Urate oxidase mutant with high activity, high degree of humanization, and low immunogenicity

Abstract

Provided is a urate oxidase mutant with high activity, a high degree of humanization, and low immunogenicity. A first urate oxidase mutant is a human urate oxidase mutant obtained by mutating an inactive human urate oxidase, wherein the mutant has amino acid substitutions at positions 83, 119, 121, 151, 222, 232, 233, 240 and 252. A second urate oxidase mutant further comprises amino acid substitutions at positions 208 and 219 on the basis of the first urate oxidase mutant. A third urate oxidase mutant further comprises an amino acid substitution at position 112 on the basis of the second urate oxidase mutant. Further provided are urate oxidase mutants that comprise additional amino acid substitutions at other positions on the basis of the three urate oxidase mutants. The urate oxidase mutants with high activity, a high degree of humanization, and low immunogenicity maintain a high homology to the theoretical amino acid sequence of the human urate oxidase, while retaining a high activity, thereby meeting the clinical need for low immunogenicity. The urate oxidase mutants can be used for preparing a drug for treating hyperuricemia and gout.

Description

A highly active, highly humanized, and low immunogenic uricase mutant Technical Field

[0001] This invention belongs to the field of biotechnology and relates to modified uricase, particularly to a uricase mutant with high activity, high humanization rate, and low immunogenicity. Background Technology

[0002] The main clinical treatments for hyperuricemia and gout include allopurinol, xanthine oxidase inhibitors, benzbromarone, probenecid, and colchicine. These drugs generally carry multi-organ toxicity risks, severely limiting their use. Uric acid oxidase, a key enzyme in the final stage of purine metabolism, catalyzes the conversion of uric acid into the more soluble allantoin, exhibiting a more desirable uric acid-lowering effect. Currently, recombinant uricases approved clinically for the treatment of hyperuricemia are mainly derived from heterologous species, such as Aspergillus flavus and pig-baboon chimeras. Due to their heterologous nature, they often cause severe allergic reactions in patients. Although PEG-modified uricase shows improvements in half-life and stability, patients may still develop anti-drug antibodies (ADAs) and experience infusion reactions, limiting its widespread clinical application. Therefore, it is primarily used to treat acute, severe, and life-threatening refractory hyperuricemia. Immunogenicity has become a major limitation in the application of uricase. Developing uricases with low immunogenicity has become a hot research area in recombinant uricase modification.

[0003] Due to complex missense mutations and accumulation during evolution, the human urate oxidase gene has become a pseudogene. Although short peptides of urate oxidase have been found in human sweat glands, they do not exhibit urate oxidase activity. JIANG et al. reported obtaining a therapeutic urate oxidase with low immunogenicity by resurrecting the human-derived urate oxidase pseudogene (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.). This literature shows that by performing multiple sequence alignment analysis on the inactive human uricase gene and mutating 15 highly conserved sites, a "revived" uricase rHU15 with 95.06% amino acid sequence identity with human uricase was obtained, but its specific activity was only 2.30 U / mg. Further mutations at more conserved sites yielded a mutant rHU19 with 19 sites (resulting in 93.75% amino acid sequence identity with human uricase), achieving a specific activity of 8.29 U / mg. This literature did not test the immunogenicity of the mutant. Other literature reports that even baboon uricase with 93.75% amino acid sequence identity with human uricase may still exhibit significant immunogenicity (Reference 2: Xiong Runsong. Cloning and Molecular Modification of Baboon Uric Acid Oxidase Gene [D]. Beijing University of Chemical Technology, 2012.). Compared to baboon uricase, rHU19 has lower sequence identity, suggesting that rHU19 is more similar to heterologous uricase than human uricase, and therefore still exhibits significant immunogenicity. Because the human uricase pseudogene is revived through reversion mutations at all highly conserved sites, it is essentially dehumanized, increasing the heterologity of the revived enzyme and thus increasing immunogenicity.

[0004] Human uricase, a protein that never existed in the body as a complete protein molecule during human development, presents a dual challenge in its reactivation strategy: First, restoring human uricase activity through mutation analysis of the conservation of amino acid sequences of isoenzymes from different species actually involves dehumanization of the protein amino acid sequence, and while repairing conserved sites restores enzyme activity, it also enhances immunogenicity; Second, when the humanization rate of the amino acid sequence reaches a certain threshold (e.g., 95%), further increasing the humanization rate leads to a significant decrease in enzyme activity, or even loss of activity.

[0005] Therefore, the current strategy of functional reactivation of human uricase pseudogenes through homology comparison and conserved amino acid mutations faces challenges and difficulties. It is necessary to find new strategies to obtain human uricase reactivation mutants with high activity and high amino acid sequence humanization rate. Summary of the Invention

[0006] The primary objective of this invention is to provide a uricase mutant with high activity, high humanization rate, and low immunogenicity. This uricase mutant maintains high homology with the theoretical amino acid sequence of human uricase, while also maintaining high activity and exhibiting low immunogenicity.

[0007] The first highly active, highly humanized, and low immunogenic uricase mutant provided by this invention is a human uricase mutant obtained by mutation of inactive human uricase with the amino acid sequence SEQ ID NO. 1, which has amino acid substitutions at positions 83, 119, 121, 151, 222, 232, 233, 240, and 252.

[0008] Preferably, the amino acid substitution at position 83 is glycine (G) replacing glutamic acid (E), the amino acid substitution at position 119 is arginine (R) replacing histidine (H), the amino acid substitution at position 121 is glutamic acid (E) replacing glycine (G), the amino acid substitution at position 151 is proline (P) replacing glutamine (Q), the amino acid substitution at position 222 is phenylalanine (F) replacing serine (S), the amino acid substitution at position 232 is serine (S) replacing leucine (L), the amino acid substitution at position 233 is proline (P) replacing threonine (T), the amino acid substitution at position 240 is tyrosine (Y) replacing cysteine ​​(C), and the amino acid substitution at position 252 is glutamic acid (E) replacing alanine (A); the amino acid sequence of the mutated uricase mutant is SEQ ID NO.2.

[0009] The second highly active, highly humanized, and low immunogenic uricase mutant provided by this invention is based on the first uricase mutant and has amino acid substitutions at positions 208 and 219.

[0010] Preferably, the amino acid substitution at position 208 is glutamic acid (E) replacing lysine (K) and the amino acid substitution at position 219 is leucine (L) replacing methionine (M); the amino acid sequence of the mutated uricase mutant is SEQ ID NO.3.

[0011] The third highly active, highly humanized, and low immunogenic uricase mutant provided by this invention is based on the second uricase mutant and has a substitution at amino acid position 112.

[0012] Preferably, the amino acid substitution at position 112 is valine (V) replacing methionine (M); the amino acid sequence of the mutated uricase mutant is SEQ ID NO.4.

[0013] The fourth uricase mutant with high activity, high humanization rate, and low immunogenicity provided by this invention is based on the first uricase mutant and has an additional amino acid substitution at position 84.

[0014] Preferably, the 84th amino acid substitution is to replace isoleucine (I) with cysteine ​​(C), histidine (H), asparagine (N), or arginine (R), and the amino acid sequence of the mutated uricase mutant is SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO.8.

[0015] The fifth highly active, highly humanized, and low immunogenic uricase mutant provided by this invention is based on the first uricase mutant and has an additional amino acid substitution at position 85.

[0016] Preferably, the 85th amino acid substitution is cysteine ​​(C) replacing lysine (K); the amino acid sequence of the mutated uricase mutant is SEQ ID NO. 9.

[0017] The sixth highly active, highly humanized, and low immunogenic uricase mutant provided by this invention is based on the first uricase mutant and has an additional amino acid substitution at position 86.

[0018] Preferably, the 86th amino acid substitution is a substitution of serine (S) with cysteine ​​(C) or aspartic acid (D); the amino acid sequences of the mutated uricase mutant are SEQ ID NO. 10 and SEQ ID NO. 11.

[0019] The seventh highly active, highly humanized, and low immunogenic uricase mutant provided by this invention is based on the second uricase mutant described above, and also has a substitution at the 84th amino acid position.

[0020] Preferably, the 84th amino acid substitution is to replace isoleucine (I) with cysteine ​​(C), histidine (H), asparagine (N), or arginine (R); the amino acid sequence of the mutated uricase mutant is SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, or SEQ ID NO.15.

[0021] The eighth highly active, highly humanized, and low immunogenic uricase mutant provided by this invention is based on the second uricase mutant described above, and also has an 85th amino acid substitution.

[0022] Preferably, the 85th amino acid substitution is cysteine ​​(C) replacing lysine (K); the amino acid sequence of the mutated human uricase mutant is SEQ ID NO. 16.

[0023] The ninth highly active, highly humanized, and low immunogenic uricase mutant provided by this invention is based on the second uricase mutant described above, and also has a substitution at the 86th amino acid position.

[0024] Preferably, the 86th amino acid substitution is a substitution of serine (S) with cysteine ​​(C) or aspartic acid (D); the amino acid sequences of the mutated uricase mutant are SEQ ID NO. 17 and SEQ ID NO. 18.

[0025] The tenth highly active, highly humanized, and low immunogenic uricase mutant provided by this invention is based on the third uricase mutant described above, and also has a substitution at the 84th amino acid position.

[0026] Preferably, the 84th amino acid substitution is to replace isoleucine (I) with cysteine ​​(C), histidine (H), asparagine (N), or arginine (R); the amino acid sequence of the mutated uricase mutant is SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, or SEQ ID NO.22.

[0027] The eleventh highly active, highly humanized, and low immunogenic uricase mutant provided by this invention is based on the third uricase mutant described above, and also has an 85th amino acid substitution.

[0028] Preferably, the 85th amino acid substitution is cysteine ​​(C) replacing lysine (K); the amino acid sequence of the mutated uricase mutant is SEQ ID NO. 23.

[0029] The twelfth highly active, highly humanized, and low immunogenic uricase mutant provided by this invention is based on the third uricase mutant described above, and also has a substitution at the 86th amino acid position.

[0030] Preferably, the 86th amino acid substitution is a substitution of serine (S) with cysteine ​​(C) or aspartic acid (D); the amino acid sequences of the mutated uricase mutant are SEQ ID NO. 24 and SEQ ID NO. 25.

[0031] A second objective of this invention is to provide a DNA molecule that encodes a highly active, highly humanized, and low-immunogenic uricase mutant as described herein.

[0032] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 2, the nucleotide sequence of the uricase mutant is SEQ ID NO. 26.

[0033] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 3, the nucleotide sequence of the uricase mutant is SEQ ID NO. 27.

[0034] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 4, the nucleotide sequence of the uricase mutant is SEQ ID NO. 28.

[0035] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 5, the nucleotide sequence of the uricase mutant is SEQ ID NO. 29.

[0036] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 6, the nucleotide sequence of the uricase mutant is SEQ ID NO. 30.

[0037] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 7, the nucleotide sequence of the uricase mutant is SEQ ID NO. 31.

[0038] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 8, the nucleotide sequence of the uricase mutant is SEQ ID NO. 32.

[0039] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 9, the nucleotide sequence of the uricase mutant is SEQ ID NO. 33.

[0040] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 10, the nucleotide sequence of the uricase mutant is SEQ ID NO. 34.

[0041] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 11, the nucleotide sequence of the uricase mutant is SEQ ID NO. 35.

[0042] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 12, the nucleotide sequence of the uricase mutant is SEQ ID NO. 36.

[0043] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 13, the nucleotide sequence of the uricase mutant is SEQ ID NO. 37.

[0044] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 14, the nucleotide sequence of the uricase mutant is SEQ ID NO. 38.

[0045] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 15, the nucleotide sequence of the uricase mutant is SEQ ID NO. 39.

[0046] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 16, the nucleotide sequence of the uricase mutant is SEQ ID NO. 40.

[0047] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 17, the nucleotide sequence of the uricase mutant is SEQ ID NO. 41.

[0048] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 18, the nucleotide sequence of the uricase mutant is SEQ ID NO. 42.

[0049] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 19, the nucleotide sequence of the uricase mutant is SEQ ID NO. 43.

[0050] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 20, the nucleotide sequence of the uricase mutant is SEQ ID NO. 44.

[0051] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 21, the nucleotide sequence of the uricase mutant is SEQ ID NO. 45.

[0052] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 22, the nucleotide sequence of the uricase mutant is SEQ ID NO. 46.

[0053] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 23, the nucleotide sequence of the uricase mutant is SEQ ID NO. 47.

[0054] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 24, the nucleotide sequence of the uricase mutant is SEQ ID NO. 48.

[0055] When the amino acid sequence of the uricase mutant described in this invention is SEQ ID NO. 25, the nucleotide sequence of the uricase mutant is SEQ ID NO. 49.

[0056] A third object of the present invention is to provide a carrier containing the DNA molecule described herein.

[0057] A fourth object of the present invention is to provide a host cell containing the DNA molecule described in the present invention, or containing the vector described in the present invention.

[0058] Both the aforementioned vectors and host cells can be prepared using techniques known in the art.

[0059] The fifth objective of this invention is to provide a method for producing a highly active, highly humanized, and low-immunogenic uricase mutant.

[0060] The method for producing the highly active, highly humanized, and low immunogenic uricase mutant of the present invention includes: culturing the host cells of the present invention under conditions suitable for the expression of human uricase mutants, and isolating the highly active, highly humanized, and low immunogenic uricase mutant from the culture medium.

[0061] When the DNA molecule described in this invention is inserted into the vector with a suitable orientation and the correct reading frame, or is transformed 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 bacteriophages, plasmids, or granules; 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 this invention include viral vectors, plasmids, granules, or oligonucleotides.

[0062] The preferred host of this invention is a prokaryotic system such as Escherichia coli; the preferred protein expression method of this invention is expression by bacteria.

[0063] This invention involves site-directed mutagenesis of the inactive human uricase pseudogene (hUOX gene). The GENBANK accession number for the human uricase pseudogene sequence is NR_003927.2. By changing the terminators at positions 33 and 187 to R, the amino acid sequence of the inactive human uricase (SEQ ID NO. 1) is obtained.

[0064] Based on SEQ ID NO.1, the inventors carried out reduction modifications to obtain the first uricase mutant mut1 (SEQ ID NO.2), the second uricase mutant mut2 (SEQ ID NO.3), and the third uricase mutant mut3 (SEQ ID NO.4). Their homology with the theoretical amino acid sequence of human uricase is 96.38%, 95.72%, and 95.39%, respectively, and their specific activities are 8.19 U / mg, 7.32 U / mg, and 7.15 U / mg, respectively. They have a higher humanization rate than the uricases reported in the literature. While breaking the 95% homology threshold reported in the literature, they also show higher enzyme activity, and are therefore more suitable for developing human uricases for clinical treatment and drugs for treating hyperuricemia and gout in clinical practice.

[0065] Based on the first uricase mutant mut1, the inventors obtained a fourth uricase mutant through site-directed mutagenesis that maintains good activity and a high degree of humanization even after immunoattenuation design. This fourth mutant has four substitutions at amino acid position 84, namely rhUOX. mut1 / I84C (SEQ ID NO. 5), rhUOX mut1 / I84H (SEQ ID NO. 6), rhUOX mut1 / I84N (SEQ ID NO. 7) and rhUOX mut1 / I84R(SEQ ID NO. 8). They showed 96.05% homology with the theoretical amino acid sequence of human uricase, exhibiting a higher humanization rate than previously reported uricases. Their specific activities were 7.998 U / mg, 7.254 U / mg, 7.439 U / mg, and 7.922 U / mg, respectively, exceeding the 95% homology threshold reported in the literature while demonstrating high enzyme activity. Experiments showed that they possessed low immunogenicity, similar to that of human serum albumin (HSA). This is because the hotspot amino acids of the HSA protein surface antigen peptide were further mutated, rendering them unrecognizable and thus reducing immunogenicity.

[0066] Based on the first uricase mutant mut1, the inventors obtained a fifth uricase mutant with good activity and a high degree of humanization after site-directed mutagenesis, which was designed to be immunoattenuated. This fifth mutant has a substitution at amino acid position 85, namely rhUOX. mut1 / K85C (SEQ ID NO. 9). This mutant shares 96.05% homology with the theoretical amino acid sequence of human uricase, exhibiting a higher humanization rate than previously reported uricases. Its specific activity is 7.766 U / mg, exceeding the 95% homology threshold reported in the literature while demonstrating high enzyme activity. Experiments show that this mutant has low immunogenicity, similar to that of human serum albumin (HSA). This is because the hotspot amino acids of its protein surface antigen peptide are further mutated, rendering them unrecognizable and thus reducing immunogenicity.

[0067] Based on the first uricase mutant mut1, the inventors obtained a sixth uricase mutant through site-directed mutagenesis that maintains good activity and a high degree of humanization even after immunoattenuation design. This sixth mutant has two substitutions at amino acid position 86, namely rhUOX. mut1 / S86C (SEQ ID NO. 10) and rhUOX mut1 / S86D (SEQ ID NO. 11) These enzymes share 96.05% homology with the theoretical amino acid sequence of human uricase, exhibiting a higher humanization rate than previously reported uricases. Their specific activities are 7.609 U / mg and 8.25 U / mg, respectively, exceeding the 95% homology threshold reported in the literature while demonstrating high enzyme activity. Experiments show that they possess low immunogenicity, similar to that of human serum albumin (HSA). This is because the hotspot amino acids of the HSA protein surface antigen peptide are further mutated, rendering them unrecognizable and thus reducing immunogenicity.

[0068] Based on the second uricase mutant mut2, the inventors obtained a seventh uricase mutant through site-directed mutagenesis that maintains good activity and a high degree of humanization even after immunoattenuation design. This seventh mutant has four substitutions at amino acid position 84, namely rhUOX. mut2 / I84C (SEQ ID NO. 12), rhUOX mut2 / I84H (SEQ ID NO. 13), rhUOX mut2 / I84N (SEQ ID NO. 14) and rhUOX mut2 / I84R (SEQ ID NO. 15). They showed 95.39% homology with the theoretical amino acid sequence of human uricase, exhibiting a higher humanization rate than previously reported uricases. Their specific activities were 7.472 U / mg, 7.591 U / mg, 7.388 U / mg, and 7.615 U / mg, respectively, exceeding the 95% homology threshold reported in the literature while demonstrating high enzyme activity. Experiments showed that they possessed low immunogenicity, similar to that of human serum albumin (HSA). This is because the hotspot amino acids of the HSA protein surface antigen peptide were further mutated, rendering them unrecognizable and thus reducing immunogenicity.

[0069] Based on the second uricase mutant mut2, the inventors obtained an eighth uricase mutant with good activity and a high degree of humanization after immunoattenuation design through site-directed mutagenesis. This eighth mutant has a substitution at amino acid position 85, namely rhUOX. mut2 / K85C (SEQ ID NO. 16). This mutant shares 95.39% homology with the theoretical amino acid sequence of human uricase, exhibiting a higher humanization rate than previously reported uricases. Its specific activity is 7.685 U / mg, exceeding the 95% homology threshold reported in the literature while demonstrating high enzyme activity. Experiments show that this mutant has low immunogenicity, similar to that of human serum albumin (HSA). This is because the hotspot amino acids of its protein surface antigen peptide are further mutated, rendering them unrecognizable and thus reducing immunogenicity.

[0070] Based on the second uricase mutant mut2, the inventors obtained a ninth uricase mutant through site-directed mutagenesis that maintains good activity and a high degree of humanization even after immunoattenuation design. This ninth mutant has two substitutions at amino acid position 86, namely rhUOX. mut2 / S86C (SEQ ID NO. 17) and rhUOX mut2 / S86D(SEQ ID NO. 18) These enzymes share 95.39% homology with the theoretical amino acid sequence of human uricase, exhibiting a higher humanization rate than previously reported uricases. Their specific activities are 7.85 U / mg and 7.64 U / mg, respectively, exceeding the reported 95% homology threshold while demonstrating high enzyme activity. Experiments show that they possess low immunogenicity, similar to that of human serum albumin (HSA). This is attributed to further mutations in the hotspot amino acids of the HSA protein surface antigen peptide, rendering it unrecognizable and thus reducing immunogenicity.

[0071] Based on the third uricase mutant mut3, the inventors obtained a tenth uricase mutant through site-directed mutagenesis that maintains good activity and a high degree of humanization even after immunoattenuation design. This tenth mutant has four substitutions at amino acid position 84, namely rhUOX. mut3 / I84C (SEQ ID NO. 19), rhUOX mut3 / I84H (SEQ ID NO. 20), rhUOX mut3 / I84N (SEQ ID NO. 21) and rhUOX mut3 / I84R (SEQ ID NO. 22). They showed 95.06% homology with the theoretical amino acid sequence of human uricase, and their specific activities were 7.36 U / mg, 7.55 U / mg, 7.41 U / mg, and 7.47 U / mg, respectively, exceeding the reported 95% homology threshold while exhibiting high enzyme activity. Experiments showed that they possessed low immunogenicity, similar to that of human serum albumin (HSA). This is because the hotspot amino acids of the HSA protein surface antigen peptide were further mutated, rendering them unrecognizable and thus reducing immunogenicity.

[0072] Based on the third uricase mutant mut3, the inventors obtained an eleventh uricase mutant through site-directed mutagenesis that maintains good activity and a high degree of humanization even after immunoattenuation design. This eleventh mutant has a substitution at amino acid position 85, namely rhUOX. mut3 / K85C (SEQ ID NO. 23). This mutant shares 95.06% homology with the theoretical amino acid sequence of human uricase and exhibits a specific activity of 7.825 U / mg, exceeding the reported 95% homology threshold while demonstrating high enzyme activity. Experiments show that this mutant possesses low immunogenicity, similar to that of human serum albumin (HSA). This is because the hotspot amino acids of its protein surface antigen peptide are further mutated, rendering them unrecognizable and thus reducing immunogenicity.

[0073] Based on the third uricase mutant mut3, the inventors obtained a twelfth uricase mutant through site-directed mutagenesis that maintains good activity and a high degree of humanization even after immunoattenuation design. This twelfth mutant has two substitutions at amino acid position 86, namely rhUOX. mut3 / S86C (SEQ ID NO. 24) and rhUOX mut3 / S86D (SEQ ID NO. 25) These enzymes share 95.06% homology with the theoretical amino acid sequence of human uricase oxidase, and exhibit specific activities of 7.72 U / mg and 7.95 U / mg, respectively, exceeding the reported 95% homology threshold while demonstrating high enzyme activity. Experiments show that they possess low immunogenicity, similar to that of human serum albumin (HSA). This is because the hotspot amino acids of the HSA protein surface antigen peptide are further mutated, rendering them unrecognizable and thus reducing immunogenicity.

[0074] In summary, the highly active, highly humanized, and low-immunogenic uricase mutant of the present invention maintains high homology with the theoretical amino acid sequence of human uricase while maintaining high activity, which better meets the clinical needs of low-immunogenic uricase. It is suitable for long-term clinical use in the treatment of hyperuricemia and gout, and can be used to prepare drugs for the treatment of hyperuricemia and gout. Attached Figure Description

[0075] Figure 1 is an SDS-PAGE electrophoresis diagram of the human uricase mutant protein described in this invention, wherein 1: rhUOX wt ; 2: rHU15 mutant; 3: mut1 mutant; 4: mut2 mutant; 5: mut3 mutant; M is Marker.

[0076] Figure 2 shows the rhUOX described in this invention. wt The uric acid degradation activities of rHU15, mut1, mut2 and mut3.

[0077] Figure 3 is an SDS-PAGE protein electrophoresis image of the human uricase mutant described in this invention, wherein 1: rhUOX wt ;2:rhUOX mut1 / I84C Mutant; 3: rhUOX mut1 / I84H Mutant; 4: rhUOX mut1 / I84N Mutant; 5: rhUOX mut1 / I84R Mutant; M stands for Marker.

[0078] Figure 4 shows the human uricase mutant rhUOX described in this invention. wt rhUOX mut1 / I84C rhUOX mut1 / I84HrhUOX mut1 / I84N rhUOX mut1 / I84R Its activity in degrading uric acid.

[0079] Figure 5 shows the human uricase mutant rhUOX described in this invention. mut1 / I84C rhUOX mut1 / I84H rhUOX mut1 / I84N and rhUOX mut1 / I84R The results of immunogenicity assays of human mononuclear cells containing uricase (AfUOX) and human serum albumin (HAS) derived from Aspergillus flavus.

[0080] Figure 6 shows the human uricase mutant rhUOX described in this invention. mut1 / I84C rhUOX mut1 / I84H rhUOX mut1 / I84N and rhUOX mut1 / I84R The in vivo efficacy results are as follows: A: efficacy results in male mice; B: efficacy results in female mice.

[0081] Figure 7 is an SDS-PAGE protein electrophoresis diagram of the human uricase mutant described in this invention, wherein 1: rhUOX wt ;3:rhUOX mut1 / K85C M stands for Marker.

[0082] Figure 8 shows the human uricase mutant rhUOX described in this invention. wt rhUOX mut1 / K85C Its activity in degrading uric acid.

[0083] Figure 9 shows the human uricase mutant rhUOX described in this invention. mut1 / K85C The results of immunogenicity assays of human mononuclear cells containing uricase (AfUOX) and human serum albumin (HAS) derived from Aspergillus flavus.

[0084] Figure 10 shows the human uricase mutant rhUOX described in this invention. mut1 / K85C The in vivo efficacy results are as follows: A: efficacy results in male mice; B: efficacy results in female mice.

[0085] Figure 11 is an SDS-PAGE protein electrophoresis diagram of the human uricase mutant described in this invention, wherein 1: rhUOX wt ;2:rhUOX mut1 / S86C ;3:rhUOX mut1 / S86D M stands for Marker.

[0086] Figure 12 shows the human uricase mutant rhUOX described in this invention. wtrhUOX mut1 / S86C and rhUOX mut1 / S86D Its activity in degrading uric acid.

[0087] Figure 13 shows the human uricase mutant rhUOX described in this invention. mut1 / S86C and rhUOX mut1 / S86D The results of immunogenicity assays of human mononuclear cells containing uricase (AfUOX) and human serum albumin (HAS) derived from Aspergillus flavus.

[0088] Figure 14 shows the human uricase mutant rhUOX described in this invention. mut1 / S86C and rhUOX mut1 / S86D The in vivo efficacy results are as follows: A: efficacy results in male mice; B: efficacy results in female mice.

[0089] Figure 15 is an SDS-PAGE protein electrophoresis diagram of the human uricase mutant described in this invention, wherein 1: rhUOX wt ;2:rhUOX mut2 / I84C Mutant; 3: rhUOX mut2 / I84H Mutant; 4: rhUOX mut2 / I84N Mutant; 5: rhUOX mut2 / I84R Mutant; M stands for Marker.

[0090] Figure 16 shows the human uricase mutant rhUOX described in this invention. wt rhUOX 14 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N rhUOX mut2 / I84R Its activity in degrading uric acid.

[0091] Figure 17 shows the human uricase mutant rhUOX described in this invention. mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N and rhUOX mut2 / I84R The results of immunogenicity assays of human mononuclear cells containing uricase (AfUOX) and human serum albumin (HAS) derived from Aspergillus flavus.

[0092] Figure 18 shows the human uricase mutant rhUOX described in this invention. mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N and rhUOX mut2 / I84R The in vivo efficacy results are as follows: A: efficacy results in male mice; B: efficacy results in female mice.

[0093] Figure 19 is an SDS-PAGE electrophoresis diagram of the human uricase mutant protein described in this invention, wherein 1: rhUOX wt ;2:rhUOX mut2 / K85C M stands for Marker.

[0094] Figure 20 shows the human uricase mutant rhUOX described in this invention. wt rhUOX mut2 / K85C Its activity in degrading uric acid.

[0095] Figure 21 shows the human uricase mutant rhUOX described in this invention. mut2 / K85C The results of immunogenicity assays of human mononuclear cells containing uricase (AfUOX) and human serum albumin (HAS) derived from Aspergillus flavus.

[0096] Figure 22 shows the human uricase mutant rhUOX. mut2 / K85C The in vivo efficacy results are as follows: A: efficacy results in male mice; B: efficacy results in female mice.

[0097] Figure 23 is an SDS-PAGE electrophoresis diagram of the human uricase mutant protein described in this invention, wherein 1: rhUOX wt ;2:rhUOX mut2 / S86C ;3:rhUOX mut2 / S86D M stands for Marker.

[0098] Figure 24 shows the human uricase mutant rhUOX described in this invention. wt rhUOX mut2 / S86C and rhUOX mut2 / S86D Its activity in degrading uric acid.

[0099] Figure 25 shows the human uricase mutant rhUOX described in this invention. mut2 / S86C and rhUOX mut2 / S86D The results of immunogenicity assays of human mononuclear cells containing uricase (AfUOX) and human serum albumin (HAS) derived from Aspergillus flavus.

[0100] Figure 26 shows the human uricase mutant rhUOX described in this invention. wt rhUOX mut2 / S86C and rhUOX mut2 / S86D The in vivo efficacy results are as follows: A: efficacy results in male mice; B: efficacy results in female mice.

[0101] Figure 27 is an SDS-PAGE electrophoresis diagram of the human uricase mutant protein described in this invention, 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 stands for Marker.

[0102] Figure 28 shows the human uricase mutant rhUOX described in this invention. wt rhUOX mut3 / I84C rhUOX mut3 / I84H rhUOX mut3 / I84N rhUOX mut3 / I84R Its activity in degrading uric acid.

[0103] Figure 29 shows the human uricase mutant rhUOX described in this invention. mut3 / I84C rhUOX mut3 / I84H rhUOX mut3 / I84N and rhUOX mut3 / I84R The results of immunogenicity assays of human mononuclear cells containing uricase (AfUOX) and human serum albumin (HAS) derived from Aspergillus flavus.

[0104] Figure 30 shows the human uricase mutant rhUOX. wt rhUOX mut3 / I84C rhUOX mut3 / I84H rhUOX mut3 / I84N rhUOX mut3 / I84R The in vivo efficacy results are as follows: A: efficacy results in male mice; B: efficacy results in female mice.

[0105] Figure 31 is an SDS-PAGE electrophoresis diagram of the human uricase mutant protein described in this invention, wherein 1: rhUOX wt ;2:rhUOX mut3 / K85C M stands for Marker.

[0106] Figure 32 shows the human uricase mutant rhUOX described in this invention. wt rhUOX mut3 / K85C Its activity in degrading uric acid.

[0107] Figure 33 shows the human uricase mutant rhUOX described in this invention. mut3 / K85C The results of immunogenicity assays of human mononuclear cells containing uricase (AfUOX) and human serum albumin (HAS) derived from Aspergillus flavus.

[0108] Figure 34 shows the human uricase mutant rhUOX described in this invention. wt rhUOX mut3 / K85CThe in vivo efficacy results are as follows: A: efficacy results in male mice; B: efficacy results in female mice.

[0109] Figure 35 is an SDS-PAGE electrophoresis diagram of the human uricase mutant protein described in this invention, wherein 1: rhUOX wt ;2:rhUOX mut3 / S86C ;3:rhUOX mut3 / S86D M stands for Marker.

[0110] Figure 36 shows the human uricase mutant rhUOX described in this invention. wt rhUOX mut3 / S86C and rhUOX mut3 / S86D Its activity in degrading uric acid.

[0111] Figure 37 shows the human uricase mutant rhUOX described in this invention. mut3 / S86C and rhUOX mut3 / S86D The results of immunogenicity assays of human mononuclear cells containing uricase (AfUOX) and human serum albumin (HAS) derived from Aspergillus flavus.

[0112] Figure 38 shows the human uricase mutant rhUOX described in this invention. wt rhUOX mut3 / S86C and rhUOX mut3 / S86D The in vivo efficacy results are as follows: A: efficacy results in male mice; B: efficacy results in female mice. Embodiments of the present invention

[0113] Unless otherwise stated, the terms used herein have the meanings commonly understood by those skilled in the art. Definitions of some specific terms used in this invention are provided below.

[0114] rhUOX wt "This indicates the inactive human uricase oxidase whose pseudogenes at positions 33 and 187 have been restored to arginine (R). The gene is represented by the italicized "rhuox" wt "indicates". rhUOX wt The preferred amino acid sequence is SEQ ID NO.1.

[0115] “rHU15” indicates rhUOX wt For the control of human uricase reactivated by amino acid mutation, see reference 1 by JIANG et al., whose gene is represented by italicized “rhu15”.

[0116] “mut1” or “rhUOX” mut1"" indicates the first uric acid oxidase mutant described in this invention, namely mutant mut1, whose gene is represented by italicized "mut1" or "rhuox". mut1 The amino acid sequence of mutant mut1 is preferably SEQ ID NO.2.

[0117] "mut2" or "rhUOX" mut2 This indicates the second uric acid oxidase mutant described in this invention, namely mutant mut2, whose gene is indicated by italics "mut2" or "rhuox". mut2 The amino acid sequence of the mutant mut2 is preferably SEQ ID NO.3.

[0118] “mut3” or “rhUOX” mut3 This indicates the third uric acid oxidase mutant described in this invention, namely the mut3 mutant, whose gene is represented by italicized "mut3" or "rhuox". mut3 The amino acid sequence of the mutant mut3 is preferably SEQ ID NO.4.

[0119] rhUOX mut1 / I84C rhUOX mut1 / I84H rhUOX mut1 / I84N and rhUOX mut1 / I84R "" indicates the four types of the fourth type of uric acid oxidase mutant described in this invention, whose genes are in italics "rhuox mut1 / I84C rhuox mut1 / I84H rhuox mut1 / I84N rhuox mut1 / I84R "indicates". rhUOX mut1 / I84C rhUOX mut1 / I84H rhUOX mut1 / I84N and rhUOX mut1 / I84R The preferred amino acid sequences of the mutants are SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0120] rhUOX mut1 / K85C This indicates the fifth uric acid oxidase mutant described in this invention, whose gene is represented in italics as "rhuoxmut1 / K85C". mut1 / K85C The amino acid sequence of the mutant is SEQ ID NO.9.

[0121] rhUOX mut1 / S86C rhUOX mut1 / S86DThis indicates two types of the sixth type of uric acid oxidase mutant described in this invention, whose genes are represented in italics as "rhuoxmut1 / S86C" and "rhuoxmut1 / S86D". mut1 / S86C The amino acid sequence of the mutant is SEQ ID NO.10, rhUOX mut1 / S86D The amino acid sequence of the mutant is SEQ ID NO.11.

[0122] rhUOX mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N and rhUOX mut2 / I84R "" indicates the four types of the seventh type of uric acid oxidase mutant described in this invention, whose genes are in italics "rhuox mut2 / I84C rhuox mut2 / I84H rhuox mut2 / I84N rhuox mut2 / I84R "indicates". rhUOX mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N and rhUOX mut2 / I84R The amino acid sequences of the mutants are SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively.

[0123] rhUOX mut2 / K85C "" indicates the eighth uric acid oxidase mutant described in this invention, whose gene is in italics "rhuox mut2 / K85C "indicates". rhUOX mut2 / K85C The amino acid sequences of the mutants are SEQ ID NO.16.

[0124] rhUOX mut2 / S86C rhUOX mut2 / S86D This indicates two types of the ninth type of uric acid oxidase mutant described in this invention, whose genes are represented by italics "rhuox". mut2 / S86C rhuox mut2 / S86D "indicates". rhUOX mut2 / S86C The amino acid sequence of the mutant is SEQ ID NO.17, rhUOX mut2 / S86D The amino acid sequence of the mutant is SEQ ID NO.18.

[0125] rhUOX mut3 / I84C rhUOX mut3 / I84H rhUOX mut3 / I84N and rhUOX mut3 / I84R "" indicates the four types of the tenth uric acid oxidase mutant described in this invention, whose genes are in italics "rhuoxmut3 / I84C rhuox mut3 / I84H rhuox mut3 / I84N rhuox mut3 / I84R "indicates". rhUOX mut3 / I84C rhUOX mut3 / I84H rhUOX mut3 / I84N and rhUOX mut3 / I84R The amino acid sequences of the mutants are SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22, respectively.

[0126] rhUOX mut3 / K85C "" indicates the eleventh uric acid oxidase mutant described in this invention, whose gene is in italics "rhuox mut3 / K85C "indicates". rhUOX mut3 / K85C The amino acid sequence of the mutant is SEQ ID NO.23.

[0127] rhUOX mut3 / S86C and rhUOX mut3 / S86D This indicates two types of the twelfth uric acid oxidase mutant described in this invention, whose genes are represented by italics "rhuox". mut3 / S86C rhuox mut3 / S86D "indicates". rhUOX mut3 / S86C The amino acid sequence of the mutant is SEQ ID NO.24, rhUOX. mut3 / S86D The amino acid sequence of the mutant is SEQ ID NO.25.

[0128] Example 1: rhuox wt The synthesis of the rhu15, mut1, mut2 and mut3 genes was transformed into Escherichia coli Rosetta (DE3) and the recombinant protein was induced to express.

[0129] This invention uses a human uricase pseudogene sequence (GenBank accession number NR_003927.2) to modify and obtain a human uricase mutant, rhuox. wt The mutant genes, rhu15, mut1, mut2, and mut3, were synthesized by BGI Genomics. The genes were cloned between the NcoI and XhoI regions of the pET28a plasmid (Invitrogen), and named pET28a-huox. wt pET28a-rhu15, pET28a-mut1, and pET28a-mut3.

[0130] The recipient bacteria in this experiment was *Escherichia coli* Rosetta (DE3), using pET28a-huox. wt After transformation with recombinant plasmids pET28a-rhu15, pET28a-mut1, and pET28a-mut3, preliminary screening was performed using kanamycin (Kan) resistant plates. Then, single clones from the Kan plates were picked and cultured in 5 mL of LB liquid medium (containing 50 mg / mL Kan) for 12-16 h. The plasmids were then extracted for PCR verification, and positive clones were further screened.

[0131] Positive recombinant proteins were inoculated into 5 mL of LB medium (50 mg / mL Kan) and cultured overnight at 37°C and 200 rpm. The revived bacterial culture was inoculated at a 1% inoculation rate into 500 mL of LB medium (50 mg / mL Kan) and cultured at 37°C and 200 rpm until the OD600 reached 0.6. 60 μM lactose was added, and expression was induced for 12 h at 30°C and 200 rpm. After induction, the bacterial cells were collected and resuspended in pH 10.0 carbonate buffer (1:20 by volume). The cells were then disrupted using an ultrasonic homogenizer, centrifuged at 6000 g for 10 min, and the supernatant was collected to obtain the recombinant protein.

[0132] rhUOX will be obtained from positive clone recombinants wt The rHU15, mut1, mut2, and mut3 mutant recombinant proteins were used in experiments in Examples 2 and 3 below.

[0133] Example 2: rhUOX wt SDS-PAGE electrophoresis detection of recombinant proteins with rHU15, mut1, mut2 and mut3 mutants

[0134] The SDS-PAGE electrophoresis detection in this embodiment includes the following steps:

[0135] (1) Prepare 10 mL of 10% separating gel, mix well and pour the gel into the glass plate with a micropipette until it is 2-3 cm away from the top edge of the short glass plate. Then seal the gel surface with distilled water. You can 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 use filter paper to absorb the excess water.

[0136] (2) Prepare 4 mL of 5% concentrated gel and pour it evenly onto the separating gel. Insert a comb of the appropriate size while avoiding air bubbles. Polymerize for 30 min until the gel solidifies.

[0137] (3) Set up the electrophoresis tank, fill the tank with electrophoresis solution, the volume of which should be greater than half the volume of the electrophoresis tank. Transfer the prepared gel into the electrophoresis tank and carefully remove the comb.

[0138] (4) Spot the sample sequentially, and the amount of sample should not be too much. 15 μL per well is appropriate.

[0139] (5) When starting electrophoresis, set the voltage to 90 V for gel running. When the indicator reaches the stacking gel, change the voltage to 120 V to continue electrophoresis. Stop electrophoresis when the target band reaches the middle position.

[0140] (6) Carefully peel off the gel, stain with Coomassie Brilliant Blue R-250 for 30 min, and then decolorize with destaining solution until the background is light and the protein bands are clear.

[0141] (7) Gel imaging and observation of results.

[0142] The SDS-PAGE protein electrophoresis results are shown in Figure 1. Lane 1 contains denatured rhUOX. wt Protein samples were collected in lanes 2, 3, 4, and 5. Lane 2 contained the target protein sample of mutant rHU15, lane 3 contained the target protein sample of mutant mut1, lane 4 contained the target protein sample of mutant mut2, and lane 5 contained the target protein sample of mutant mut3. The target protein was approximately 34 kDa, indicating that the target gene was successfully expressed in *E. coli* Rosetta and can be used for subsequent experiments.

[0143] Example 3: rhUOX wt Characterization of the active properties of recombinant proteins with rHU15, mut1, mut2 and mut3 mutants

[0144] At 37℃, 20 μL of purified protein of appropriate concentration was added to 600 μL of 0.1 M uric acid solution (pH 8.0). After reacting for 10 min, an equal volume of pure methanol was added to terminate the reaction. Enzyme activity was calculated by measuring the decrease in UV absorbance at 293 nm using UV spectrophotometry. The control group consisted of inactivated uricase. The experimental results are shown in Figure 2.

[0145] Enzyme activity unit definition: The amount of enzyme required to catalyze the oxidation of 1 µmol of uric acid per minute is defined as 1 unit.

[0146] E (U / mg) = (△OD) 293 ×V1×D) / (12.3×V2×T×C)

[0147] In the formula, 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.

[0148] Regarding the number, location, homology, and specific enzyme activity of point mutations, a comparison of the urate oxidase mutant described in this invention with references 1 and 2 mentioned in the "Background Art" section is shown in Table 1 below:

[0149] Table 1:

[0150]

[0151] As shown in Table 1, the mutants mut1 (SEQ ID NO.2), mut2 (SEQ ID NO.3), and mut3 (SEQ ID NO.4) described in this invention share 96.38%, 95.72%, and 95.39% homology with the theoretical amino acid sequence of human uricase, respectively. The specific activities of each mutant are 8.19 U / mg, 7.32 U / mg, and 7.15 U / mg, respectively, maintaining high activity while reducing the number of mutation sites. These uricase mutants exhibit higher humanization rates and activities than rHU15 (Reference 1), exceeding the reported 95% homology threshold while displaying higher enzyme activity. Therefore, they are more suitable for long-term clinical use in treating hyperuricemia and gout, and can be used to prepare drugs for treating hyperuricemia and gout.

[0152] Example 4: rhuox wt rhuox mut1 rhuox mut1 / I84C rhuox mut1 / I84H rhuox mut1 / I84N rhuox mut1 / I84R Gene synthesis was performed, and the recombinant protein was induced to be expressed in E. coli Rosetta (DE3).

[0153] This invention uses the human uricase pseudogene sequence rhuox. wt (GenBank accession number NR_003927.2), the human uricase mutant rhuox was obtained through modification. mut1 / I84C rhuox mut1 / I84H rhuox mut1 / I84N rhuox mut1 / I84R The aforementioned gene was synthesized by BGI Genomics Co., Ltd. and cloned between NcoI and XhoI in the pET28a plasmid (Invitrogen). The recombinant plasmid was named pET28a-huox. wt pET28a-huox mut1 / I84C pET28a-huox mut1 / I84H pET28a-huox mut1 / I84N and pET28a-huox mut1 / I84R .

[0154] The recipient bacteria in this experiment was *Escherichia coli* Rosetta (DE3), using pET28a-huox. wt pET28a-huox mut1 / I84CpET28a-huox mut1 / I84H pET28a-huox mut1 / I84N and pET28a-huox mut1 / I84R After transformation of the recombinant plasmid, preliminary screening was performed using kanamycin (Kan) resistant plates. Then, single clones from the Kan plates were picked and cultured in 5 mL of LB liquid medium (containing 50 mg / mL Kan) for 12-16 h. The plasmids were then extracted for PCR verification, and positive clones were further screened.

[0155] The positive recombinant was inoculated into 5 mL of LB medium (50 mg / mL Kan) and incubated overnight at 37°C and 200 rpm. The revived bacterial culture was then inoculated at 1% into 500 mL of LB medium (50 mg / mL Kan) and incubated at 37°C and 200 rpm until OD reached [value missing]. 600 The concentration was 0.6, 60 μM lactose was added, and expression was induced at 30℃ and 200 rpm for 12 h. After induction, the bacterial cells were collected, resuspended in carbonate buffer (pH 10.0, mass-to-volume ratio 1:20), and the cells were disrupted by sonication. After centrifugation at 6000g for 10 min, the supernatant was collected to obtain the recombinant protein.

[0156] Example 5: rhUOX wt rhUOX mut1 / I84C rhUOX mut1 / I84H rhUOX mut1 / I84N and rhUOX mut1 / I84R SDS-PAGE electrophoresis detection of mutant recombinant proteins

[0157] The SDS-PAGE electrophoresis detection method in this embodiment is the same as in Embodiment 2.

[0158] The SDS-PAGE protein electrophoresis results are shown in Figure 3. Lane 1 contains denatured rhUOX. wt Protein samples, lanes 2-5 contain the mutant rhUOX. mut1 / I84C rhUOX mut1 / I84H rhUOX mut1 / I84N and rhUOX mut1 / I84R The target protein sample. The target protein is approximately 34 kDa in size, indicating that the target gene was successfully expressed in *E. coli* Rosetta and can be used for subsequent experiments.

[0159] Example 6: rhUOX mut1 / I84C rhUOX mut1 / I84H rhUOX mut1 / I84N and rhUOX mut1 / I84R Characterization of the active substances of mutants

[0160] At 37°C, take 20 μL of the purified protein (rhUOX). mut1 / I84C rhUOX mut1 / I84H rhUOX mut1 / I84N and rhUOX mut1 / I84R In 600 μL of 0.1 M uric acid solution (pH 8.0), the reaction was stopped by adding an equal volume of pure methanol after 10 min. Enzyme activity was calculated by measuring the decrease in UV absorbance at 293 nm using UV spectrophotometry. The control group consisted of inactivated uric acid oxidase. The experimental results are shown in Figure 4. mut1 / I84C rhUOX mut1 / I84H rhUOX mut1 / I84N and rhUOX mut1 / I84R The specific activities of the enzymes were 7.998 U / mg, 7.254 U / mg, 7.439 U / mg and 7.922 U / mg, respectively, and these mutants all had high activity.

[0161] Enzyme activity unit definition: The amount of enzyme required to catalyze the oxidation of 1 µmol of uric acid per minute is defined as 1 unit.

[0162] E (U / mg) = (△OD) 293 ×V1×D) / (12.3×V2×T×C)

[0163] In the formula, 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.

[0164] Example 7: rhUOX mut1 / I84C rhUOX mut1 / I84H rhUOX mut1 / I84N and rhUOX mut1 / I84R Immunogenicity analysis of human PBMC cells with mutants

[0165] Human serum albumin (HAS) and aflatoxin-derived uricase (AfUOX) were used as control groups, while rhUOX was used as the control group. mut1 / I84C rhUOX mut1 / I84H rhUOX mut1 / I84N and rhUOX mut1 / I84R For the experimental group, 50 µg / mL of the above protein and PWM (20 µg / mL) were added to each well of a 24-well cell culture plate at a rate of 100 µL (5 µg), followed by PBMC cell suspension (1×10⁻⁶). 6500 µL per well (dose of 10 µg / mL). Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. After incubation, centrifuge at 3000 rpm for 15 min and collect cells. Add 500 μL of cells to each well of the corresponding protein-coated cell culture plate. Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. Collect the culture supernatant after incubation. Analyze immunogenicity by detecting anti-uricase IgG using indirect ELISA.

[0166] Indirect ELISA steps:

[0167] (1) Antibody coating: Coating the protein samples (HSA, AfUOX, rhUOX) with antibodies. mut1 / I84C rhUOX mut1 / I84H rhUOX mut1 / I84N and rhUOX mut1 / I84R Add 100 µL (5 μg) to each well of a 96-well plate, with two replicates for each sample. Incubate overnight at 4 °C, then wash three times with PBST and pat dry.

[0168] (2) Blocking: Block with ELISA blocking solution for 1 h.

[0169] (3) Washing: Wash 3 times with PBST and pat dry.

[0170] (4) Incubation: Add 100 µL of the supernatant (HSA, AfUOX, rhUOX) obtained from the above cell culture to each well. mut1 / I84C rhUOX mut1 / I84H rhUOX mut1 / I84N and rhUOX mut1 / I84R The blank control was PBS, and the mixture was incubated at 37 °C for 1.5 h.

[0171] (5) Washing: Wash three times with detergent. Pat dry.

[0172] (6) HRP-goat anti-human IgG incubation: Add 100 µL of diluted HRP-goat anti-human IgG (1:3000, PBST, pH7.4) to each well and incubate at 37 °C for 1 h.

[0173] (7) Washing: Wash three times with detergent. Pat dry.

[0174] (8) Colorimetric reaction: Add 100 µL of freshly prepared TMB working solution to each well and incubate at room temperature in the dark for 15 min. Add 50 µL of stop solution (2 M H2SO4) to each well. Read the OD values ​​using a microplate reader. 450value.

[0175] A test well is positive if its A450 is greater than or equal to 2.1 times that of the negative control: P / N=OD 450 of test serum / OD 450 of negative control serum (i.e., P / N≥2.1 is positive).

[0176] The results are shown in Figure 5, the mutant rhUOX mut1 / I84C , rhUOX mut1 / I84H , rhUOX mut1 / I84N and rhUOX mut1 / I84R have low immunogenicity, which is similar to that of HSA.

[0177] Example 8: rhUOX mut1 / I84C , rhUOX mut1 / I84H , rhUOX mut1 / I84N and rhUOX mut1 / I84R Efficacy verification of mutant urate oxidase gene in KO mice in vivo

[0178] C57BL / 6J mice (UOX- / -) were used in the experiment, 48 mice, half male and half female, 5-8 weeks old, 25-30g, SPF grade, purchased from Cyagen Biosciences Inc., housed in the SPF laboratory animal room of Experimental Animal Management Center of Jinan University, room temperature 20-25°C, experimental animal use license: SYXK (Guangdong) 2022-0174. C57BL / 6J mice were given clean drinking water and allowed free access to food before the experiment, and were used for the experiment after 3 days of feeding.

[0179] A normal control group (with normal serum level), a hyperuricemia control group, and experimental groups (rhUOX mut1 / I84C , rhUOX mut1 / I84H , rhUOX mut1 / I84N and rhUOX mut1 / I84R ). The normal control group consisted of wild-type C57BL / 6J mice, and the other groups were tested with urate oxidase knockout KO mice, with 12 mice in each group (6 males and 6 females). The normal control group and the hyperuricemia control group were given an equal volume of normal saline, while the experimental group was administered with reference to the dosage of rasburicase (2U / kg). Administration was via intraperitoneal injection, and blood was collected from the orbital vein at 0, 0.5, 1, 3, and 6h after administration. Serum treatment was the same as described above, and the collected serum was tested for uric acid content by HPLC. The results are shown in Figure 6. In the pharmacodynamic experiment of hyperuricemic KO mice, under the experimental conditions, the immunogenicity-attenuated rhUOX mut1 / I84C , rhUOX mut1 / I84H , rhUOX mut1 / I84N and rhUOX mut1 / I84RIt can maintain the blood uric acid level of KO mice at a normal level for at least 3 hours, and can still exert the effect of lowering uric acid after 6 hours.

[0180] HPLC detection conditions

[0181] 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℃; injection volume: 20 uL; mobile phase: A 0.1% formic acid-water; B pure methanol.

[0182] Regarding the number, location, homology, specific enzyme activity, and immunogenicity of point mutations, the rhUOX urate oxidase mutant described in this invention... mut1 / I84C rhUOX mut1 / I84H rhUOX mut1 / I84N and rhUOX mut1 / I84R A comparison with References 1 and 2 mentioned in the "Background Art" section is shown in Table 2 below.

[0183] Table 2:

[0184]

[0185] As shown in Table 2, the human uricase mutant rhUOX described in this invention... mut1 / I84C rhUOX mut1 / I84H rhUOX mut1 / I84N and rhUOX mut1 / I84R The homology of the amino acid sequence with human uricase was 96.05%. The specific activities of the mutants were 8.098 U / mg, 8.254 U / mg, 7.439 U / mg, and 7.922 U / mg, respectively. While reducing the number of mutation sites, they retained high activity and a high humanization rate. These mutants showed higher humanization rates and activities than rHU15 (Reference 1). Experiments showed that these mutants had low immunogenicity, similar to that of HSA, because the hotspot amino acids of their protein surface antigen peptide were further mutated, making them unrecognizable and thus reducing immunogenicity. Therefore, the human uricase mutant rhUOX described in this invention... mut1 / I84C rhUOX mut1 / I84H rhUOX mut1 / I84N and rhUOX mut1 / I84R It better meets the clinical needs of low-immunogenic uricase, is suitable for long-term clinical use in the treatment of hyperuricemia and gout, and can be used to prepare drugs for the treatment of hyperuricemia and gout.

[0186] Example 9: rhuox wtThe synthesis of the rhuoxmut1 / K85C gene was performed, and the recombinant protein was induced to be expressed in E. coli Rosetta (DE3).

[0187] This invention uses the human uricase pseudogene sequence rhuox. wt (GenBank accession number NR_003927.2) The human uricase mutant rhuoxmut1 / K85C was obtained through modification. The above gene was synthesized by BGI Genomics Co., Ltd. and cloned between NcoI and XhoI of the pET28a plasmid (Invitrogen). The recombinant plasmid was named pET28a-huox. wt and pET28a-huoxmut1 / K85C.

[0188] The recipient bacteria in this experiment was *Escherichia coli* Rosetta (DE3), using pET28a-huox. wt After transformation with the pET28a-huoxmut1 / K85C recombinant plasmid, preliminary screening was performed using kanamycin (Kan) resistant plates. Then, single clones from the Kan plates were picked and cultured in 5 mL of LB liquid medium (containing 50 mg / mL Kan) for 12-16 h. The plasmids were then extracted for PCR verification, and positive clones were further screened.

[0189] The positive recombinant was inoculated into 5 mL of LB medium (50 mg / mL Kan) and incubated overnight at 37°C and 200 rpm. The revived bacterial culture was then inoculated at 1% into 500 mL of LB medium (50 mg / mL Kan) and incubated at 37°C and 200 rpm until OD reached [value missing]. 600 The concentration was 0.6, 60 μM lactose was added, and expression was induced at 30℃ and 200 rpm for 12 h. After induction, the bacterial cells were collected, resuspended in carbonate buffer (pH 10.0, mass-to-volume ratio 1:20), and the cells were disrupted by sonication. After centrifugation at 6000g for 10 min, the supernatant was collected to obtain the recombinant protein.

[0190] Example 10: rhUOX wt and rhUOX mut1 / K85C SDS-PAGE electrophoresis detection of mutant recombinant proteins

[0191] The SDS-PAGE electrophoresis detection method in this embodiment is the same as in Embodiment 2.

[0192] The SDS-PAGE protein electrophoresis results are shown in Figure 7. Lane 1 contains denatured rhUOX. wt Protein sample, lane 2 contains the mutant rhUOX mut1 / K85CThe target protein sample. The target protein is approximately 34 kDa in size, indicating that the target gene was successfully expressed in *E. coli* Rosetta and can be used for subsequent experiments.

[0193] Example 11: rhUOX mut1 / K85C Characterization of the active substances of mutants

[0194] Take 20 μL of purified protein rhUOX at 37℃. mut1 / K85C The reaction was terminated by adding an equal volume of pure methanol after reacting in 600 μL of 0.1 M uric acid solution (pH 8.0) for 10 min. Enzyme activity was calculated by measuring the decrease in UV absorbance at 293 nm using UV spectrophotometry. The control group consisted of inactivated uric acid oxidase. The experimental results are shown in Figure 8. mut1 / K85C The specific enzyme activity is 7.766 U / mg, indicating that this mutant exhibits high activity.

[0195] Enzyme activity unit definition: The amount of enzyme required to catalyze the oxidation of 1 µmol of uric acid per minute is defined as 1 unit.

[0196] E (U / mg) = (△OD) 293 ×V1×D) / (12.3×V2×T×C)

[0197] In the formula, 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.

[0198] Example 12: rhUOX mut1 / K85C Immunogenicity analysis of human PBMC cells with mutants

[0199] Human serum albumin (HAS) and aflatoxin-derived uricase (AfUOX) were used as control groups, while rhUOX was used as the control group. mut1 / K85C For the experimental group, 50 µg / mL of the above protein and PWM (20 µg / mL) were added to each well of a 24-well cell culture plate at a rate of 100 µL (5 µg), followed by PBMC cell suspension (1×10⁻⁶). 6 500 µL per well (dose of 10 µg / mL). Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. After incubation, centrifuge at 3000 rpm for 15 min and collect cells. Add 500 μL of cells to each well of the corresponding protein-coated cell culture plate. Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. Collect the culture supernatant after incubation. Analyze immunogenicity by detecting anti-uricase IgG using indirect ELISA.

[0200] Indirect ELISA steps:

[0201] (1) Antibody coating: The protein samples (HSA, AfUOX and rhUOX) are coated with antibodies. mut1 / K85C Add 100 µL (5 μg) to each well of a 96-well plate, with two replicates for each sample. Incubate overnight at 4 °C, then wash three times with PBST and pat dry.

[0202] (2) Blocking: Block with ELISA blocking solution for 1 h.

[0203] (3) Washing: Wash 3 times with PBST and pat dry.

[0204] (4) Incubation: Add 100 µL of the supernatant (HSA, AfUOX and rhUOX) obtained from the above cell culture to each well. mut1 / K85C The blank control was PBS, and the mixture was incubated at 37 °C for 1.5 h.

[0205] (5) Washing: Wash three times with detergent. Pat dry.

[0206] (6) HRP-goat anti-human IgG incubation: Add 100 µL of diluted HRP-goat anti-human IgG (1:3000, PBST, pH7.4) to each well and incubate at 37 °C for 1 h.

[0207] (7) Washing: Wash three times with detergent. Pat dry.

[0208] (8) Colorimetric reaction: Add 100 µL of freshly prepared TMB working solution to each well and incubate at room temperature in the dark for 15 min. Add 50 µL of stop solution (2 M H2SO4) to each well. Read the OD values ​​using a microplate reader. 450 value.

[0209] A positive result is defined as a test well having an A450 value that is 2.1 times greater than or equal to that of the negative control well: P / N = OD 450 Serum to be tested / OD 450 Negative control serum (i.e., P / N ≥ 2.1 is considered positive).

[0210] The results are shown in Figure 9, for the mutant rhUOX. mut1 / K85C It has low immunogenicity, similar to that of HSA.

[0211] Example 13: rhUOX mut1 / K85C In vivo efficacy verification of the mutant uricase gene in KO mice

[0212] In the experiment, 48 C57BL / 6J mice (UOX- / -), half male and half female, aged 5-8 weeks, weighing 25-30g, SPF grade, were purchased from Cyagen Biosciences Co., Ltd., and raised in the SPF grade laboratory animal room of the Experimental Animal Management Center of Jinan University. The room temperature is 20-25°C, and the laboratory animal use license is SYXK (Guangdong) 2022-0174. C57BL / 6J mice were given clean drinking water before the experiment, had free access to food and water, and were raised for 3 days before being used for the experiment.

[0213] A normal control group (with normal serum level), a hyperuricemia control group, and an experimental group (rhUOX mut1 / K85C ) were set up respectively. The normal control group consisted of wild-type C57BL / 6J mice, while the other groups used urate oxidase knockout KO mice for the experiment, with 12 mice in each group (6 males and 6 females). The normal control group and the hyperuricemia control group were given an equal volume of normal saline, while the experimental group was administered with reference to the dosage of rasburicase (2U / kg). Administration was performed via intraperitoneal injection, and blood was collected from the orbital vein at 0, 0.5, 1, 3, and 6 hours after administration. Serum treatment was the same as described above, and the collected serum was tested for uric acid content by HPLC. The results are shown in Figure 10. In the pharmacodynamic experiment on hyperuricemic KO mice, under the experimental conditions, the immunogenicity-attenuated rhUOX mut1 / K85C can maintain the blood uric acid level of KO mice at a normal level for at least 3 hours, and can still exert a uric acid-lowering effect after 6 hours.

[0214] The HPLC detection conditions are the same as those in Example 8.

[0215] With respect to the number and positions of point mutations, homology, specific enzyme activity, immunogenicity and other aspects, the urate oxidase mutant rhUOX described in the present invention mut1 / K85C The comparison with Document 1 and Document 2 mentioned in the "Background Art" is shown in Table 3 below.

[0216] Table 3:

[0217]

[0218] It can be seen from Table 3 that the human-derived urate oxidase mutant rhUOX described in the present invention mut1 / K85C has 96.05% homology with the theoretical amino acid sequence of human-derived urate oxidase, and the specific enzyme activity is 7.766 U / mg. It retains high activity while reducing the number of mutation sites, and maintains a high humanization rate. The mutant is higher than rHU15 (Document 1) in both humanization rate and activity. Experiments show that the mutant has low immunogenicity, which is similar to that of HSA. The reason is that the hotspot amino acids of the antigenic peptide on the protein surface are further mutated so that they cannot be recognized, thereby reducing immunogenicity. Therefore, the human-derived urate oxidase mutant rhUOX described in the present invention mut1 / K85CIt better meets the clinical needs of low-immunogenic uricase, is suitable for long-term clinical use in the treatment of hyperuricemia and gout, and can be used to prepare drugs for the treatment of hyperuricemia and gout.

[0219] Example 14: rhuox wt The synthesis of rhuoxmut1 / S86C and rhuoxmut1 / S86D genes was performed, and the recombinant proteins were induced to express in E. coli Rosetta (DE3).

[0220] This invention uses the human uricase pseudogene sequence rhuox. wt (GenBank accession number NR_003927.2), the human uricase mutant rhUOX was obtained through modification. mut1 / S86C and rhUOX mut1 / S86D The aforementioned gene was synthesized by BGI Genomics Co., Ltd. and cloned between NcoI and XhoI in the pET28a plasmid (Invitrogen). The recombinant plasmid was named pET28a-huox. wt pET28a-huoxmut1 / S86C and pET28a-huoxmut1 / S86D.

[0221] The recipient bacteria in this experiment was *Escherichia coli* Rosetta (DE3), using pET28a-huox. wt After transformation with pET28a-huoxmut1 / S86C and pET28a-huoxmut1 / S86D recombinant plasmids, preliminary screening was performed using kanamycin (Kan) resistant plates. Then, single clones from the Kan plates were picked and cultured in 5 mL of LB liquid medium (containing 50 mg / mL Kan) for 12-16 h. The plasmids were then extracted for PCR verification, and positive clones were further screened.

[0222] The positive recombinant was inoculated into 5 mL of LB medium (50 mg / mL Kan) and incubated overnight at 37°C and 200 rpm. The revived bacterial culture was then inoculated at 1% into 500 mL of LB medium (50 mg / mL Kan) and incubated at 37°C and 200 rpm until OD reached [value missing]. 600 The concentration was 0.6, 60 μM lactose was added, and expression was induced at 30℃ and 200 rpm for 12 h. After induction, the bacterial cells were collected, resuspended in carbonate buffer (pH 10.0, mass-to-volume ratio 1:20), and the cells were disrupted by sonication. After centrifugation at 6000g for 10 min, the supernatant was collected to obtain the recombinant protein.

[0223] Example 15: rhUOX wt rhUOXmut1 / S86C and rhUOX mut1 / S86D SDS-PAGE electrophoresis detection of mutant recombinant proteins

[0224] The SDS-PAGE electrophoresis detection method in this embodiment is the same as in Embodiment 2.

[0225] The SDS-PAGE protein electrophoresis results are shown in Figure 11. Lane 1 contains denatured rhUOX. wt Protein sample, lane 2 contains the mutant rhUOX mut1 / S86C The target protein sample, lane 3, contains the mutant rhUOX. mut1 / S86D The target protein sample. The target protein is approximately 34 kDa in size, indicating that the target gene was successfully expressed in *E. coli* Rosetta and can be used for subsequent experiments.

[0226] Example 16: rhUOX mut1 / S86C and rhUOX mut1 / S86D Characterization of the active substances of mutants

[0227] Take 20 μL of purified protein rhUOX at 37℃. mut1 / S86C rhUOX mut1 / S86D The reaction was terminated by adding an equal volume of pure methanol after reacting in 600 μL of 0.1 M uric acid solution (pH 8.0) for 10 min. Enzyme activity was calculated by measuring the decrease in UV absorbance at 293 nm using UV spectrophotometry. The control group consisted of inactivated uric acid oxidase. The experimental results are shown in Figure 12. mut1 / S86C and rhUOX mut1 / S86D The specific activities of the enzymes were 7.609 U / mg and 8.25 U / mg, respectively, and these mutants all exhibited high activity.

[0228] Enzyme activity unit definition: The amount of enzyme required to catalyze the oxidation of 1 µmol of uric acid per minute is defined as 1 unit.

[0229] E (U / mg) = (△OD) 293 ×V1×D) / (12.3×V2×T×C)

[0230] In the formula, 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.

[0231] Example 17: rhUOX mut1 / S86C and rhUOX mut1 / S86D Immunogenicity analysis of human PBMC cells with mutants

[0232] Human serum albumin (HAS) and aflatoxin-derived uricase (AfUOX) were used as control groups, while rhUOX was used as the control group. mut1 / S86Cand rhUOX mut1 / S86D For the experimental group, 50 µg / mL of the above protein and PWM (20 µg / mL) were added to each well of a 24-well cell culture plate at a rate of 100 µL (5 µg), followed by PBMC cell suspension (1×10⁻⁶). 6 500 µL per well (dose of 10 µg / mL). Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. After incubation, centrifuge at 3000 rpm for 15 min and collect cells. Add 500 μL of cells to each well of the corresponding protein-coated cell culture plate. Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. Collect the culture supernatant after incubation. Analyze immunogenicity by detecting anti-uricase IgG using indirect ELISA.

[0233] Indirect ELISA steps:

[0234] (1) Antibody coating: Coating the protein samples (HSA, AfUOX, rhUOX) with antibodies. mut1 / S86C and rhUOX mut1 / S86D Add 100 µL (5 μg) to each well of a 96-well plate, with two replicates for each sample. Incubate overnight at 4 °C, then wash three times with PBST and pat dry.

[0235] (2) Blocking: Block with ELISA blocking solution for 1 h.

[0236] (3) Washing: Wash 3 times with PBST and pat dry.

[0237] (4) Incubation: Add 100 µL of the supernatant (HSA, AfUOX, rhUOX) obtained from the above cell culture to each well. mut1 / S86C and rhUOX mut1 / S86D The blank control was PBS, and the mixture was incubated at 37 °C for 1.5 h.

[0238] (5) Washing: Wash three times with detergent. Pat dry.

[0239] (6) HRP-goat anti-human IgG incubation: Add 100 µL of diluted HRP-goat anti-human IgG (1:3000, PBST, pH7.4) to each well and incubate at 37 °C for 1 h.

[0240] (7) Washing: Wash three times with detergent. Pat dry.

[0241] (8) Color development by reaction: add 100 µL of freshly prepared TMB working solution to each well, and react for 15 min at room temperature in the dark. Add 50 µL of stop solution (2 M H₂SO₄) to each well. Read its OD with a microplate reader 450 value.

[0242] A result is determined as positive when the A₄₅₀ of the test well is greater than or equal to 2.1 times that of the negative control well: P / N=OD 450 of test serum / OD 450 of negative control serum (that is, P / N≥2.1 is positive).

[0243] The results are shown in Figure 13, the mutant rhUOX mut1 / S86C and rhUOX mut1 / S86D have low immunogenicity, with no significant difference from the HSA control group (P>0.05).

[0244] Example 18: Efficacy verification of rhUOX mut1 / S86C and rhUOX mut1 / S86D mutant urate oxidase in urate oxidase gene knockout mice in vivo

[0245] C57BL / 6J mice (UOX- / -) were used in the experiment, 48 mice, half male and half female, 5-8 weeks old, 25-30 g, SPF grade, purchased from Cyagen Biosciences Co., Ltd., housed in the SPF grade experimental animal room of the Experimental Animal Management Center of Jinan University, with room temperature of 20-25°C. The experimental animal use license: SYXK (Guangdong) 2022-0174. C57BL / 6J mice were given clean drinking water before the experiment, fed ad libitum, and used for the experiment after 3 days of feeding.

[0246] A normal control group (with normal serum level), a hyperuricemia control group, and experimental groups (rhUOX mut1 / S86C and rhUOX mut1 / S86D ) were set up respectively. The normal control group was wild-type C57BL / 6J mice, and the other groups used urate oxidase knockout KO mice for the experiment, with 12 mice in each group (6 males and 6 females). The normal control group and the hyperuricemia control group were given an equal volume of normal saline, while the experimental groups were administered with reference to the dosage of rasburicase (2 U / kg). Administration was performed by intraperitoneal injection, and orbital venous blood collection was performed at 0, 0.5, 1, 3, and 6 h after administration. Serum treatment was the same as described above, and the collected serum was tested for uric acid content by HPLC. The results are shown in Figure 14. In the pharmacodynamic experiment of hyperuricemic KO mice, under the experimental conditions, the low-immunogenicity rhUOX mut1 / S86C and rhUOX mut1 / S86D can maintain the blood uric acid level of KO mice at a normal level for at least 3 hours, and can still exert the uric acid-lowering effect after 6 hours.

[0247] HPLC detection conditions were the same as those in Example 8.

[0248] Regarding the number, location, homology, specific enzyme activity, and immunogenicity of point mutations, the rhUOX urate oxidase mutant described in this invention... mut1 / S86C and rhUOX mut1 / S86D A comparison with References 1 and 2 mentioned in the "Background Art" section is shown in Table 4 below.

[0249] Table 4:

[0250]

[0251] As shown in Table 4, the rhUOX of the human uricase mutant described in this invention... mut1 / S86C and rhUOX mut1 / S86D The mutants showed 96.05% homology to the theoretical amino acid sequence of human uricase. Their specific activities were 7.766 U / mg and 8.25 U / mg, respectively, maintaining high activity and a high humanization rate despite reduced mutation sites. These mutants were superior to rHU15 in both humanization rate and activity (Reference 1). Experiments showed that these mutants had low immunogenicity, similar to that of HSA, because the hotspot amino acids of their protein surface antigen peptide were further mutated, rendering them unrecognizable and thus reducing immunogenicity. Therefore, the human uricase mutant rhUOX described in this invention... mut1 / S86C and rhUOX mut1 / S86D It better meets the clinical needs of low-immunogenic uricase, is suitable for long-term clinical use in the treatment of hyperuricemia and gout, and can be used to prepare drugs for the treatment of hyperuricemia and gout.

[0252] Example 19: rhuox wt rhuox mut2 / I84C rhuox mut2 / I84H rhuox mut2 / I84N rhuox mut2 / I84R Gene synthesis was performed, and the recombinant protein was induced to be expressed in E. coli Rosetta (DE3).

[0253] This invention uses the human uricase pseudogene sequence rhuox. wt (GenBank accession number NR_003927.2), the human uricase mutant rhuox was obtained through modification. mut2 / I84C rhuox mut2 / I84H rhuox mut2 / I84N and rhuox mut2 / I84R The aforementioned gene was synthesized by BGI Genomics Co., Ltd. and cloned between NcoI and XhoI in the pET28a plasmid (Invitrogen). The recombinant plasmid was named pET28a-huox.wt pET28a-huox mut2 / I84C pET28a-huox mut2 / I84H pET28a-huox mut2 / I84N and pET28a-huox mut2 / I84R .

[0254] The recipient bacteria in this experiment was *Escherichia coli* Rosetta (DE3), using pET28a-huox. wt pET28a-huox mut2 / I84C pET28a-huox mut2 / I84H pET28a-huox mut2 / I84N and pET28a-huox mut2 / I84R After transformation of the recombinant plasmid, preliminary screening was performed using kanamycin (Kan) resistant plates. Then, single clones from the Kan plates were picked and cultured in 5 mL of LB liquid medium (containing 50 mg / mL Kan) for 12-16 h. The plasmids were then extracted for PCR verification, and positive clones were further screened.

[0255] The positive recombinant was inoculated into 5 mL of LB medium (50 mg / mL Kan) and incubated overnight at 37°C and 200 rpm. The revived bacterial culture was then inoculated at 1% into 500 mL of LB medium (50 mg / mL Kan) and incubated at 37°C and 200 rpm until OD reached [value missing]. 600 The concentration was 0.6, 60 μM lactose was added, and expression was induced at 30℃ and 200 rpm for 12 h. After induction, the bacterial cells were collected, resuspended in carbonate buffer (pH 10.0, mass-to-volume ratio 1:20), and the cells were disrupted by sonication. After centrifugation at 6000g for 10 min, the supernatant was collected to obtain the recombinant protein.

[0256] Example 20: rhUOX wt rhUOX mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N and rhUOX mut2 / I84R SDS-PAGE electrophoresis detection of mutant recombinant proteins

[0257] The SDS-PAGE electrophoresis detection method in this embodiment is the same as in Embodiment 2.

[0258] The SDS-PAGE protein electrophoresis results are shown in Figure 15. Lane 1 contains denatured rhUOX. wt Protein samples, lanes 2-5 contain the mutant rhUOX. mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N and rhUOXmut2 / I84R The target protein sample. The target protein is approximately 34 kDa in size, indicating that the target gene was successfully expressed in *E. coli* Rosetta and can be used for subsequent experiments.

[0259] Example 21: rhUOX mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N and rhUOX mut2 / I84R Characterization of the active substances of mutants

[0260] At 37°C, take 20 μL of the purified protein (rhUOX). mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N and rhUOX mut2 / I84R In 600 μL of 0.1 M uric acid solution (pH 8.0), the reaction was stopped by adding an equal volume of pure methanol after 10 min. Enzyme activity was calculated by measuring the decrease in UV absorbance at 293 nm using UV spectrophotometry. The control group consisted of inactivated uric acid oxidase. The experimental results are shown in Figure 16. mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N and rhUOX mut2 / I84R The specific activities of the enzymes were 7.472 U / mg, 7.591 U / mg, 7.388 U / mg, and 7.615 U / mg, respectively, and all of these mutants exhibited high activity.

[0261] Enzyme activity unit definition: The amount of enzyme required to catalyze the oxidation of 1 µmol of uric acid per minute is defined as 1 unit.

[0262] E (U / mg) = (△OD) 293 ×V1×D) / (12.3×V2×T×C)

[0263] In the formula, 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.

[0264] Example 22: rhUOX mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N and rhUOX mut2 / I84R Immunogenicity analysis of human PBMC cells with mutants

[0265] Human serum albumin (HAS) and aflatoxin-derived uricase (AfUOX) were used as control groups, while rhUOX was used as the control group. mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N and rhUOXmut2 / I84R For the experimental group, 50 µg / mL of the above protein and PWM (20 µg / mL) were added to each well of a 24-well cell culture plate at a rate of 100 µL (5 µg), followed by PBMC cell suspension (1×10⁻⁶). 6 500 µL per well (dose of 10 µg / mL). Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. After incubation, centrifuge at 3000 rpm for 15 min and collect cells. Add 500 μL of cells to each well of the corresponding protein-coated cell culture plate. Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. Collect the culture supernatant after incubation. Analyze immunogenicity by detecting anti-uricase IgG using indirect ELISA.

[0266] Indirect ELISA steps:

[0267] (1) Antibody coating: Coating the protein samples (HSA, AfUOX, rhUOX) with antibodies. mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N and rhUOX mut2 / I84R Add 100 µL (5 μg) to each well of a 96-well plate, with two replicates for each sample. Incubate overnight at 4 °C, then wash three times with PBST and pat dry.

[0268] (2) Blocking: Block with ELISA blocking solution for 1 h.

[0269] (3) Washing: Wash 3 times with PBST and pat dry.

[0270] (4) Incubation: Add 100 µL of the supernatant (HSA, AfUOX, rhUOX) obtained from the above cell culture to each well. mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N and rhUOX mut2 / I84R The blank control was PBS, and the mixture was incubated at 37 °C for 1.5 h.

[0271] (5) Washing: Wash three times with detergent. Pat dry.

[0272] (6) HRP-goat anti-human IgG incubation: Add 100 µL of diluted HRP-goat anti-human IgG (1:3000, PBST, pH7.4) to each well and incubate at 37 °C for 1 h.

[0273] (7) Washing: Wash three times with detergent. Pat dry.

[0274] (8) Color development reaction: add 100 µL of freshly prepared TMB working solution to each well, and react for 15 min at room temperature in the dark. Add 50 µL of stop solution (2 M H₂SO₄) to each well. Read the OD with a microplate reader 450 value.

[0275] A result is determined as positive when the A₄₅₀ of the test well is greater than or equal to 2.1 times that of the negative control well: P / N=OD 450 of test serum / OD 450 of negative control serum (that is, P / N≥2.1 is positive).

[0276] The results are shown in Figure 17, the mutant rhUOX mut2 / I84C , rhUOX mut2 / I84H , rhUOX mut2 / I84N and rhUOX mut2 / I84R have reduced immunogenicity, with P / N values similar to that of HSA.

[0277] Example 23: rhUOX mut2 / I84C , rhUOX mut2 / I84H , rhUOX mut2 / I84N and rhUOX mut2 / I84R Efficacy verification of mutant urate oxidase gene in KO mice

[0278] C57BL / 6J mice (UOX- / -) were used in the experiment, 48 individuals, half male and half female, 5-8 weeks old, 25-30g, SPF grade, purchased from Cyagen Biosciences Co., Ltd., and housed in the SPF grade experimental animal room of the Experimental Animal Management Center of Jinan University, with room temperature of 20-25°C. The laboratory animal use license is: SYXK (Guangdong) 2022-0174. Before the experiment, C57BL / 6J mice were given clean drinking water, fed ad libitum, and housed for 3 days before being used for the experiment.

[0279] A normal control group (with normal serum levels), a hyperuricemia control group, and test groups (rhUOX mut2 / I84C , rhUOX mut2 / I84H , rhUOX mut2 / I84N and rhUOX mut2 / I84RThe normal control group consisted of wild-type C57BL / 6J mice, while other groups used uricase knockout KO mice, with 12 mice in each group (6 males and 6 females). The normal control and high-uric acid control groups received an equal volume of physiological saline, while the experimental groups received the same dose of rhUOX (2 U / kg). Administration was via intraperitoneal injection, and blood samples were collected from the orbital vein at 0, 0.5, 1, 3, and 6 hours after administration. Serum processing was the same as above, and the uric acid content of the collected serum was determined by HPLC. The results are shown in Figure 18. In the pharmacodynamic experiment of high-uric acid KO mice, under the experimental conditions, the immunogenicity of the attenuated rhUOX was significantly reduced. mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N and rhUOX mut2 / I84R It can maintain the blood uric acid level of KO mice at a normal level for at least 3 hours, and can still exert the effect of lowering uric acid after 6 hours.

[0280] The HPLC detection conditions were the same as in Example 8.

[0281] Regarding the number, location, homology, specific enzyme activity, and immunogenicity of point mutations, the rhUOX urate oxidase mutant described in this invention... mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N and rhUOX mut2 / I84R A comparison with References 1 and 2 mentioned in the "Background Art" section is shown in Table 5 below.

[0282] Table 5:

[0283]

[0284] As shown in Table 5, the human uricase mutant rhUOX described in this invention... mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84N and rhUOX mut2 / I84R The mutants showed 95.39% homology to the theoretical amino acid sequence of human uricase. Their specific activities were 7.472 U / mg, 7.591 U / mg, 7.388 U / mg, and 7.615 U / mg, respectively. While reducing the number of mutation sites, they retained high activity and a high humanization rate. These mutants were superior to rHU15 in both humanization rate and activity (Reference 1). Experiments showed that these mutants had low immunogenicity, similar to that of HSA, because the hotspot amino acids of their protein surface antigen peptide were further mutated, making them unrecognizable and thus reducing immunogenicity. Therefore, the human uricase mutant rhUOX described in this invention... mut2 / I84C rhUOX mut2 / I84H rhUOX mut2 / I84Nand rhUOX mut2 / I84R It better meets the clinical needs of low-immunogenic uricase, is suitable for long-term clinical use in the treatment of hyperuricemia and gout, and can be used to prepare drugs for the treatment of hyperuricemia and gout.

[0285] Example 24: rhuox wt and rhuox mut2 / K85C Gene synthesis was performed, and the recombinant protein was induced to be expressed in E. coli Rosetta (DE3).

[0286] This invention uses the human uricase pseudogene sequence rhuox. wt (GenBank accession number NR_003927.2), the human uricase mutant rhuox was obtained through modification. mut2 / K85C The aforementioned gene was synthesized by BGI Genomics Co., Ltd. and cloned between NcoI and XhoI in the pET28a plasmid (Invitrogen). The recombinant plasmid was named pET28a-huox. wt pET28a-huox mut2 / K85C .

[0287] The recipient bacteria in this experiment was *Escherichia coli* Rosetta (DE3), using pET28a-huox. wt pET28a-huox mut2 / K85C After transformation of the recombinant plasmid, preliminary screening was performed using kanamycin (Kan) resistant plates. Then, single clones from the Kan plates were picked and cultured in 5 mL of LB liquid medium (containing 50 mg / mL Kan) for 12-16 h. The plasmids were then extracted for PCR verification, and positive clones were further screened.

[0288] The positive recombinant was inoculated into 5 mL of LB medium (50 mg / mL Kan) and incubated overnight at 37°C and 200 rpm. The revived bacterial culture was then inoculated at 1% into 500 mL of LB medium (50 mg / mL Kan) and incubated at 37°C and 200 rpm until OD reached [value missing]. 600 The concentration was 0.6, 60 μM lactose was added, and expression was induced at 30℃ and 200 rpm for 12 h. After induction, the bacterial cells were collected, resuspended in carbonate buffer (pH 10.0, mass-to-volume ratio 1:20), and the cells were disrupted by sonication. After centrifugation at 6000g for 10 min, the supernatant was collected to obtain the recombinant protein.

[0289] Example 25: rhUOX wt and rhUOX mut2 / K85C SDS-PAGE electrophoresis detection of mutant recombinant proteins

[0290] The SDS-PAGE electrophoresis detection method in this embodiment is the same as in Embodiment 2.

[0291] The SDS-PAGE protein electrophoresis results are shown in Figure 19. Lane 1 contains denatured rhUOX. wt Protein sample, lane 2 contains the mutant rhUOX mut2 Lane 3 is a mutant rhUOX mut2 / K85C The target protein sample. The target protein is approximately 34 kDa in size, indicating that the target gene was successfully expressed in *E. coli* Rosetta and can be used for subsequent experiments.

[0292] Example 26: rhUOX mut2 / K85C Characterization of the active substances of mutants

[0293] Take 20 μL of purified protein rhUOX at 37℃. mut2 / K85C The reaction was terminated by adding an equal volume of pure methanol after reacting in 600 μL of 0.1 M uric acid solution (pH 8.0) for 10 min. Enzyme activity was calculated by measuring the decrease in UV absorbance at 293 nm using UV spectrophotometry. The control group consisted of inactivated uric acid oxidase. The experimental results are shown in Figure 20. mut2 / K85C The specific enzyme activity is 7.685 U / mg, indicating that this mutant exhibits high activity.

[0294] Enzyme activity unit definition: The amount of enzyme required to catalyze the oxidation of 1 µmol of uric acid per minute is defined as 1 unit.

[0295] E (U / mg) = (△OD) 293 ×V1×D) / (12.3×V2×T×C)

[0296] In the formula, 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.

[0297] Example 27: rhUOX mut2 / K85C Immunogenicity analysis of human PBMC cells with mutants

[0298] Human serum albumin (HAS) and aflatoxin-derived uricase (AfUOX) were used as control groups, while rhUOX was used as the control group. mut2 / K85C For the experimental group, 50 µg / mL of the above protein and PWM (20 µg / mL) were added to each well of a 24-well cell culture plate at a rate of 100 µL (5 µg), followed by PBMC cell suspension (1×10⁻⁶). 6500 µL per well (dose of 10 µg / mL). Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. After incubation, centrifuge at 3000 rpm for 15 min and collect cells. Add 500 μL of cells to each well of the corresponding protein-coated cell culture plate. Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. Collect the culture supernatant after incubation. Analyze immunogenicity by detecting anti-uricase IgG using indirect ELISA.

[0299] Indirect ELISA steps:

[0300] (1) Antibody coating: The protein samples (HSA, AfUOX and rhUOX) are coated with antibodies. mut2 / K85C Add 100 µL (5 μg) to each well of a 96-well plate, with two replicates for each sample. Incubate overnight at 4 °C, then wash three times with PBST and pat dry.

[0301] (2) Blocking: Block with ELISA blocking solution for 1 h.

[0302] (3) Washing: Wash 3 times with PBST and pat dry.

[0303] (4) Incubation: Add 100 µL of the supernatant (HSA, AfUOX and rhUOX) obtained from the above cell culture to each well. mut2 / K85C The blank control was PBS, and the mixture was incubated at 37 °C for 1.5 h.

[0304] (5) Washing: Wash three times with detergent. Pat dry.

[0305] (6) HRP-goat anti-human IgG incubation: Add 100 µL of diluted HRP-goat anti-human IgG (1:3000, PBST, pH7.4) to each well and incubate at 37 °C for 1 h.

[0306] (7) Washing: Wash three times with detergent. Pat dry.

[0307] (8) Colorimetric reaction: Add 100 µL of freshly prepared TMB working solution to each well and incubate at room temperature in the dark for 15 min. Add 50 µL of stop solution (2 M H2SO4) to each well. Read the OD values ​​using a microplate reader. 450 value.

[0308] A positive result is defined as a test well having an A450 value that is 2.1 times greater than or equal to that of the negative control well: P / N = OD 450 Serum to be tested / OD 450 Negative control serum (i.e., P / N ≥ 2.1 is considered positive).

[0309] The results are shown in Figure 21, the mutant rhUOX mut2 / K85C has low immunogenicity, with no significant difference compared with HSA (P>0.05).

[0310] Example 28: In vivo efficacy verification of mutant urate oxidase gene KO mice for rhUOX mut2 / K85C Efficacy verification of mutant urate oxidase gene KO mice in vivo

[0311] In the experiment, 48 C57BL / 6J mice (UOX- / -), half male and half female, aged 5-8 weeks, weighing 25-30g, SPF grade, were purchased from Cyagen Biosciences Co., Ltd., and housed in the SPF grade experimental animal room of the Experimental Animal Management Center of Jinan University, with room temperature of 20-25°C. The laboratory animal use license is SYXK (Guangdong) 2022-0174. C57BL / 6J mice were given clean drinking water and free access to food before the experiment, and were used for the experiment after 3 days of breeding.

[0312] A normal control group (with normal serum level), a hyperuricemia control group, and an experimental group (rhUOX mut2 / K85C ) were set up respectively. The normal control group consisted of wild-type C57BL / 6J mice, while other groups used urate oxidase knockout KO mice for the experiment, with 12 mice in each group (6 males and 6 females). The normal control group and the hyperuricemia control group were given an equal volume of normal saline, while the experimental group was administered with reference to the dosage of rasburicase (2 U / kg). Administration was performed via intraperitoneal injection, and blood was collected from the orbital vein at 0, 0.5, 1, 3, and 6 hours after administration. Serum treatment was performed as described above, and the collected serum was tested for uric acid content by HPLC. The results are shown in Figure 22. In the pharmacodynamic experiment of hyperuricemic KO mice, under the experimental conditions, the immunogenicity-attenuated rhUOX mut2 / K85C can maintain the serum uric acid level of KO mice at a normal level for at least 3 hours, and can still exert the uric acid-lowering effect after 6 hours.

[0313] The HPLC detection conditions are the same as in Example 8.

[0314] Regarding the number and position of point mutations, homology, specific enzyme activity, immunogenicity and other aspects, the urate oxidase mutant rhUOX described in the present invention mut2 / K85C The comparison with Document 1 and Document 2 mentioned in "Background Art" is shown in Table 6 below.

[0315] Table 6:

[0316]

[0317] It can be seen from Table 6 that the human urate oxidase mutant rhUOX described in the present invention mut2 / K85CThe mutant exhibits 95.39% homology with the theoretical amino acid sequence of human uricase and a specific activity of 7.685 U / mg. While reducing the number of mutation sites, it retains high activity and a high humanization rate. This mutant demonstrates higher humanization rate and activity than rHU15 (Reference 1). Experiments show that this mutant has low immunogenicity, similar to that of HSA, because the hotspot amino acids of its protein surface antigen peptide are further mutated, rendering them unrecognizable and thus reducing immunogenicity. Therefore, the human uricase mutant rhUOX described in this invention... mut2 / K85C It better meets the clinical needs of low-immunogenic uricase, is suitable for long-term clinical use in the treatment of hyperuricemia and gout, and can be used to prepare drugs for the treatment of hyperuricemia and gout.

[0318] Example 29: rhuox wt rhuox mut2 / S86C rhuox mut2 / S86D Gene synthesis was performed, and the recombinant protein was induced to be expressed in E. coli Rosetta (DE3).

[0319] This invention uses the human uricase pseudogene sequence rhuox. wt (GenBank accession number NR_003927.2), the human uricase mutant rhuox was obtained through modification. mut2 / S86C rhuox mut2 / S86D The aforementioned gene was synthesized by BGI Genomics Co., Ltd. and cloned between NcoI and XhoI in the pET28a plasmid (Invitrogen). The recombinant plasmid was named pET28a-huox. wt pET28a-huox mut2 / S86C and pET28a-huox mut2 / S86D .

[0320] The recipient bacteria in this experiment was *Escherichia coli* Rosetta (DE3), using pET28a-huox. wt pET28a-huox mut2 / S86C and pET28a-huox mut2 / S86D After transformation of the recombinant plasmid, preliminary screening was performed using kanamycin (Kan) resistant plates. Then, single clones from the Kan plates were picked and cultured in 5 mL of LB liquid medium (containing 50 mg / mL Kan) for 12-16 h. The plasmids were then extracted for PCR verification, and positive clones were further screened.

[0321] The positive recombinant was inoculated into 5 mL of LB medium (50 mg / mL Kan) and incubated overnight at 37°C and 200 rpm. The revived bacterial culture was then inoculated at 1% into 500 mL of LB medium (50 mg / mL Kan) and incubated at 37°C and 200 rpm until OD reached [value missing]. 600 The concentration was 0.6, 60 μM lactose was added, and expression was induced at 30℃ and 200 rpm for 12 h. After induction, the bacterial cells were collected, resuspended in carbonate buffer (pH 10.0, mass-to-volume ratio 1:20), and the cells were disrupted by sonication. After centrifugation at 6000g for 10 min, the supernatant was collected to obtain the recombinant protein.

[0322] Example 30: rhUOX wt rhUOX mut2 / S86C and rhUOX mut2 / S86D SDS-PAGE electrophoresis detection of mutant recombinant proteins

[0323] The SDS-PAGE electrophoresis detection method in this embodiment is the same as in Embodiment 2.

[0324] The SDS-PAGE protein electrophoresis results are shown in Figure 23. Lane 1 contains denatured rhUOX. wt Protein sample, lane 2 contains the mutant rhUOX mut2 Lane 3 is a mutant rhUOX mut2 / S86C Lane 4 is rhUOX mut2 / S86D The target protein sample. The target protein is approximately 34 kDa in size, indicating that the target gene was successfully expressed in *E. coli* Rosetta and can be used for subsequent experiments.

[0325] Example 31: rhUOX mut2 / S86C and rhUOX mut2 / S86D Characterization of the active substances of mutants

[0326] At 37°C, take 20 μL of the purified protein (rhUOX). mut2 / S86C and rhUOX mut2 / S86D In 600 μL of 0.1 M uric acid solution (pH 8.0), the reaction was carried out for 10 min, and then an equal volume of pure methanol was added to terminate the reaction. Enzyme activity was calculated by measuring the decrease in UV absorbance at 293 nm using UV spectrophotometry. The control group consisted of inactivated uric acid oxidase. The experimental results are shown in Figure 24. mut2 / S86C and rhUOX mut2 / S86D The specific activities of the enzymes were 7.85 U / mg and 7.64 U / mg, respectively, and these mutants all exhibited high activity.

[0327] Enzyme activity unit definition: The amount of enzyme required to catalyze the oxidation of 1 µmol of uric acid per minute is defined as 1 unit.

[0328] E (U / mg) = (△OD) 293 ×V1×D) / (12.3×V2×T×C)

[0329] In the formula, 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.

[0330] Example 32: rhUOX mut2 / S86C and rhUOX mut2 / S86D Immunogenicity analysis of human PBMC cells with mutants

[0331] Human serum albumin (HAS) and aflatoxin-derived uricase (AfUOX) were used as control groups, while rhUOX was used as the control group. mut2 / S86C and rhUOX mut2 / S86D For the experimental group, 50 µg / mL of the above protein and PWM (20 µg / mL) were added to each well of a 24-well cell culture plate at a rate of 100 µL (5 µg), followed by PBMC cell suspension (1×10⁻⁶). 6 500 µL per well (dose of 10 µg / mL). Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. After incubation, centrifuge at 3000 rpm for 15 min and collect cells. Add 500 μL of cells to each well of the corresponding protein-coated cell culture plate. Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. Collect the culture supernatant after incubation. Analyze immunogenicity by detecting anti-uricase IgG using indirect ELISA.

[0332] Indirect ELISA steps:

[0333] (1) Antibody coating: Coating the protein samples (HSA, AfUOX, rhUOX) with antibodies. mut2 / S86C and rhUOX mut2 / S86D Add 100 µL (5 μg) to each well of a 96-well plate, with two replicates for each sample. Incubate overnight at 4 °C, then wash three times with PBST and pat dry.

[0334] (2) Blocking: Block with ELISA blocking solution for 1 h.

[0335] (3) Washing: Wash 3 times with PBST and pat dry.

[0336] (4) Incubation: Add 100 µL of the supernatant (HSA, AfUOX, rhUOX) obtained from the above cell culture to each well. mut2 / S86C and rhUOXmut2 / S86D ), the blank control is PBS, and incubated at 37 °C for 1.5 h.

[0337] (5) Washing: Wash three times with washing solution. Pat dry.

[0338] (6) Incubation with HRP-goat anti-human IgG: Add 100 µL of diluted HRP-goat anti-human IgG (1:3000, in PBST, pH 7.4) to each well, and incubate at 37 °C for 1 h.

[0339] (7) Washing: Wash three times with washing solution. Pat dry.

[0340] (8) 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 with a microplate reader 450 value.

[0341] A result is considered positive when the A₄₅₀ of the test well is greater than or equal to 2.1 times that of the negative control: P / N=OD 450 of test serum / OD 450 of negative control serum (that is, P / N≥2.1 is positive).

[0342] The results are shown in Figure 25, the mutant rhUOX mut2 / S86C and rhUOX mut2 / S86D have reduced immunogenicity, with no significant difference from HSA (P>0.05).

[0343] Example 33: rhUOX mut2 / S86C and rhUOX mut2 / S86D Efficacy verification of mutant urate oxidase in KO mice in vivo

[0344] C57BL / 6J mice (UOX- / -) were used in the experiment, 48 mice, half male and half female, 5-8 weeks old, 25-30 g, SPF grade, purchased from Cyagen Biosciences Co., Ltd., housed in the SPF grade laboratory animal room of the Experimental Animal Management Center of Jinan University, with room temperature of 20-25°C. The laboratory animal use license is SYXK (Guangdong) 2022-0174. C57BL / 6J mice were given clean drinking water and allowed free access to food before the experiment, and were used for the experiment after 3 days of feeding.

[0345] A normal control group (with normal serum levels), a hyperuricemia control group, and experimental groups (rhUOX mut2 / S86C and rhUOX mut2 / S86DThe normal control group consisted of wild-type C57BL / 6J mice, while other groups used uricase knockout KO mice, with 12 mice in each group (6 males and 6 females). The normal control and high-uric acid control groups received an equal volume of physiological saline, while the experimental groups received the same dose of rhUOX (2 U / kg). The drugs were administered via intraperitoneal injection, and blood samples were collected from the orbital vein at 0, 0.5, 1, 3, and 6 hours after administration. Serum processing was the same as above, and the uric acid content of the collected serum was determined by HPLC. The results are shown in Figure 26. In the pharmacodynamic experiment of high-uric acid KO mice, under the experimental conditions, the low immunogenicity of rhUOX... mut2 / S86C and rhUOX mut2 / S86D It can maintain the blood uric acid level of KO mice at a normal level for at least 3 hours, and can still exert the effect of lowering uric acid after 6 hours.

[0346] The HPLC detection conditions were the same as in Example 8.

[0347] Regarding the number, location, homology, specific enzyme activity, and immunogenicity of point mutations, the rhUOX urate oxidase mutant described in this invention... mut2 / S86C and rhUOX mut2 / S86D A comparison with References 1 and 2 mentioned in the "Background Art" section is shown in Table 7 below.

[0348] Table 7:

[0349]

[0350] As shown in Table 7, the rhUOX of the human uricase mutant described in this invention... mut2 / S86C and rhUOX mut2 / S86D The mutants showed 95.39% homology to the theoretical amino acid sequence of human uricase. Their specific activities were 7.85 U / mg and 7.64 U / mg, respectively, maintaining high activity and a high humanization rate despite reduced mutation sites. These mutants were superior to rHU15 in both humanization rate and activity (Reference 1). Experiments showed that these mutants exhibited low immunogenicity, similar to that of HSA, because the hotspot amino acids of their protein surface antigen peptide were further mutated, rendering them unrecognizable and thus reducing immunogenicity. Therefore, the human uricase mutant rhUOX described in this invention... mut2 / S86C and rhUOX mut2 / S86D It better meets the clinical needs of low-immunogenic uricase, is suitable for long-term clinical use in the treatment of hyperuricemia and gout, and can be used to prepare drugs for the treatment of hyperuricemia and gout.

[0351] Example 34: rhuox wt rhuox mut3 / I84C rhuox mut3 / I84H rhuoxmut3 / I84N rhuox mut3 / I84R Gene synthesis was performed, and the recombinant protein was induced to be expressed in E. coli Rosetta (DE3).

[0352] This invention uses the human uricase pseudogene sequence rhuox. wt (GenBank accession number NR_003927.2), the human uricase mutant rhuox was obtained through modification. mut3 / I84C rhuox mut3 / I84H rhuox mut3 / I84N rhuox mut3 / I84R The aforementioned gene was synthesized by BGI Genomics Co., Ltd. and cloned between NcoI and XhoI in the pET28a plasmid (Invitrogen). The recombinant plasmid was named pET28a-huox. wt pET28a-huox mut3 / I84C pET28a-huox mut3 / I84H pET28a-huox mut3 / I84N pET28a-huox mut3 / I84R .

[0353] The recipient bacteria in this experiment was *Escherichia coli* Rosetta (DE3), using 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, preliminary screening was performed using kanamycin (Kan) resistant plates. Then, single clones from the Kan plates were picked and cultured in 5 mL of LB liquid medium (containing 50 mg / mL Kan) for 12-16 h. The plasmids were then extracted for PCR verification, and positive clones were further screened.

[0354] The positive recombinant was inoculated into 5 mL of LB medium (50 mg / mL Kan) and incubated overnight at 37°C and 200 rpm. The revived bacterial culture was then inoculated at 1% into 500 mL of LB medium (50 mg / mL Kan) and incubated at 37°C and 200 rpm until OD reached [value missing]. 600 The concentration was 0.6, 60 μM lactose was added, and expression was induced at 30℃ and 200 rpm for 12 h. After induction, the bacterial cells were collected, resuspended in carbonate buffer (pH 10.0, mass-to-volume ratio 1:20), and the cells were disrupted by sonication. After centrifugation at 6000g for 10 min, the supernatant was collected to obtain the recombinant protein.

[0355] Example 35: rhUOX wt rhUOX mut3 / I84C rhUOX mut3 / I84H rhUOX mut3 / I84N and rhUOX mut3 / I84R SDS-PAGE electrophoresis detection of mutant recombinant proteins

[0356] The SDS-PAGE electrophoresis detection method in this embodiment is the same as in Embodiment 2.

[0357] The SDS-PAGE protein electrophoresis results are shown in Figure 27. Lane 1 contains denatured rhUOX. wt Protein samples, lanes 2-5 contain the mutant rhUOX. mut3 / I84C rhUOX mut3 / I84H rhUOX mut3 / I84N and rhUOX mut3 / I84R The target protein sample. The target protein is approximately 34 kDa in size, indicating that the target gene was successfully expressed in *E. coli* Rosetta and can be used for subsequent experiments.

[0358] Example 36: rhUOX mut3 / I84C rhUOX mut3 / I84H rhUOX mut3 / I84N and rhUOX mut3 / I84R Characterization of the active substances of mutants

[0359] 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 0.1 M uric acid solution (pH 8.0), the reaction was carried out for 10 min, and then an equal volume of pure methanol was added to terminate the reaction. Enzyme activity was calculated by measuring the decrease in UV absorbance at 293 nm using UV spectrophotometry. The control group consisted of inactivated uric acid oxidase. The experimental results are shown in Figure 28. mut3 / I84C rhUOX mut3 / I84H rhUOX mut3 / I84N and rhUOX mut3 / I84R The specific activities of the enzymes were 7.36 U / mg, 7.55 U / mg, 7.41 U / mg, and 7.47 U / mg, respectively, and all of these mutants exhibited high activity.

[0360] Enzyme activity unit definition: The amount of enzyme required to catalyze the oxidation of 1 µmol of uric acid per minute is defined as 1 unit.

[0361] E (U / mg) = (△OD) 293 ×V1×D) / (12.3×V2×T×C)

[0362] In the formula, 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.

[0363] Example 37: rhUOX mut3 / I84C rhUOX mut3 / I84H rhUOX mut3 / I84N and rhUOX mut3 / I84R Immunogenicity analysis of human PBMC cells with mutants

[0364] Human serum albumin (HAS) and aflatoxin-derived uricase (AfUOX) were used as control groups, while rhUOX was used as the control group. mut3 / I84C rhUOX mut3 / I84H rhUOX mut3 / I84N and rhUOX mut3 / I84R For the experimental group, 50 µg / mL of the above protein and PWM (20 µg / mL) were added to each well of a 24-well cell culture plate at a rate of 100 µL (5 µg), followed by PBMC cell suspension (1×10⁻⁶). 6 500 µL per well (dose of 10 µg / mL). Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. After incubation, centrifuge at 3000 rpm for 15 min and collect cells. Add 500 μL of cells to each well of the corresponding protein-coated cell culture plate. Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. Collect the culture supernatant after incubation. Analyze immunogenicity by detecting anti-uricase IgG using indirect ELISA.

[0365] Indirect ELISA steps:

[0366] (1) Antibody coating: Coating the protein samples (HSA, AfUOX, rhUOX) with antibodies. mut3 / I84C rhUOX mut3 / I84H rhUOX mut3 / I84N and rhUOX mut3 / I84R Add 100 µL (5 μg) to each well of a 96-well plate, with two replicates for each sample. Incubate overnight at 4 °C, then wash three times with PBST and pat dry.

[0367] (2) Blocking: Block with ELISA blocking solution for 1 h.

[0368] (3) Washing: Wash 3 times with PBST and pat dry.

[0369] (4) Incubation: Add 100 µL of the supernatant (HSA, AfUOX, rhUOX) obtained from the above cell culture to each well. mut3 / I84C rhUOX mut3 / I84H rhUOX mut3 / I84N and rhUOX mut3 / I84R The blank control was PBS, and the mixture was incubated at 37 °C for 1.5 h.

[0370] (5) Washing: Wash three times with detergent. Pat dry.

[0371] (6) HRP-goat anti-human IgG incubation: Add 100 µL of diluted HRP-goat anti-human IgG (1:3000, PBST, pH7.4) to each well and incubate at 37 °C for 1 h.

[0372] (7) Washing: Wash three times with detergent. Pat dry.

[0373] (8) Colorimetric reaction: Add 100 µL of freshly prepared TMB working solution to each well and incubate at room temperature in the dark for 15 min. Add 50 µL of stop solution (2 M H2SO4) to each well. Read the OD values ​​using a microplate reader. 450 value.

[0374] A positive result is defined as a test well having an A450 value that is 2.1 times greater than or equal to that of the negative control well: P / N = OD 450 Serum to be tested / OD 450 Negative control serum (i.e., P / N ≥ 2.1 is considered positive).

[0375] The results are shown in Figure 29, for the mutant rhUOX. mut3 / I84C rhUOX mut3 / I84H rhUOX mut3 / I84N and rhUOX mut3 / I84R It has low immunogenicity, comparable to that of HSA.

[0376] Example 38: rhUOX mut3 / I84C rhUOX mut3 / I84H rhUOX mut3 / I84N and rhUOX mut3 / I84R In vivo efficacy verification of the mutant uricase gene in KO mice

[0377] In the experiment, 48 C57BL / 6J mice (UOX- / -), half male and half female, aged 5-8 weeks, weighing 25-30 g, of SPF grade, were purchased from Cyagen Biosciences Co., Ltd., and raised in the SPF-grade experimental animal room of the Experimental Animal Management Center of Jinan University, with room temperature of 20-25°C. The license for the use of experimental animals is SYXK (Guangdong) 2022-0174. Before the experiment, C57BL / 6J mice were given clean drinking water, allowed free access to food, and housed for 3 days before being used for the experiment.

[0378] A normal control group (with normal serum uric acid level), a hyperuricemia control group, and an experimental group (rhUOX mut3 / I84C , rhUOX mut3 / I84H , rhUOX mut3 / I84N and rhUOX mut3 / I84R ). The normal control group consisted of wild-type C57BL / 6J mice, while the other groups used urate oxidase knockout KO mice for the experiment, with 12 mice in each group (6 males and 6 females). The normal control group and the hyperuricemia control group were administered an equal volume of normal saline, while the experimental group was administered the drug with reference to the dosage of rasburicase (2 U / kg). The drug was administered via intraperitoneal injection, and blood was collected from the orbital vein at 0, 0.5, 1, 3, and 6 hours after administration. Serum treatment was performed as described above, and the collected serum was tested for uric acid content by HPLC. The results are shown in Figure 30. In the pharmacodynamic experiment of hyperuricemic KO mice, under the experimental conditions, the immunogenically attenuated rhUOX mut3 / I84C , rhUOX mut3 / I84H , rhUOX mut3 / I84N and rhUOX mut3 / I84R can maintain the blood uric acid level of KO mice at a normal level for at least 3 hours, and can still exert the uric acid-lowering effect after 6 hours.

[0379] The HPLC detection conditions were the same as those in Example 8.

[0380] Regarding the number and positions of point mutations, homology, specific enzyme activity, immunogenicity and other aspects, the urate oxidase mutant rhUOX described in the present invention mut3 / I84C , rhUOX mut3 / I84H , rhUOX mut3 / I84N and rhUOX mut3 / I84R are compared with Document 1 and Document 2 mentioned in the "Background Art", as shown in Table 8 below.

[0381] Table 8:

[0382]

[0383] It can be seen from Table 8 that the human-derived urate oxidase mutant rhUOX described in the present invention mut3 / I84C , rhUOX mut3 / I84H , rhUOX mut3 / I84N and rhUOXmut3 / I84R The homology of the theoretical amino acid sequence of human uricase oxidase was 95.06% (identical to rHU15 reported in Reference 1). The specific activities of these mutants were 7.36 U / mg, 7.55 U / mg, 7.41 U / mg, and 7.47 U / mg, respectively (significantly higher than rHU15 reported in Reference 1), maintaining high activity and a high humanization rate while reducing the number of mutation sites. Experiments showed that these mutants had low immunogenicity, similar to that of HSA, because the hotspot amino acids of their protein surface antigen peptide were further mutated, making them unrecognizable and thus reducing immunogenicity. Therefore, the human uricase mutant rhUOX described in this invention... mut3 / I84C rhUOX mut3 / I84H rhUOX mut3 / I84N and rhUOX mut3 / I84R It better meets the clinical needs of low-immunogenic uricase, is suitable for long-term clinical use in the treatment of hyperuricemia and gout, and can be used to prepare drugs for the treatment of hyperuricemia and gout.

[0384] Example 39: rhuox wt rhuox mut3 / K85C Gene synthesis was performed, and the recombinant protein was induced to be expressed in E. coli Rosetta (DE3).

[0385] This invention uses the human uricase pseudogene sequence rhuox. wt (GenBank accession number NR_003927.2), the human uricase mutant rhuox was obtained through modification. mut3 / K85C The aforementioned gene was synthesized by BGI Genomics Co., Ltd. and cloned between NcoI and XhoI in the pET28a plasmid (Invitrogen). The recombinant plasmid was named pET28a-huox. wt pET28a-huox mut3 / K85C .

[0386] The recipient bacteria in this experiment was *Escherichia coli* Rosetta (DE3), using pET28a-huox. wt pET28a-huox mut3 / K85C After transformation of the recombinant plasmid, preliminary screening was performed using kanamycin (Kan) resistant plates. Then, single clones from the Kan plates were picked and cultured in 5 mL of LB liquid medium (containing 50 mg / mL Kan) for 12-16 h. The plasmids were then extracted for PCR verification, and positive clones were further screened.

[0387] The positive recombinant was inoculated into 5 mL of LB medium (50 mg / mL Kan) and incubated overnight at 37°C and 200 rpm. The revived bacterial culture was then inoculated at 1% into 500 mL of LB medium (50 mg / mL Kan) and incubated at 37°C and 200 rpm until OD reached [value missing]. 600 The concentration was 0.6, 60 μM lactose was added, and expression was induced at 30℃ and 200 rpm for 12 h. After induction, the bacterial cells were collected, resuspended in carbonate buffer (pH 10.0, mass-to-volume ratio 1:20), and the cells were disrupted by sonication. After centrifugation at 6000g for 10 min, the supernatant was collected to obtain the recombinant protein.

[0388] Example 40: rhUOX wt and rhUOX mut3 / K85C SDS-PAGE electrophoresis detection of mutant recombinant proteins

[0389] The SDS-PAGE electrophoresis detection method in this embodiment is the same as in Embodiment 2.

[0390] The SDS-PAGE protein electrophoresis results are shown in Figure 31. Lane 1 contains denatured rhUOX. wt Protein sample, lane 2 contains the mutant rhUOX mut3 / K85C The target protein sample. The target protein is approximately 34 kDa in size, indicating that the target gene was successfully expressed in *E. coli* Rosetta and can be used for subsequent experiments.

[0391] Example 41: rhUOX mut3 / K85C Characterization of the active substances of mutants

[0392] Take 20 μL of purified protein rhUOX at 37℃. mut3 / K85C The reaction was terminated by adding an equal volume of pure methanol after reacting in 600 μL of 0.1 M uric acid solution (pH 8.0) for 10 min. Enzyme activity was calculated by measuring the decrease in UV absorbance at 293 nm using UV spectrophotometry. The control group consisted of inactivated uric acid oxidase. The experimental results are shown in Figure 32. mut3 / K85C The specific enzyme activity was 7.825 U / mg, and the mutant exhibited high activity.

[0393] Enzyme activity unit definition: The amount of enzyme required to catalyze the oxidation of 1 µmol of uric acid per minute is defined as 1 unit.

[0394] E (U / mg) = (△OD) 293 ×V1×D) / (12.3×V2×T×C)

[0395] In the formula, 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.

[0396] Example 42: rhUOX mut3 / K85C Immunogenicity analysis of human PBMC cells with mutants

[0397] Human serum albumin (HAS) and aflatoxin-derived uricase (AfUOX) were used as control groups, while rhUOX was used as the control group. mut3 / K85C For the experimental group, 50 µg / mL of the above protein and PWM (20 µg / mL) were added to each well of a 24-well cell culture plate at a rate of 100 µL (5 µg), followed by PBMC cell suspension (1×10⁻⁶). 6 500 µL per well (dose of 10 µg / mL). Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. After incubation, centrifuge at 3000 rpm for 15 min and collect cells. Add 500 μL of cells to each well of the corresponding protein-coated cell culture plate. Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. Collect the culture supernatant after incubation. Analyze immunogenicity by detecting anti-uricase IgG using indirect ELISA.

[0398] Indirect ELISA steps:

[0399] (1) Antibody coating: Coating the protein samples (HSA, AfUOX, rhUOX) with antibodies. mut3 and rhUOX mut3 / K85C Add 100 µL (5 μg) to each well of a 96-well plate, with two replicates for each sample. Incubate overnight at 4 °C, then wash three times with PBST and pat dry.

[0400] (2) Blocking: Block with ELISA blocking solution for 1 h.

[0401] (3) Washing: Wash 3 times with PBST and pat dry.

[0402] (4) Incubation: Add 100 µL of the supernatant (HSA, AfUOX, rhUOX) obtained from the above cell culture to each well. mut3 and rhUOX mut3 / K85C The blank control was PBS, and the mixture was incubated at 37 °C for 1.5 h.

[0403] (5) Washing: Wash three times with detergent. Pat dry.

[0404] (6) Incubation with HRP-goat anti-human IgG: Add 100 µL of diluted HRP-goat anti-human IgG (1:3000, in PBST, pH 7.4) to each well, and incubate at 37 °C for 1 h.

[0405] (7) Washing: Wash three times with washing solution. Pat dry.

[0406] (8) 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 with a microplate reader 450 value.

[0407] A result is considered positive if A450 of the test well is greater than or equal to 2.1 times that of the negative control well: P / N = OD 450 of test serum / OD 450 of negative control serum (i.e., P / N ≥ 2.1 is positive).

[0408] The results are shown in Figure 33, the mutant rhUOX mut3 / K85C has low immunogenicity, which is comparable to that of HSA.

[0409] Example 43: rhUOX mut3 / K85C Efficacy verification of mutant urate oxidase gene in KO mice in vivo

[0410] In the experiment, 48 C57BL / 6J mice (UOX- / -), half male and half female, aged 5-8 weeks, weighing 25-30 g, SPF grade, were purchased from Cyagen Biosciences Co., Ltd., and housed in the SPF grade experimental animal room of the Experimental Animal Management Center of Jinan University. The room temperature is 20-25 °C. The license for the use of experimental animals is SYXK (Guangdong) 2022-0174. C57BL / 6J mice were given clean drinking water and fed ad libitum for 3 days before the experiment, and then used for experiments.

[0411] A normal control group (normal serum level), a hyperuricemia control group, and an experimental group (rhUOX mut3 and rhUOX mut3 / K85C ) are set up respectively. The normal control group consists of wild-type C57BL / 6J mice, while the other groups use urate oxidase knockout (KO) mice for the experiment, with 12 mice in each group (6 males and 6 females). The normal control group and the hyperuricemia control group are given an equal volume of normal saline, while the experimental group is administered with reference to the dosage of rasburicase (2 U / kg). Administration is performed by intraperitoneal injection, and blood is collected from the orbital vein at 0, 0.5, 1, 3, and 6 hours after administration. Serum treatment is the same as described above. The collected serum is tested for uric acid content by HPLC. The results are shown in Figure 34. In the pharmacodynamic experiment of hyperuricemia KO mice, under the experimental conditions, the immunogenicity-attenuated rhUOX mut3 / K85CIt can maintain the blood uric acid level of KO mice at a normal level for at least 3 hours, and can still exert the effect of lowering uric acid after 6 hours.

[0412] The HPLC detection conditions were the same as in Example 8.

[0413] Regarding the number, location, homology, specific enzyme activity, and immunogenicity of point mutations, the rhUOX urate oxidase mutant described in this invention... mut3 / K85C A comparison with References 1 and 2 mentioned in the "Background Art" section is shown in Table 9 below.

[0414] Table 9:

[0415]

[0416] As shown in Table 9, the human uricase mutant rhUOX described in this invention... mut3 / K85C The mutant showed 95.06% homology to the theoretical amino acid sequence of human uricase (identical to rHU15 reported in Reference 1), and its specific activity was 7.825 U / mg (significantly higher than rHU15 reported in Reference 1). It retained high activity and a high humanization rate while reducing the number of mutation sites. Experiments showed that this mutant had low immunogenicity, similar to that of HSA, because the hotspot amino acids of its protein surface antigen peptide were further mutated, making them unrecognizable and thus reducing immunogenicity. Therefore, the human uricase mutant rhUOX described in this invention... mut3 / K85C It better meets the clinical needs of low-immunogenic uricase, is suitable for long-term clinical use in the treatment of hyperuricemia and gout, and can be used to prepare drugs for the treatment of hyperuricemia and gout.

[0417] Example 44: rhuox wt rhuox mut3 / S86C rhuox mut3 / S86D Gene synthesis was performed, and the recombinant protein was induced to be expressed in E. coli Rosetta (DE3).

[0418] This invention uses the human uricase pseudogene sequence rhuox. wt (GenBank accession number NR_003927.2), the human uricase mutant rhuox was obtained through modification. mut3 / S86C rhuox mut3 / S86D The aforementioned gene was synthesized by BGI Genomics Co., Ltd. and cloned between NcoI and XhoI in the pET28a plasmid (Invitrogen). The recombinant plasmid was named pET28a-huox. wt pET28a-huox mut3 / S86C and pET28a-huoxmut3 / S86D .

[0419] The recipient bacteria in this experiment was *Escherichia coli* Rosetta (DE3), using pET28a-huox. wt pET28a-huox mut3 / S86C and pET28a-huox mut3 / S86D After transformation of the recombinant plasmid, preliminary screening was performed using kanamycin (Kan) resistant plates. Then, single clones from the Kan plates were picked and cultured in 5 mL of LB liquid medium (containing 50 mg / mL Kan) for 12-16 h. The plasmids were then extracted for PCR verification, and positive clones were further screened.

[0420] The positive recombinant was inoculated into 5 mL of LB medium (50 mg / mL Kan) and incubated overnight at 37°C and 200 rpm. The revived bacterial culture was then inoculated at 1% into 500 mL of LB medium (50 mg / mL Kan) and incubated at 37°C and 200 rpm until OD reached [value missing]. 600 The concentration was 0.6, 60 μM lactose was added, and expression was induced at 30℃ and 200 rpm for 12 h. After induction, the bacterial cells were collected, resuspended in carbonate buffer (pH 10.0, mass-to-volume ratio 1:20), and the cells were disrupted by sonication. After centrifugation at 6000g for 10 min, the supernatant was collected to obtain the recombinant protein.

[0421] Example 45: rhUOX wt rhUOX mut3 / S86C and rhUOX mut3 / S86D SDS-PAGE electrophoresis detection of mutant recombinant proteins

[0422] The SDS-PAGE electrophoresis detection method in this embodiment is the same as in Embodiment 2.

[0423] The SDS-PAGE protein electrophoresis results are shown in Figure 35. Lane 1 contains denatured rhUOX. wt Protein sample, lane 2 contains the mutant rhUOX mut3 / S86C Lane 3 is rhUOX mut3 / S86D The target protein sample. The target protein is approximately 34 kDa in size, indicating that the target gene was successfully expressed in *E. coli* Rosetta and can be used for subsequent experiments.

[0424] Example 46: rhUOX mut3 / S86C and rhUOX mut3 / S86D Characterization of the active substances of mutants

[0425] At 37°C, take 20 μL of the purified protein (rhUOX). mut3 / S86C and rhUOXmut3 / S86D In 600 μL of 0.1 M uric acid solution (pH 8.0), the reaction was carried out for 10 min, and then an equal volume of pure methanol was added to terminate the reaction. Enzyme activity was calculated by measuring the decrease in UV absorbance at 293 nm using UV spectrophotometry. The control group consisted of inactivated uric acid oxidase. The experimental results are shown in Figure 36. mut3 / S86C and rhUOX mut3 / S86D The specific activities of the enzymes were 7.72 U / mg and 7.95 U / mg, respectively, and both mutants exhibited high activity.

[0426] Enzyme activity unit definition: The amount of enzyme required to catalyze the oxidation of 1 µmol of uric acid per minute is defined as 1 unit.

[0427] E (U / mg) = (△OD) 293 ×V1×D) / (12.3×V2×T×C)

[0428] In the formula, 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.

[0429] Example 47: rhUOX mut3 / S86C and rhUOX mut3 / S86D Immunogenicity analysis of human PBMC cells with mutants

[0430] Human serum albumin (HAS) and aflatoxin-derived uricase (AfUOX) were used as control groups, while rhUOX was used as the control group. mut3 / S86C and rhUOX mut3 / S86D For the experimental group, 50 µg / mL of the above protein and PWM (20 µg / mL) were added to each well of a 24-well cell culture plate at a rate of 100 µL (5 µg), followed by PBMC cell suspension (1×10⁻⁶). 6 500 µL per well (dose of 10 µg / mL). Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. After incubation, centrifuge at 3000 rpm for 15 min and collect cells. Add 500 μL of cells to each well of the corresponding protein-coated cell culture plate. Incubate at 37°C in a 5% CO2 incubator for 7 days, changing half the medium every 2-3 days. Collect the culture supernatant after incubation. Analyze immunogenicity by detecting anti-uricase IgG using indirect ELISA.

[0431] Indirect ELISA steps:

[0432] (1) Antibody coating: Coating the protein samples (HSA, AfUOX, rhUOX) with antibodies. mut3 / S86C and rhUOX mut3 / S86DAdd 100 µL (5 μg) to each well of a 96-well plate, with two replicates for each sample. Incubate overnight at 4 °C, then wash three times with PBST and pat dry.

[0433] (2) Blocking: Block with ELISA blocking solution for 1 h.

[0434] (3) Washing: Wash 3 times with PBST and pat dry.

[0435] (4) Incubation: Add 100 µL of the supernatant (HSA, AfUOX, rhUOX) obtained from the above cell culture to each well. mut3 / S86C and rhUOX mut3 / S86D The blank control was PBS, and the mixture was incubated at 37 °C for 1.5 h.

[0436] (5) Washing: Wash three times with detergent. Pat dry.

[0437] (6) HRP-goat anti-human IgG incubation: Add 100 µL of diluted HRP-goat anti-human IgG (1:3000, PBST, pH7.4) to each well and incubate at 37 °C for 1 h.

[0438] (7) Washing: Wash three times with detergent. Pat dry.

[0439] (8) Colorimetric reaction: Add 100 µL of freshly prepared TMB working solution to each well and incubate at room temperature in the dark for 15 min. Add 50 µL of stop solution (2 M H2SO4) to each well. Read the OD values ​​using a microplate reader. 450 value.

[0440] A positive result is defined as a test well having an A450 value that is 2.1 times greater than or equal to that of the negative control well: P / N = OD 450 Serum to be tested / OD 450 Negative control serum (i.e., P / N ≥ 2.1 is considered positive).

[0441] The results are shown in Figure 37, for the mutant rhUOX. mut3 / S86C and rhUOX mut3 / S86D It has low immunogenicity, and its immunogenicity is not significantly different from that of HSA (P>0.05).

[0442] Example 48: rhUOX mut3 / S86C and rhUOX mut3 / S86D In vivo efficacy verification of the mutant uricase gene in KO mice

[0443] In the experiment, 48 C57BL / 6J mice (UOX- / -), half male and half female, aged 5-8 weeks, weighing 25-30g, SPF grade, were purchased from Cyagen Biosciences Co., Ltd., and raised in the SPF grade experimental animal room of the Experimental Animal Management Center of Jinan University. The room temperature was 20-25°C, and the laboratory animal use license was SYXK (Guangdong) 2022-0174. C57BL / 6J mice were given clean drinking water and free access to food before the experiment, and were used for the experiment after 3 days of feeding.

[0444] A normal control group (with normal serum level), a hyperuricemia control group, and an experimental group (rhUOX mut3 / S86C and rhUOX mut3 / S86D ) were set up respectively. The normal control group consisted of wild-type C57BL / 6J mice, while the other groups used urate oxidase knockout KO mice for the experiment, with 12 mice in each group (6 males and 6 females). The normal control group and the hyperuricemia control group were given an equal volume of normal saline, while the experimental group was administered with reference to the dosage of rasburicase (2U / kg). Administration was performed via intraperitoneal injection, and blood was collected from the orbital vein at 0, 0.5, 1, 3, and 6 hours after administration. Serum treatment was the same as above, and the collected serum was tested for uric acid content by HPLC. The results are shown in Figure 38. In the pharmacodynamic experiment on hyperuricemic KO mice, under the experimental conditions, the immunogenically attenuated rhUOX mut3 / S86C and rhUOX mut3 / S86D can maintain the blood uric acid level of KO mice at a normal level for at least 3 hours, and can still exert the uric acid-lowering effect after 6 hours.

[0445] The HPLC detection conditions were the same as those in Example 8.

[0446] Regarding the number and positions of point mutations, homology, specific enzyme activity, immunogenicity and other aspects, the urate oxidase mutant rhUOX described in the present invention mut3 / S86C and rhUOX mut3 / S86D compared with Document 1 and Document 2 mentioned in the "Background Art" are shown in Table 10 below.

[0447] Table 10:

[0448]

[0449] It can be seen from Table 10 that the humanized urate oxidase mutant rhUOX described in the present invention mut3 / S86C and rhUOX mut3 / S86DThe homology of the amino acid sequence with human uricase was 95.06% (identical to rHU15 reported in Reference 1). These mutants exhibited specific activities of 7.72 U / mg and 7.95 U / mg, respectively (significantly higher than rHU15 reported in Reference 1), maintaining high activity and a high humanization rate while reducing the number of mutation sites. Experiments showed that these mutants had low immunogenicity, similar to that of HSA, because the hotspot amino acids of their protein surface antigen peptide were further mutated, rendering them unrecognizable and thus reducing immunogenicity. Therefore, the human uricase mutant rhUOX described in this invention... mut3 / S86C and rhUOX mut3 / S86D It better meets the clinical needs of low-immunogenic uricase, is suitable for long-term clinical use in the treatment of hyperuricemia and gout, and can be used to prepare drugs for the treatment of hyperuricemia and gout.

Claims

A highly active, highly humanized, and low immunogenic uricase mutant, characterized by: The mutant is a human uricase mutant obtained by mutating inactive human uricase with the amino acid sequence SEQ ID NO. 1, which has amino acid substitutions at positions 83, 119, 121, 151, 222, 232, 233, 240, and 252. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 1 is characterized in that: The amino acid substitutions at position 83 are: glycine (G) replacing glutamic acid (E); at position 119, arginine (R) replacing histidine (H); at position 121, glutamic acid (E) replacing glycine (G); at position 151, proline (P) replacing glutamine (Q); at position 222, phenylalanine (F) replacing serine (S); at position 232, serine (S) replacing leucine (L); at position 233, proline (P) replacing threonine (T); at position 240, tyrosine (Y) replacing cysteine ​​(C); and at position 252, glutamic acid (E) replacing alanine (A). The amino acid sequence of the uricase mutant is SEQ ID NO.

2. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 1 is characterized in that: It also has amino acid substitutions at positions 208 and 219. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 3 is characterized in that: The amino acid substitution at position 208 is glutamic acid (E) replacing lysine (K), and the amino acid substitution at position 219 is leucine (L) replacing methionine (M); the amino acid sequence of the uricase mutant is SEQ ID NO.

3. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 3 is characterized in that: It also has a substitution at amino acid position 112. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 5 is characterized in that: The amino acid substitution at position 112 is valine (V) replacing methionine (M); the amino acid sequence of the uricase mutant is SEQ ID NO.

4. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 1 is characterized in that: It also has a substitution at the 84th amino acid position. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 7 is characterized in that: The 84th amino acid substitution is the substitution of isoleucine (I) with cysteine ​​(C), histidine (H), asparagine (N), or arginine (R), and the amino acid sequence of the site-directed mutagenesis-modified human uricase mutant is SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO.

8. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 1 is characterized in that: It also has a substitution at the 85th amino acid position. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 9 is characterized in that: The 85th amino acid substitution is cysteine ​​(C) replacing lysine (K); the amino acid sequence of the site-directed mutagenesis-modified human uricase mutant is SEQ ID NO.

9. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 1 is characterized in that: It also has a substitution at the 86th amino acid position. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 11 is characterized in that: The 86th amino acid substitution is a substitution of serine (S) with cysteine ​​(C) or aspartic acid (D); the amino acid sequences of the site-directed mutagenesis-modified human uricase mutant are SEQ ID NO. 10 and SEQ ID NO.

11. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 3 is characterized in that: It also has a substitution at the 84th amino acid position. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 13 is characterized in that: The 84th amino acid substitution is the substitution of isoleucine (I) with cysteine ​​(C), histidine (H), asparagine (N), or arginine (R), and the amino acid sequence of the site-directed mutagenesis-modified human uricase mutant is SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, or SEQ ID NO.

15. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 3 is characterized in that: It also has a substitution at the 85th amino acid position. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 15 is characterized in that: The 85th amino acid substitution is cysteine ​​(C) replacing lysine (K); the amino acid sequence of the site-directed mutagenesis-modified human uricase mutant is SEQ ID NO.

16. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 3 is characterized in that: It also has a substitution at the 86th amino acid position. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 17 is characterized in that: The 86th amino acid substitution is a substitution of serine (S) with cysteine ​​(C) or aspartic acid (D); the amino acid sequences of the site-directed mutagenesis-modified human uricase mutant are SEQ ID NO. 17 and SEQ ID NO.

18. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 5 is characterized in that: It also has a substitution at the 84th amino acid position. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 19 is characterized in that: The 84th amino acid substitution is the substitution of isoleucine (I) with cysteine ​​(C), histidine (H), asparagine (N), or arginine (R), and the amino acid sequence of the site-directed mutagenesis-modified human uricase mutant is SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, or SEQ ID NO.

22. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 5 is characterized in that: It also has a substitution at the 85th amino acid position. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 21 is characterized in that: The 85th amino acid substitution is cysteine ​​(C) replacing lysine (K); the amino acid sequence of the site-directed mutagenesis-modified human uricase mutant is SEQ ID NO.

23. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 5 is characterized in that: It also has a substitution at the 86th amino acid position. The uricase mutant with high activity, high humanization rate, and low immunogenicity according to claim 23 is characterized in that: The 86th amino acid substitution is a substitution of serine (S) with cysteine ​​(C) or aspartic acid (D); the amino acid sequences of the site-directed mutagenesis-modified human uricase mutant are SEQ ID NO. 24 and SEQ ID NO.

25. A DNA molecule characterized by: It encodes a highly active, highly humanized, and low immunogenic uricase mutant as described in any one of claims 1 to 24. The DNA molecule according to claim 25 is characterized in that: Their nucleotide sequences are SEQ ID NO. 26 to SEQ ID NO. 49, respectively. A carrier, characterized in that: It contains the DNA molecule as described in claim 25 or 26. A host cell, characterized by: It contains the DNA molecule as described in claim 25 or 26, or the vector as described in claim 27. A method for producing a highly active, highly humanized, and low immunogenic uricase mutant as described in any one of claims 1 to 24, characterized in that, include: The host cells of claim 28 are cultured under conditions suitable for uricase expression, and the uricase mutant is isolated from the culture medium. The use of the highly active, highly humanized, and low immunogenic uricase mutant as described in any one of claims 1 to 24 in the preparation of a medicament for the treatment of hyperuricemia and gout.

Images