Novel PH20 mutant enzyme

Abstract

The present disclosure provides a new PH20 mutant enzyme which shows significantly enhanced aggregation stability, can be used for the development of drug subcutaneous injection preparations, facilitates the diffusion of drugs in subcutaneous tissues, reduces subcutaneous administration volume limitations, and alleviates swelling or pain caused by large-volume subcutaneous injection.

Description

Technical Field

[0001] The present disclosure relates to the fields of genetic engineering technology and enzymology, and particularly to obtaining a recombinant novel PH20 mutant enzyme using gene mutation technology. The mutated enzyme can be used for the development of subcutaneous injection preparations of antibody drugs to promote the dispersion of drugs in subcutaneous tissues. Background Art

[0002] The human skin consists of three main structures: the epidermis, dermis, and subcutaneous tissue. The subcutaneous tissue has advantages such as abundant capillaries and lymphatic vessels, and low-level protein degradation activity, making it an ideal location for the administration of biological agents.

[0003] Hyaluronic acid is a mucopolysaccharide in which N-acetyl-D-glucosamine and D-glucuronic acid are linked by β-1,4 glycosidic bonds to form disaccharide units, and the disaccharide units are further linked by β-1,3 glycosidic bonds to form a linear high-molecular polysaccharide structure. Hyaluronic acid is an important component of the extracellular matrix in subcutaneous tissues and has the effect of restricting the diffusion of water and other extracellular substances. When treating diseases by subcutaneous injection, hyaluronic acid will hinder the diffusion of drug molecules.

[0004] Hyaluronidase (HAase) is a general term for enzymes that can hydrolyze hyaluronic acid, which can reduce the activity and viscosity of hyaluronic acid in the body, thereby increasing the permeability of fluids in tissues. Hyaluronidase is widely used as a penetration enhancer for drugs, co-formulated with drug molecules to promote the dispersion and absorption of drugs after subcutaneous injection.

[0005] In recent years, protein drugs have developed vigorously, and high-dose drug products with concentration ranges from dozens of milligrams to hundreds of milligrams per milliliter have been developed. The application of recombinant human hyaluronidase (rHuPH20) as a delivery carrier to promote the subcutaneous delivery of protein drugs has also increased. In high-concentration protein drugs, proteins may aggregate more due to high concentrations, thereby reducing the therapeutic effect of the drug and the drug stability. The combined use of rHuPH20 will also cause aggregation, which in turn affects the stability of protein drugs. Patent WO2020022791A1 obtained the rHuPH20 mutant HM7 by multi-point amino acid mutation, and the aggregation temperature (T agg ) increased by 11.5 °C, but the activity only remained at about 15%. In WO2021150079A1, multi-point amino acid mutation and truncation were used to obtain the rHuPH20 mutant HM280, and the aggregation temperature T agg was increased to 59 °C and the enzyme activity was retained. However, a large-scale modification of the protein sequence of the enzyme will cause greater perturbation to the enzyme structure, which may bring immunogenicity problems. Therefore, there is an urgent need to obtain a recombinant hyaluronidase with retained enzyme activity and improved stability with as few modifications as possible. SUMMARY OF THE INVENTION

[0007] The present disclosure provides a mutant of recombinant human hyaluronidase PH20, which exhibits enhanced enzymatic activity and stability compared to wild-type PH20, can rapidly degrade hyaluronic acid, and can be used to promote the dispersion and absorption of drugs after subcutaneous injection.

[0008] The recombinant PH20 mutant enzyme of the present disclosure is derived from human hyaluronidase PH20, and the sequence of its complete wild-type enzyme is shown as SEQ ID NO:1. The PH20 mutant enzyme in the present disclosure is a fragment from the 36th to the 482nd position of SEQ ID NO:1, and the sequence of the fragment is SEQ ID NO:2.

[0009] The PH20 mutant enzyme of the present disclosure is preferably generated by site-directed mutagenesis, including one or more amino acid substitutions in the unmutated PH20 polypeptide, wherein: the unmutated PH20 polypeptide is the polypeptide shown as SEQ ID NO:1 or a truncated polypeptide at its N-terminus or C-terminus, and the truncated polypeptide contains at least the polypeptide shown as SEQ ID NO:3; the PH20 mutant enzyme has hyaluronic acid hydrolysis activity and an increased aggregation temperature relative to wild-type PH20; the positions of the substitutions are based on the amino acid sequence shown as SEQ ID NO:2, and the substitutions include T417V, E31M, I46K, and / or R248Q. In some preferred embodiments, the substitutions are T417V, E31M, and / or I46K. In some more preferred embodiments, the substitution is T417V.

[0010] In some preferred embodiments, the PH20 mutant enzyme of the present disclosure has the following amino acid mutations in an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NOs: 1-3: T417V, E31M, I46K, and / or R248Q, preferably T417V. In some preferred embodiments, the PH20 mutant enzyme of the present disclosure contains at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acid mutations compared to the unmutated PH20.

[0011] Domains and fragments containing the mutation sites of the PH20 mutant enzyme of the present disclosure and having hyaluronidase activity are included in the present disclosure, and any such domains and fragments can be fused with other proteins, including but not limited to HSA, Fc, oleosin, etc. The protein of the mutant can be modified or truncated, including but not limited to the N-terminus and C-terminus.

[0012] Based on the function of the PH20 mutant enzyme of the present disclosure described above, it can be used for the hydrolysis of hyaluronic acid and further for the development of subcutaneous drug formulations.

[0013] In some embodiments, the present disclosure provides a nucleic acid encoding the PH20 mutant enzyme described in the present disclosure.

[0014] In some embodiments, the present disclosure provides a vector comprising the nucleic acid described in the present disclosure.

[0015] In some embodiments, the present disclosure provides a host cell comprising the nucleic acid or the vector described in the present disclosure.

[0016] The present disclosure also provides a pharmaceutical composition comprising the mutant enzyme of the present disclosure. In some embodiments, the pharmaceutical composition further comprises other pharmaceutically active ingredients and / or pharmaceutically acceptable excipients. Preferably, the pharmaceutically active ingredient is an immunoglobulin, a recombinant protein, a synthetic polypeptide, RNA, DNA, or a chemical drug.

[0017] Furthermore, the present disclosure also provides the use of the mutant enzyme in the preparation of a pharmaceutical composition, which can be used to treat diseases, disorders or conditions related to acetyl hyaluronic acid. Preferably, it is used to treat tumors, the accumulation of glycosaminoglycans in the brain, cardiovascular diseases, ophthalmic diseases, pulmonary diseases, cellulitis, proliferative disorders, elevated interstitial tissue pressure, intervertebral disc pressure, or edema.

[0018] In some embodiments, the mutant enzyme is used for delivering a therapeutic agent or for increasing the penetration of a chemotherapeutic agent into a solid tumor.

[0019] In some embodiments, the present disclosure provides a treatment method of administering an effective amount of the mutant enzyme or the pharmaceutical composition of the present disclosure to a subject to treat diseases, disorders or conditions related to acetyl hyaluronic acid, or to treat diseases or conditions with the accumulation of hyaluronidase substrates.

[0020] Preferably, the pharmaceutical composition is formulated for oral administration, or for intravenous (IV), subcutaneous, intramuscular, intratumoral, intradermal, topical, transdermal, rectal, or subepidermal injection.

[0021] Preferably, the pharmaceutical composition is formulated in a form for subcutaneous injection, and the amount of the mutant enzyme in the pharmaceutical composition is sufficient to make the pharmaceutical composition therapeutically effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1Results of SDS-PAGE purification of PH20 WT and mutants. Purification was performed using Polar MC30-NiExcel. Legend: Marker is the molecular weight marker; CS is the supernatant; FT is the flow-through; and the eluted proteins of each mutant of the present disclosure. Detailed Description

[0023] Term

[0024] Unless otherwise defined, the techniques used in the present disclosure are conventional techniques in immunology, molecular biology, microbiology, cell biology, genetic engineering, and protein engineering, and the academic terms used in the present disclosure have the same meaning as commonly understood by those skilled in the art to which the present disclosure pertains. All publications and patent documents mentioned in the present disclosure can be regarded as a reference for those with ordinary knowledge in the relevant technical field.

[0025] The three-letter and single-letter codes for amino acids used herein are as described in J. Biol. Chem., 243, p3558 (1968).

[0026] The mutant enzymes of the present disclosure are obtained by recombinant DNA technology. The terms "mutant" or "mutant enzyme" can be used interchangeably and both refer to the recombinant enzyme after mutation, whose chemical nature is still a protein molecule and has sequence characteristics different from those of the wild-type enzyme.

[0027] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure can be described in terms of ranges. Unless otherwise specified, it should be understood that the use of numerical ranges or descriptions in terms of ranges is only for the purpose of simplicity and convenience, and should not be considered as a strict limitation on the scope of the present disclosure. Therefore, the description in terms of ranges should be considered as specifically disclosing all possible sub-ranges and all possible specific numerical points within that range, as if these sub-ranges and numerical points were explicitly written herein. The above principle applies equally regardless of the width of the numerical values. When a range is described, the range includes the endpoints of the range.

[0028] When referring to measurable values, the term "about" means a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0029] As used herein, the term "hyaluronidase" refers to a general term for enzymes that can hydrolyze hyaluronic acid, and is an enzyme that can reduce the activity of hyaluronic acid in the body and thus improve the liquid penetration ability in tissues. Many pathological processes are often accompanied by changes in hyaluronidase and hyaluronic acid, which can alter the distribution of some drugs and physiologically active substances in the body. Hyaluronidases include hyaluronidase precursors, mature hyaluronidases, active truncated forms, allelic variants and species variants, splice variant-encoded variants and other variants, including polypeptides having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the polypeptide shown in one of SEQ NO:1-3.

[0030] PH20 is a class of hyaluronidases that are known to be expressed in sperm of different species, including those of any origin, such as human, chimpanzee, cynomolgus monkey, etc. As used herein, "PH20" narrowly refers only to human PH20, and its various truncated forms, such as the full-length sequence of 509 residues shown in SEQ ID NO:1, wherein residues 1-35 are the signal peptide and residues 491-509 are the GPI anchor sequence; SEQ ID NO:2 shows the sequence of residues 36-482, which is the full-length sequence with the signal peptide removed and a certain truncation at the C-terminus, and is the sequence currently commercially available from Halozyme sequence. The article by Frost (Gregory I Frost; Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration; Expert Opinion on Drug Delivery, 2007, 4(4), 427-440) discloses that the truncated form of residues 36-467 (relative to SEQ ID NO:1) has enzymatic activity, so SEQ ID NO:3 is the shortest truncated form of the currently published wild-type rHuPH20, which removes residues 1-35 and residues 468-509 of the full-length sequence SEQ ID NO:1.

[0031] The term "recombinant human hyaluronidase (rHuPH20)" in this article refers to human hyaluronidase PH20 expressed by genetic engineering means, which can be in truncated form, can contain various mutations, and can also contain those with chemical or post-translational modifications as well as those without chemical or post-translational modifications. Such modifications include, but are not limited to, pegylation, albuminization, glycosylation, farnesylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art.

[0032] The term "WT" refers to wild-type hyaluronidase, that is, an enzyme that has not been artificially modified or has no mutations in the amino acids in the sequence, and in the examples herein, it narrowly refers only to the enzyme represented by the sequence shown in SEQ ID NO:2.

[0033] The term "unmutated" includes WT and also includes various truncated forms of the sequence that can achieve the hydrolytic activity of hyaluronidase, but does not include amino acid mutations. The "unmutated PH20 polypeptide" is the polypeptide shown in SEQ ID NO:1 or its truncated polypeptide at the N-terminus or C-terminus, and the truncated polypeptide contains at least the polypeptide shown in SEQ ID NO:3; that is, the group composed of any truncated form between the full-length sequence SEQ ID NO:1 and the shortest truncated sequence SEQ ID NO:3.

[0034] The term "vector" in this article refers to a self-replicating DNA molecule in genetic engineering and recombinant DNA technology, which is a tool for transferring a target gene into a recipient cell. These vectors can not only self-replicate, carry the target gene into the host cell, and enable it to be expressed or replicated in the host cell. A plasmid is a small circular DNA molecule present in bacterial cells that can replicate autonomously and is one of the most commonly used vectors. In addition to plasmid vectors, there are also viral vectors, phage vectors, etc.

[0035] The term "recombinant expression vector" in this article refers to a vector constructed by using molecular cloning technology. By inserting foreign genes into the vector, these genes can be expressed in host cells. This technology is not only very important in basic biological research but also a key tool in the biotechnology and pharmaceutical industries.

[0036] The term "transfection" in this article refers to a method of introducing a plasmid into a cell using chemical or physical means. The plasmid can be inserted into the genome or remain free in the cytoplasm, thereby enabling the cell to express the target protein. Transfection is divided into transient transfection and stable transfection. In this disclosure, transient transfection is carried out by a chemical method using liposomes, and the plasmid containing the target gene remains free in the cytoplasm.

[0037] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. The term "pharmaceutical excipient" or "pharmaceutically acceptable excipient" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle administered together with a therapeutic agent.

[0038] As used herein, the terms "disease" or "disorder" or "ailment" etc. refer to any alteration or derangement that impairs or interferes with the normal function of cells, tissues, or organs. For example, the "diseases" described include, but are not limited to: tumors, pathogen infections, autoimmune diseases, T cell dysfunctional diseases, or immune tolerance deficiencies (such as transplant rejection), etc.

[0039] As used herein, the term "treatment" refers to a clinical intervention in an attempt to modify an individual or to address a disease caused by cells, either for prevention or during the clinical pathological process. Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the progression of a disease, improving or alleviating the condition, and relieving or improving the prognosis, etc.

[0040] As used herein, "acetyl hyaluronic acid-related diseases, disorders or conditions" refers to any such disease or condition in which increased levels of acetyl hyaluronic acid are a cause, consequence or manifestation of the disease or condition. Acetyl hyaluronic acid-related diseases and conditions are associated with increased acetyl hyaluronic acid expression in tissues or cells, increased interstitial tissue pressure, reduced vascular volume and / or increased water content in tissues. Acetyl hyaluronic acid-related diseases, disorders or conditions can be treated by administering a composition comprising an acetyl hyaluronic acid-degrading enzyme such as hyaluronidase, e.g., soluble hyaluronidase, either alone or in combination with or in addition to another therapy and / or medicament. Exemplary diseases or conditions include, but are not limited to, cancers enriched in acetyl hyaluronic acid, such as tumors, including solid tumors such as advanced cancers, metastatic cancers, undifferentiated cancers, ovarian cancer, in situ carcinomas (ISC), squamous cell carcinomas (SCC), prostate cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, colon cancer and other cancers. Exemplary acetyl hyaluronic acid-related diseases and conditions are also diseases associated with increased interstitial tissue pressure, such as diseases associated with disc pressure and edema, e.g., edema due to organ transplantation, stroke, traumatic brain injury or other injuries. Exemplary acetyl hyaluronic acid-related diseases and conditions include diseases and conditions associated with increased interstitial tissue pressure, reduced vascular volume and / or increased water content in tissues, including cancer, disc pressure and edema. In one example, treatment of an acetyl hyaluronic acid-related disorder, disease or condition comprises having an improving, alleviating or other beneficial effect on one or more of increased interstitial tissue pressure (IFP), reduced vascular volume and increased water content in tissues.

[0041] The annotation of the amino acids contained in the molecules of the present disclosure conforms to the conventions in the art, and the position of the mutation is indicated by the single-character symbol of the wild-type amino acid and its number. For example, Thr at position 417 is referred to as "T417". The mutation is indicated by the single-character symbol of the wild-type amino acid, its number and the single-character symbol of the amino acid after mutation. For example, the mutation of replacing Thr at position 417 with Val is referred to as "T417V", and if the mutant has multiple mutations, the multiple mutations are indicated using the separator " / " between them.

[0042] In the present disclosure, the PH20 mutant T417 is a mutant composed of a sequence in which Thr at position 417 of the wild-type PH20 shown in SEQ ID NO:2 is replaced by other natural amino acids.

[0043] The mutants of the present disclosure do not have to have a full-length sequence, as long as the regions important for the activity of PH20 are retained, which are within the scope of the present disclosure. On this basis, recombinant proteins that contain the said fragment and appropriately add, for example, a signal peptide sequence, a purification tag (such as His-tag), a linker sequence (such as GGGS) or other functional components at the N-terminus or C-terminus to form a new fusion protein without affecting the activity of PH20 are within the scope of protection of the present disclosure. On this basis, truncated forms that contain the said fragment and are appropriately deleted at the N-terminus or C-terminus are also within the scope of the present disclosure.

[0044] In the amino acid sequence of the mutants of the present disclosure, one to several amino acids can be substituted, deleted, inserted and / or added at positions other than the positions of the following essential mutations to the extent that the enzyme activity is not affected. Any position can be selected for such amino acid changes as long as it does not affect the enzyme activity. In the present disclosure, the term "several" means 20 or fewer, preferably 10 or fewer, more preferably 5 or fewer, and most preferably 4, 3, 2 or 1.

[0045] When used in the specification of the present disclosure, a reference without a specific number can mean one or more. When used in the claims of the present disclosure, when combined with the word "comprising", a reference without a specific number can mean one or more. When used in the present disclosure, "another" can mean at least a second or more.

[0046] As used herein, "high purity" means that the target protein is separated from the contaminants that accompany it in its natural state or the contaminants generated or used in the process of obtaining the target protein. Generally, when the gray scale of the target protein band accounts for at least 90% of the total gray scale of all bands after gel electrophoresis separation, the target protein is of high purity. Preferably, in some embodiments, the gray scale of the target protein accounts for at least 95% of the total gray scale of all bands, and most preferably at least 98%.

[0047] The present disclosure further provides a recombinant gene encoding the above-mentioned PH20 mutant, a gene construct such as a plasmid or an expression vector containing the recombinant gene, a host cell transformed with the gene construct, a method for producing the mutant of the present disclosure, which includes the step of collecting the mutant of the present disclosure from a culture of the host cell, etc. The said recombinant gene, gene construct, host cell, etc. can be prepared based on the amino acid sequence of the mutant of the present disclosure according to known genetic engineering techniques.

[0048] The host cells transformed by introducing the gene encoding the mutant of the present disclosure can be cultured under appropriate conditions according to the cell type (cells commonly used for protein production, such as animal cells, plant cells, Escherichia coli, yeast, etc., can be appropriately selected), and the mutant of the present disclosure can be collected from the culture. The collection of the mutant is carried out by appropriately combining conventional purification techniques based on the physical properties of the protein. To facilitate the collection, a gene construct can be designed to express the mutant in the form of a tag peptide such as GST, which is pre-connected to the mutant to enable collection using the affinity for the tag peptide. The tag peptide can be removed after purification, but when it has no effect on the enzymatic activity of the mutant, the mutant with the tag peptide connected thereto can be used for reactions such as hyaluronic acid hydrolysis. The mutant of the present disclosure includes such an amino acid sequence that contains a tag peptide connected thereto.

[0049] The features and advantages of the present disclosure are more fully demonstrated by the following non-limiting examples.

[0050] Example

[0051] Materials and Methods

[0052] In the present disclosure, ExpiFectamine CHO reagent and ExpiCHO expression medium were purchased from Thermo Fisher, horse serum was purchased from Gibco brand, phosphate buffer solution was purchased from Adamas Life brand, hyaluronidase standard was purchased from the National Institutes for Food and Drug Control, hyaluronic acid was purchased from TCI company, and the remaining reagents were all conventional reagents produced by Merck, Sigma, Sinopharm Group, etc. without special instructions. The fillers, chromatographic columns, and instruments used in the present disclosure include: mPES hollow fiber column (10 kDa, Ripley Jin (Shanghai) Biotechnology Co., Ltd.), Polar MC30-Ni Excel 1 mL (Sepax) chromatographic column, gel imager (Gel Doc EZ Imager, Bio-RAD), PR.NT.Plex (Nano Temper) protein stability analyzer.

[0053] Construction of Recombinant Plasmids for Site-Directed Mutagenesis of Recombinant PH20 Wild-Type Enzyme and Its Mutant Enzymes

[0054] The cDNA encoding the amino acid sequence of human hyaluronidase PH20 was amplified by PCR, and a His-tag protein (6×His) was ligated to the C-terminus, and then inserted between XbaI and AflII in the multiple cloning site region of the vector plasmid pCDNA3.4. The codons of the mutant amino acids were introduced with primers containing mutations, and recombinant plasmids containing the nucleic acid sequences of wild-type PH20 and its mutants were produced by Escherichia coli DH5α strain.

[0055] Expression and Purification of Recombinant PH20 Wild-Type Enzyme and Its Mutant Enzymes

[0056] The wild-type PH20 and PH20 mutants were expressed by transient transfection in ExpiCHO cells. One day before transfection, the cells were passaged to a final viable cell density of 3E6 - 4E6 cells / mL and allowed to grow overnight. When the cell density reached 7E6 - 10E6 cells / mL and the cell viability reached 95 - 99% on the day of transfection, the recombinant plasmids of PH20 WT or mutants were transfected into ExpiCHO cells using ExpiFectamine CHO reagent. Based on the ExpiCHO expression medium, continuous culture was carried out in a shaker at 37°C and 5% CO2 with an oscillation rate of 130 rpm for 7 days to achieve the expression of the PH20-6×His fusion protein (hereinafter referred to as PH20 protein).

[0057] After the culture was completed, the cell supernatant containing wild-type PH20 or PH20 mutant protein was obtained by centrifugation at 3000 rpm for 30 min, replaced into the equilibration buffer (20 mM PB, 500 mM NaCl, 10 mM imidazole, pH 7.5) using an mPES hollow fiber column (10 kDa), and then the PH20 protein was purified using a Polar MC30-Ni Excel 1 mL chromatography column. The fractions containing the PH20 protein were concentrated using an Amicon centrifugal filter (10 kDa, Millipore) and stored in the storage buffer (10 mM HEPES, 130 mM NaCl, pH 7.0). The protein purity was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and a Gel Doc EZ Imager (Bio-RAD), Image Lab scanning software, and the protein concentration was quantified using a spectrophotometer (Nano-300).

[0058] Stability of Wild-Type PH20 and Its Mutants

[0059] The T was detected by a PR.NT.Plex protein stability analyzer agg . The PH20 WT and mutant proteins were replaced into a buffer of 20 mM histidine, 130 mM NaCl, pH 6.5 using an Amicon centrifugal filter (10 kDa, Millipore), diluted to 0.5 mg / mL, centrifuged, and then aspirated into a capillary chip and placed in the detection cell for detection. The heating program was set as: 30°C - 95°C, 0.5°C / min. After the detection was completed, analysis software was used for analysis.

[0060] Turbidimetric Assay of Enzyme Activity of Wild-Type PH20 and Its Mutants

[0061] Prepare the following reagents: (1) Acetate buffer solution - Dissolve 14.0 g of potassium acetate and 25.0 mL of glacial acetic acid in water to make 1000 mL of solution. (2) Phosphate buffer solution - Purchased from Adamas Life. (3) Enzyme dilution stock solution - 500 mL of phosphate buffer solution plus 500 mL of water. (4) Enzyme dilution working solution - Add 33 mg of hydrolyzed gelatin to 50 mL of the enzyme dilution stock solution - use within 2 hours after preparation. (5) Sample stabilization buffer solution (SSB) - Add 125 μL of 20% human serum albumin solution and 50 μL of 1 M calcium chloride solution to 50 mL of the enzyme dilution working solution and mix well. (6) Serum stock solution - Dilute 1 volume of horse serum with 9 volumes of acetate buffer solution. Adjust the pH to 3.1 with 4 N hydrochloric acid and keep the solution at room temperature for 18 to 24 h. Store the solution at 4 °C and use within 30 days. (7) Serum working solution - Add 10 mL of the serum stock solution to 30 L of acetate buffer solution and return to room temperature after preparation. (8) Hyaluronic acid stock solution - Prepare an aqueous solution of sodium hyaluronate at a concentration of 5.0 mg / mL. (9) Hyaluronic acid working solution - Add 0.75 mL of the hyaluronic acid stock solution to 4.25 mL of phosphate buffer solution. (10) Standard stock solution - Prepare an aqueous solution of the national standard hyaluronidase (STD) at a concentration of 1000 units / mL in a container, aliquot into 50 μL portions, and store at -20 °C. (11) Standard working solution - Add 40 μL of the standard stock solution to 960 μL of cold enzyme dilution working solution to obtain a solution with a known concentration of 40 units / mL and use immediately for determination after preparation.

[0062] Dilute all enzyme samples in a Low Protein Binding 96-well plate according to the following instructions.

[0063] a) The maximum sensitivity range of this assay is between 10 - 30 units / mL. To obtain results within this range, the assay needs to be repeated. To minimize the number of repetitions, first determine the approximate total units / mL of the sample, and then select a dilution factor (an integer) so that the final concentration is approximately 20 units / mL.

[0064] b) The minimum sample volume required to perform the assay is described as follows: FPLC fraction = 50 μL, tissue culture supernatant = 1 mL, purified / concentrated / final step material = 10 μL.

[0065] c) For serially diluted samples, perform a 1:10 dilution in triplicate on a low-protein-binding 96-well plate by pipetting 360 μL of the SSB solution and 40 μL of the sample into each well.

[0066] Prepare standard solutions (40, 32, 24, 16, 8, 4, 0 units / mL) using the enzyme dilution working solution and STD for plotting the standard curve.

[0067] Reaction plate: Pipette the hyaluronic acid working solution into a flat-bottom 96-well microtiter plate at a volume of 30 μL per well.

[0068] Preheating stage: Place the low-protein-binding 96-well plate containing the diluted samples, standards, and controls, and the flat-bottom 96-well plate containing the hyaluronic acid working solution on a heat block and preheat them at 37 °C for 5 minutes.

[0069] Initiate the reaction by adding the enzyme to the substrate: Add 30 μL of the preheated enzyme solution from the enzyme standard solution and the sample plate to be tested into the wells of a 96-well flat-bottom plate (containing the substrate) respectively, mix well for the hydrolysis reaction, and the reaction conditions are 37 °C for 6 min.

[0070] Terminate the reaction: Pipette 240 μL of the serum working solution into each reaction well and mix well, and measure the absorbance at 640 nM. Generate a linear curve fitting from the standard curve, from which the samples to be tested can be inferred.

[0071] Example 1 Preparation of Recombinant Proteins of Wild-Type PH20 and PH20 Mutants

[0072] Human hyaluronidase PH20 (EC 3.2.1.35) belongs to the glycoside hydrolase family 56 (GH56), with a full length of 509 amino acid residues, as shown in SEQ ID NO:1. Among them, positions 1-35 are the signal peptide, and positions 491-509 are the GPI anchor sequence, which can randomly hydrolyze the β-1,4 glycosidic bond between N-acetyl-D-glucosamine and D-glucuronic acid in hyaluronic acid. The tertiary structure of human PH20 has not been clearly resolved. Based on the structural simulation of the homologous human Hyal1 (PDB ID: 2PE4) and bee venom hyaluronidase (PDB ID: 1FCV) in the same family, the catalytic domain of human PH20 presents a (β / α)8 barrel configuration, that is, composed of eight repeats of one β-strand and one α-helix, and D146 and E148 (relative to the sequence position of SEQ ID NO:1) are the catalytic residues.

[0073] Referring to the binding mode of bee venom hyaluronidase (PDB ID: 1FCV) and the substrate hyaluronic acid, in order to improve the stability of PH20 without affecting its activity, residues that are far from the substrate, located on the protein surface, located in flexible loop joints or α-helices interacting with surface loops are selected as the cleavage sites. In the embodiments of the present disclosure, SEQ ID NO: 2 is used as the basis of WT, and site-directed mutations are performed at positions E31, I46, R248, and T417 corresponding to the amino acid sequence of SEQ ID NO: 2, including but not limited to E31M, I46K, R248Q, and T417V. Wild-type human hyaluronidase PH20 and its mutants are recombinantly expressed in ExpiCHO cells by adding His-tag protein at the C-terminus, and can be purified through a Polar MC30-Ni Excel 1 mL chromatography column to obtain high-purity PH20 protein, as Figure 1 shown.

[0074] Example 2 Detection of Aggregation Temperature of Wild-Type PH20 and Its Mutants

[0075] The aggregation temperature (T agg ) is an indicator of protein stability. In the co-formulation development of protein drugs and PH20, PH20 is used as a permeation enhancer for drugs to promote the dispersion and absorption of drugs after subcutaneous injection. The improvement of PH20 stability can reduce the risk of enzyme aggregation and inactivation during the storage of co-formulations, and ensure the dispersion and absorption effect of drugs after injection. Protein aggregation is usually irreversible, and aggregation tends to form larger aggregates. After PH20 aggregates, it may affect the activity of drug proteins through enzyme-protein drug interactions and increase the risk of immunogenicity. In addition, the increase of PH20 T agg is beneficial to reducing the risk of increased viscosity of high-concentration co-formulation drugs, allowing higher-concentration drug administration, and can also relieve the pain at the injection site of patients caused by high-viscosity formulations. In the present disclosure, the aggregation temperatures of PH20 WT and its mutants are measured, and the results are shown in Table 1. The T agg of PH20 WT is 46.7 °C, which is consistent with the PH20 WT T agg of 46.5 °C in patent WO2020022791A1. Compared with WT, the T agg of E31M and I46K is increased to 48 °C and above, indicating an appropriate improvement in stability; the aggregation temperature of the mutant R248Q is decreased by 8.7 °C; surprisingly, the aggregation temperature of the mutant T417V is increased to 64.2 °C, which is 17.5 °C higher than that of WT. Historical data shows that the T agg of the HM4 mutant in patent WO2020022791A1 is 56.5 °C, and the T aggis 59°C. In the present disclosure, the T417V mutant is the one with the highest T among human hyaluronidase PH20 and its mutants agg mutant.

[0076] Table 1 Aggregation temperature of hyaluronidase PH20 WT and each mutant

[0077]

[0078] Example 3 Detection of Enzyme Activity of Wild-Type PH20 and Its Mutants

[0079] The activity of hyaluronidase was detected by the turbidimetry method of the Chinese Pharmacopoeia. The principle of the turbidimetry method is based on the formation of a precipitate between hyaluronic acid and serum under acidic conditions. When hyaluronic acid is decomposed, the precipitate in the mixture with serum will decrease. Therefore, hyaluronic acid was used as a substrate to detect the activity of hyaluronidase, and the activity of hyaluronidase was calculated by measuring the absorbance of the mixed solution of hyaluronic acid and serum under acidic conditions. The enzyme activity data of PH20 WT and its mutants are shown in Table 2. Among them, the enzyme activities of mutants R248Q and T417V are comparable to that of WT, and mutants E31M and I46K have significantly improved enzyme activities, with activities being 165% and 178% of WT respectively.

[0080] Table 2 Enzyme activity of hyaluronidase and each mutant

[0081]

[0082]

[0083] Combined with the aggregation temperature detection results in Example 2, in the present disclosure, a T417V single-point mutant with enzyme activity equivalent to that of WT and T agg being at the known best level (64.2°C) was obtained. In patent WO2020022791A1, the T of the HM4 mutant agg is 56.5°C, but the enzyme activity only remains about 15%. In WO2021150079A1, the T of the HM280 mutant agg is 59°C and retains the enzyme activity. However, this mutant is obtained by multi-point amino acid combination mutation and truncation, and such a large-scale modification of the protein sequence may bring potential immunogenicity problems. In addition, in the present disclosure, mutants E31M and I46K with T agg improved to 48.0 and 48.2°C (WT 46.7°C) and activity increased to 165% and 178% of WT were also obtained.

[0084] The present disclosure describes mutant enzymes derived from human hyaluronidase PH20, which include the mutation sites E31M, I46K, and / or T417V. Compared with the previously reported PH20 and its mutants, the aggregation temperature of T417V is 64.2 °C, the highest level currently known, which is 17.5 °C higher than that of the WT, and at the same time, it also maintains an enzyme activity comparable to that of the WT. While E31M and I46K have enhanced enzyme activities (165%, 178%), they also improve the aggregation temperature to some extent (increased by 1.3 °C and 1.5 °C respectively). Therefore, the mutant enzymes of the present disclosure have better application effects in protein drug co-formulations.

[0085] Those of ordinary skill in the art to which the present disclosure pertains should understand that the above embodiments are only for illustration, and various changes and modifications can be made to the mutant proteins without departing from the scope of the present disclosure, including but not limited to insertions and / or truncations at the N-terminus and / or C-terminus, modifications such as glycosylation, sialylation, albuminization, farnesylation, carboxylation, hydroxylation, phosphorylation, and conjugation to polymers of the protein. Importantly, it has hyaluronidase activity and contains mutations of T417V, E31M, I46K, and / or R248Q relative to the sequence of human hyaluronidase PH20 SEQ ID NO:2.

Claims

1. A PH20 mutant enzyme comprising one or more amino acid substitutions in an unmutated PH20 polypeptide, wherein: The unmutated PH20 polypeptide is the polypeptide shown in SEQ ID NO:1 or a truncated polypeptide at its N-terminus or C-terminus, and the truncated polypeptide comprises at least the polypeptide shown in SEQ ID NO:3; the PH20 mutant enzyme has hyaluronic acid hydrolysis activity and an increased aggregation temperature relative to wild-type PH20; the positions of substitution are based on the amino acid sequence shown in SEQ ID NO:2, and the substitutions include T417V, E31M, I46K, and / or R248Q.

2. A nucleic acid encoding the mutant enzyme according to claim 1.

3. A vector comprising the nucleic acid according to claim 2.

4. A host cell comprising the nucleic acid according to claim 2 or the vector according to claim 3.

5. A pharmaceutical composition comprising the mutant enzyme according to claim 1.

6. The pharmaceutical composition according to claim 5, which further comprises other pharmaceutically active ingredients and / or pharmaceutically acceptable excipients. Preferably, the pharmaceutically active ingredient is an immunoglobulin, a recombinant protein, a synthetic polypeptide, RNA, DNA, or a chemical drug.

7. Use of the mutant enzyme according to claim 1 in the preparation of a pharmaceutical composition for treating diseases, disorders, or conditions associated with acetyl hyaluronic acid. Preferably, the pharmaceutical composition is used for treating tumors, accumulation of glycosaminoglycans in the brain, cardiovascular diseases, ophthalmic conditions, pulmonary diseases, cellulitis, proliferative conditions, elevated interstitial tissue hydrostatic pressure, disc pressure, or edema.

8. Use of the mutant enzyme according to claim 1 in the preparation of a pharmaceutical composition for treating diseases or conditions with accumulation of hyaluronidase substrates.

9. Use of the mutant enzyme according to claim 1 in the preparation of a pharmaceutical composition for delivering a therapeutic agent or for increasing the penetration of a chemotherapeutic agent into a solid tumor.

10. Use according to any one of claims 7-9, wherein, The pharmaceutical composition is formulated for oral administration, or for administration by intravenous, subcutaneous, intramuscular, intratumoral, intradermal, topical, transdermal, rectal, or subepidermal injection, and the amount of the mutant enzyme in the pharmaceutical composition is sufficient to render the pharmaceutical composition therapeutically effective.

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