUOX gene). The GENBANK accession number of the human uricase pseudogene sequence is NR_003927.2. The terminators at positions 3 and 187 in the sequence are changed to R, resulting in the amino acid sequence of the inactive human uricase (SEQ ID NO.1).

[0018] Based on SEQ ID NO.1, the inventors obtained a urate oxidase mutant rhUOX that can maintain good activity and has a high degree of humanization after immunoweakening design by site-directed mutagenesis.mut2 / K85C (SEQ ID NO. 2). The mutant has a homology of 95.39% with the theoretical amino acid sequence of human uricase, which has a higher humanization rate than the existing publicly reported uricase, and its enzyme specific activity is 7.685U / mg, which shows a higher enzyme activity while breaking through the 95% homology threshold reported in the literature. Experiments show that the mutant has a lower immunogenicity, which is similar to the immunogenicity of human serum albumin (HSA), because the hotspot amino acids of the antigen peptide on its protein surface are further mutated and cannot be recognized, thereby reducing the immunogenicity. Therefore, the human uricase mutant with weakened immunogenicity and high activity described in the present invention is more in line with the clinical needs of low-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

[0019] Figure 1 The SDS-PAGE protein electrophoresis diagram of the human urate oxidase mutant of the present invention, wherein: 1: rhUOX wt ; 2: rhUOX mut2 / K85C ; M stands for Marker.

[0020] Figure 2 The rhUOX of the present invention is shown wt 、rhUOX mut2 / K85C uric acid degradation activity.

[0021] Figure 3 The rhUOX of the present invention is shown mut2 / K85C and urate oxidase from Aspergillus flavus ( A UOX) and human serum albumin (HAS) in human blood monocytes.

[0022] Figure 4 The in vivo efficacy results of human urate oxidase are shown, where A: the efficacy results in male mice; B: the efficacy results in female mice. DETAILED DESCRIPTION

[0023] 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.

[0024] “rhUOX wt " indicates an inactive human uricase in which the missense (terminator) codons at positions 33 and 187 of the human uricase pseudogene are restored to arginine (R). The gene is in italics" rhuox wt "Indicates. wtThe amino acid sequence is SEQ ID NO.1.

[0025] “rhUOX mut2 / K85C " indicates hUOX mutants with weakened immunogenicity, whose genes are in italics" rhuox mut2 / K85C "Indicates. mut2 / K85C The amino acid sequences of the mutants are SEQ ID NO.2.

[0026] Embodiment 1: rhuox wt and rhuox mut2 / K85C Gene synthesis The present invention adopts human urate oxidase pseudogene sequence rhuox wt (GenBank registration number is NR_003927.2), and the human urate oxidase mutant was obtained by transformation rhuox mut2 / 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 、pET28a-huox mut2 / K85C .

[0027] Embodiment 2: rhuox wt 、rhuox mut2 / K85C Genes were transformed into E. coli Rosetta (DE3) and recombinant protein was induced to express The recipient bacteria in this experiment was Escherichia coli Rosetta (DE3), and pET28a-huox wt 、pET28a-huox mut2 / K85C After the recombinant plasmid was transformed, a kanamycin (Kan) resistance plate was used for preliminary screening, and then a single clone on the Kan plate was picked and cultured in 5 mL of LB liquid medium (containing 50 mg / mL Kan) for 12-16 hours, and then the plasmid was extracted for PCR verification to further screen the positive clone recombinants.

[0028] The positive recombinants were inoculated into 5 mL of LB medium (50 mg / mL Kan) and cultured overnight at 37°C and 200 rpm. The revived bacterial solution was inoculated into 500 mL of LB medium (50 mg / mL Kan) at 1% and cultured at 37°C and 200 rpm until OD 600The concentration of 60 μM lactose was 0.6, and 60 μM lactose was added to induce expression at 30°C and 200 rpm for 12 h. After induction, the cells were collected and resuspended in carbonate buffer at pH 10.0 (mass-to-volume ratio was 1:20), and the cells were broken by ultrasonic disruptor, centrifuged at 6000 g for 10 min, and the supernatant was collected to obtain the recombinant protein.

[0029] Example 3: rhUOX wt and rhUOX mut2 / K85C Detection of mutant recombinant protein by SDS-PAGE electrophoresis The SDS-PAGE electrophoresis detection of the present embodiment comprises the following steps: (1) Prepare 10 mL of 10% separation gel, mix well and use a micropipette to pour the gel into the glass plate 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 polymerizing for 40 min, discard the distilled water and absorb excess water with filter paper.

[0030] (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 min.

[0031] (3) Install the electrophoresis tank and fill it with electrophoresis liquid. The volume should be larger than half of the tank volume. Move the prepared gel into the tank and carefully remove the comb.

[0032] (4) Spot samples one by one. The amount of sample spotted should not be too much. 15 μL per well is appropriate.

[0033] (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 and can be known in advance).

[0034] (6) Carefully peel off the gel and stain with Coomassie Brilliant Blue R-250 for 30 min. Then destain with destaining buffer until the background is light and the protein bands are clear.

[0035] (7) Gel imaging and observation of results. The SDS-PAGE protein electrophoresis results are as follows: Figure 1 shown.

[0036] The results of SDS-PAGE protein electrophoresis are as follows Figure 1 Lane 1 is the denatured rhUOX wt Protein samples, lane 2 is mutant rhUOX mut2 Lane 3 is the mutant rhUOX mut2 / K85CTarget protein sample. The target protein is about 34 kDa in size. This result indicates that the target gene is successfully expressed in E. coli Rosetta and can be used for subsequent experiments.

[0037] Example 4: Characterization of the activity of mutants At 37°C, take 20 μL of purified protein rhUOX mut2 / K85C In 600 μL of 0.1 M uric acid solution (pH 8.0), after 10 min of reaction, an equal volume of pure methanol was added to terminate the reaction. The enzyme activity was calculated by measuring the reduced UV absorbance at 293 nm by UV spectrophotometry. The control group was inactivated urate oxidase. The experimental results are shown in Figure 2 As shown. mut2 / K85C The specific enzyme activity was 7.685 U / mg, indicating that the mutant had high activity.

[0038] Enzyme activity unit definition: 1 unit is the amount of enzyme required to catalyze the oxidation of 1 µmol of uric acid per minute.

[0039] E (U / mg) = (△OD 293 × V1 × D) / (12.3 × V2 × T × C) 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.

[0040] Example 5: Human PBMC cell immunogenicity analysis Human serum albumin (HAS) and urate oxidase from Aspergillus flavus ( A UOX) as the control group, rhUOX mut2 / K85C The above proteins (50 μg / mL) and PWM (20 μg / mL) were added to a 24-well cell culture plate at a volume of 100 μL (5 μg) per well, and then PBMC cell suspension (1×10 6 / mL), 500 µL per well (dosage is 10 µg / mL). Place in a 37°C, 5% CO2 incubator for 7 days. After 2-3 days, change half of the medium. After the culture is completed, centrifuge at 3000 rpm for 15 minutes and collect the cells. Add the cells to the cell culture plates coated with the above proteins, 500μL per well. Also culture in a 37°C, 5% CO2 incubator for 7 days. After 2-3 days, change half of the medium. After the culture is completed, collect the culture supernatant. Anti-urate oxidase IgG was detected by indirect ELISA to analyze immunogenicity.

[0041] Indirect ELISA specific steps: (1) Antibody coating: protein samples (HSA, A UOX and rhUOX mut2 / K85C ), 100 μL (5 μg) was added to each well of a 96-well plate, and two replicates were made for each sample. After overnight at 4°C, the plates were washed three times with PBST and patted dry. (2) Blocking: Block with Elisa blocking solution for 1 h. (3) Washing: Wash three times with PBST and pat dry. (4) Incubation: Add 100 µL of the supernatant (HSA, A UOX and rhUOX mut2 / K85C ), blank control was PBS, and incubated at 37 ℃ for 1.5 h. (5) Washing: Wash three times with detergent. Pat dry. (6) HRP-goat anti-human IgG incubation: 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. (7) Washing: Wash three times with detergent. Pat dry. (8) Reaction color development: Add 100 µL of freshly prepared TMB working solution to each well and react at room temperature in the dark for 15 min. Add 50 µL of stop solution (2 M H2SO4) to each well. Read the OD value using a microplate reader. 450 value.

[0042] 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).

[0043] The results are as follows Figure 3 As shown, the mutant rhUOX mut2 / K85C It has lower immunogenicity, with no significant difference compared with HSA (P>0.05).

[0044] Example 7: In vivo efficacy verification in uricase gene KO mice The experiment used 48 C57BL / 6J mice (UOX- / -), half male and half female, 5-8 weeks old, 25-30g, SPF grade, purchased from Saiye Biotechnology Co., Ltd. and raised in the SPF grade 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 (Guangdong) 2022-0174. C57BL / 6J mice were given clean drinking water and free food before the experiment, and were used in the experiment after 3 days of feeding.

[0045] A normal control group (normal serum level), a high uric acid control group, and an experimental group (rhUOX mut2 / K85C ). The normal control group was 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 4 As shown in the pharmacodynamics experiment of hyperuricemia KO mice, under the experimental conditions, the immunogenicity of rhUOX mut2 / K85C The blood uric acid level of KO mice can be maintained at normal levels for at least 3 hours, and the uric acid-lowering effect can still be exerted after 6 hours.

[0046] HPLC detection conditions Chromatographic column: C18 chromatographic 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.

[0047] Regarding the number and position of point mutations, homology, enzyme specific activity, immunogenicity, etc., the comparison of the urate oxidase mutant described in the present invention with those in References 1 and 2 is shown in Table 1 below.

[0048] Table 1:

[0049] 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.

[0050] Reference 2: Xiong Runsong. Research on cloning and molecular modification of baboon urate oxidase gene[D]. Beijing University of Chemical Technology, 2012.

[0051] As can be seen from the above table, the present invention discloses for the first time a highly active human urate oxidase mutant rhUOX mut2 / K85CThe mutant has a homology of 95.39% with the theoretical amino acid sequence of human urate oxidase, and an enzyme specific activity of 7.685U / mg. It retains high activity while reducing mutation sites and maintains a high humanization rate. The mutant is higher than rHU15 in both humanization rate and activity (Reference 1). Experiments have shown that the mutant has low immunogenicity, which is similar to the immunogenicity of HSA. The reason is that the hotspot amino acids of the antigen peptide on the surface of its protein are further mutated and cannot be recognized, thereby reducing immunogenicity. Therefore, the human urate oxidase mutant with weakened immunogenicity and high activity described in the present invention is more in line with the clinical needs of low-immunogenic urate oxidase, 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 highly active human urate oxidase mutant with weakened immunogenicity, characterized in that: The mutant is a human uricase mutant obtained by mutation of 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, 208, 219, 222, 232, 233, 240 and 252.

2. The highly active and immunogenic human urate oxidase mutant according to claim 1, characterized in that: The amino acid substitution at position 83 is to replace glutamic acid (E) with glycine (G), the amino acid substitution at position 85 is to replace lysine (K) with cysteine ​​(C), the amino acid substitution at position 119 is to replace histidine (H) with arginine (R), the amino acid substitution at position 121 is to replace glycine (G) with glutamic acid (E), the amino acid substitution at position 151 is to replace glutamine (Q) with proline (P), the amino acid substitution at position 208 is to replace lysine (K) with glutamic acid (E), and the amino acid substitution at position 219 is to replace leucine (H). The amino acid at position 222 is substituted by methionine (M), the amino acid at position 222 is substituted by phenylalanine (F) for serine (S), the amino acid at position 232 is substituted by leucine (L) with serine (S), the amino acid at position 233 is substituted by proline (P) for threonine (T), the amino acid at position 240 is substituted by cysteine ​​(C) with tyrosine (Y), and the amino acid at position 252 is substituted by glutamic acid (E) for alanine (A); the amino acid sequence of the human urate oxidase mutant modified by site-directed mutagenesis is SEQ ID NO.

2.

3. A DNA molecule, characterized in that: It encodes the highly active and immunogenic weakened human urate oxidase mutant as described in claim 1.

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

3.

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

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

7. A method for producing a highly active human urate oxidase mutant with weakened immunogenicity according to claim 1, characterized in that: The method comprises: culturing the host cell according to claim 6 under conditions suitable for the expression of uricase, and isolating the human uricase mutant from the culture medium.

8. 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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