Novel ph20 mutant enzymes

Abstract

The present disclosure provides novel PH20 mutant enzymes that exhibit significantly enhanced aggregation stability, are useful for the development of pharmaceutical subcutaneous injection formulations, facilitate diffusion of pharmaceuticals in subcutaneous tissue, reduce the volume limitations for subcutaneous administration and alleviate swelling or pain from large volume subcutaneous injections.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of genetic engineering and the field of enzymology, in particular to obtaining a recombinant novel PH20 mutant enzyme using genetic mutation technology, the mutated enzyme can be used for the development of subcutaneous injection preparations of antibody drugs, and promotes the dispersion of drugs in subcutaneous tissue. BACKGROUND

[0002] Human skin is composed of three main structures of epidermis, dermis and subcutaneous tissue, subcutaneous tissue has the advantages of rich capillary and lymphatic vessels, low level of protein degradation activity, etc., which is an ideal location for biological drug administration.

[0003] Hyaluronic acid is a mucopolysaccharide, N-acetyl-D-glucosamine and D-glucuronic acid are connected by β-1,4 glycosidic bond to form disaccharide units, and the disaccharide units are connected by β-1,3 glycosidic bond to form a linear high molecular weight polysaccharide structure. Hyaluronic acid is an important component of the extracellular matrix in subcutaneous tissue, which has the function of limiting the diffusion of water and other extracellular substances. When drug treatment is performed 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 improving the permeability of the liquid in the tissue. Hyaluronidase is widely used as a penetration enhancer for drugs, and is co-formulated with drug molecules to promote the dispersion and absorption of drugs after subcutaneous injection.

[0005] In recent years, protein drugs have developed rapidly, and high-dose drug products with concentrations ranging from several dozen milligrams to several hundred milligrams per milliliter have been developed. The use 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 increase aggregation due to high concentration, thereby reducing drug treatment effect and drug stability. The use of rHuPH20 can also cause aggregation, which affects the stability of protein drugs. Patent WO2020022791A1 uses multiple point amino acid mutations to obtain rHuPH20 mutant HM7, the aggregation temperature (T agg ) is increased by 11.5℃, but the activity is only about 15%. WO2021150079A1 uses multiple point amino acid mutations and truncation to obtain rHuPH20 mutant HM280, the aggregation temperature T agg is increased to 59℃, and the enzyme activity is retained, but the substantial modification of the protein sequence of the enzyme will greatly disturb the structure of the enzyme, which may cause immunogenicity problems. Therefore, there is an urgent need to obtain a recombinant hyaluronidase with improved stability while retaining enzyme activity with as few modifications as possible. SUMMARY

[0006] The present disclosure provides a mutant of recombinant human hyaluronidase PH20, which has enhanced enzymatic activity and stability compared to wild-type PH20, and can rapidly degrade hyaluronic acid, and can be used to promote the dispersion and absorption of drugs after subcutaneous injection.

[0007] The recombinant PH20 mutant enzyme of the present disclosure is derived from human hyaluronidase PH20, the sequence of the complete wild-type enzyme of which is shown in SEQ ID NO: 1, and the PH20 mutant enzyme in the present disclosure is a fragment of SEQ ID NO: 1 from position 36 to position 482, the sequence of which is SEQ ID NO: 2.

[0008] The PH20 mutant enzyme of the present disclosure, preferably by site-directed mutagenesis, comprises 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 the N-terminus or C-terminus thereof, and the truncated polypeptide comprises at least the polypeptide shown in SEQ ID NO: 3; the PH20 mutant enzyme has hyaluronidase activity and an increased aggregation temperature relative to wild-type PH20; the position of the substitution is based on the arrangement of the amino acids shown in SEQ ID NO: 2, and the substitution comprises T417V, E31M, I46K and / or R248Q. In some preferred embodiments, the substitution is T417V, E31M and / or I46K. In some more preferred embodiments, the substitution is T417V.

[0009] 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 to SEQ ID NO: 1-3: T417V, E31M, I46K and / or R248Q, preferably T417V. In some preferred embodiments, the PH20 mutant enzyme of the present disclosure comprises at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 amino acid mutations compared to the unmutated PH20.

[0010] Domains and fragments comprising the mutation sites of the PH20 mutant enzyme described in the present disclosure and having hyaluronidase activity are all included in the present disclosure, and any such domain and fragment can be fused to other proteins, including but not limited to HSA, Fc, oleosin, etc. The mutant protein can be modified or truncated, including but not limited to the N-terminus, C-terminus.

[0011] Based on the aforementioned function of the PH20 mutant enzyme disclosed herein, it can be used for the hydrolysis of hyaluronic acid and further for the development of subcutaneous drug formulations.

[0012] In some embodiments, this disclosure provides a nucleic acid that encodes the PH20 mutant enzyme described herein.

[0013] In some embodiments, this disclosure provides a carrier that contains the nucleic acid described herein.

[0014] In some embodiments, this disclosure provides a host cell that contains the nucleic acid or the vector described in this disclosure.

[0015] This disclosure also provides a pharmaceutical composition comprising the mutant enzyme of this disclosure. In some embodiments, the pharmaceutical composition further comprises other pharmaceutically active ingredients and / or pharmaceutically acceptable excipients, preferably, the pharmaceutically active ingredients being immunoglobulins, recombinant proteins, synthetic peptides, RNA, DNA, or chemical drugs.

[0016] Furthermore, this disclosure also provides the use of the mutant enzyme in the preparation of pharmaceutical compositions for the treatment of diseases, disorders, or conditions related to acetylated hyaluronic acid, preferably for the treatment of tumors, glycosaminoglycan accumulation in the brain, cardiovascular diseases, ophthalmic conditions, lung diseases, cellulite, proliferative conditions, elevated interstitial fluid pressure, intervertebral disc pressure, or edema.

[0017] In some embodiments, the mutant enzyme is used to deliver therapeutic agents or to increase the penetration of chemotherapeutic agents into solid tumors.

[0018] In some embodiments, this disclosure provides a treatment method in which a subject is given an effective amount of the mutant enzyme or pharmaceutical composition of this disclosure to treat diseases, disorders or conditions related to acetylhyaluronic acid, or to treat diseases or conditions of hyaluronidase substrate accumulation.

[0019] Preferably, the pharmaceutical composition is formulated for oral, intravenous (IV), subcutaneous, intramuscular, intratumoral, intradermal, local, transdermal, rectal, or subepidermal injection.

[0020] Preferably, the pharmaceutical composition is formulated for subcutaneous injection, and the amount of mutant enzyme in the pharmaceutical composition is sufficient to make the pharmaceutical composition effective in treatment. Attached Figure Description

[0021] Figure 1These are the results of SDS-PAGE purification of PH20 WT and mutants. Purification was performed using a Polar MC30-NiExcel. Legend: Marker is the molecular weight marker; CS is the supernatant; FT is the flow-through buffer; and the eluted proteins of the various mutants disclosed herein. Detailed Implementation

[0022] Terminology

[0023] Unless otherwise defined, conventional techniques in immunology, molecular biology, microbiology, cell biology, genetic engineering, and protein engineering are used in this disclosure, and academic terms used in this disclosure have the same meanings commonly understood by one of ordinary skill in the art. All publications and patent documents mentioned in this disclosure are to be regarded as hints to those skilled in the art.

[0024] The three-letter and single-letter codes for amino acids used in this article are as described in J. Biol. Chem, 243, p3558 (1968).

[0025] The mutant enzyme disclosed herein is obtained by recombinant DNA technology. The terms "mutant" or "mutant enzyme" are used interchangeably and both refer to the mutated recombinant enzyme. Its chemical nature is still a protein molecule, and it has sequence characteristics that distinguish it from wild-type enzymes.

[0026] Some embodiments disclosed herein include numerical ranges, and certain aspects of this disclosure may be described using ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions using ranges are for purposes of brevity and convenience only and should not be considered as a strict limitation of the scope of this disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible specific numerical points within those ranges, as these subranges and numerical points have been explicitly stated herein. The above principles apply equally regardless of the breadth of the numerical values ​​described. When a range description is used, the range includes the endpoints of the range.

[0027] 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.

[0028] The term "hyaluronidase" in this article refers to a general term for enzymes that hydrolyze hyaluronic acid. It is an enzyme that reduces the activity of hyaluronic acid in the body, thereby increasing the permeability of 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. Hyaluronidase includes hyaluronidase precursors, mature hyaluronidase, active truncated forms, allelic variants and species variants, variants encoded by splice 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 with the polypeptides shown in any one of SEQ NO:1-3.

[0029] PH20 is a type of hyaluronidase known to be expressed in the sperm of various species, including those from any origin, such as humans, chimpanzees, cynomolgus monkeys, etc. In this article, "PH20" refers specifically to human PH20, and its various truncated forms, such as the full-length sequence of 509 residues shown in SEQ ID NO:1, where residues 1-35 are the signal peptide and residues 491-509 are the GPI anchoring sequence; and the sequence shown in SEQ ID NO:2, which is the full-length sequence with the signal peptide removed and a truncated C-terminus, consisting of residues 36-482, and is currently commercially available from Halozyme. The sequence of Frost. Frost’s article (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) disclosed that the truncated form of residues 36-467 (relative to SEQ ID NO:1) has enzyme activity. Therefore, SEQ ID NO:3 is the shortest truncated form of wild-type rHuPH20 currently disclosed, which removes residues 1-35 and residues 468-509 of the full-length sequence SEQ ID NO:1.

[0030] The term "recombinant human hyaluronidase (rHuPH20)" as used herein refers to human hyaluronidase PH20 expressed through genetic engineering. This expression may be truncated, may contain various mutations, and may include both chemical and post-translational modifications. Such modifications include, but are not limited to, PEGylation, albuminization, glycosylation, farnesylation, carboxylation, hydroxylation, phosphorylation, and other peptide modifications known in the art.

[0031] The term "WT" refers to wild-type hyaluronidase, which is an enzyme that has not been artificially modified or whose amino acids in the sequence have not been mutated. In the embodiments of this article, it refers only to the enzyme represented by the sequence shown in SEQ ID NO:2.

[0032] The term "unmutated" includes WT, as well as various truncated sequences capable of hyaluronidase hydrolysis activity, but excludes amino acid mutations. The "unmutated PH20 polypeptide" is the polypeptide shown in SEQ ID NO:1 or a truncated polypeptide of its N-terminus or C-terminus, and the truncated polypeptide contains at least the polypeptide shown in sequence SEQ ID NO:3; that is, the group consisting of any truncated sequence between the full-length sequence SEQ ID NO:1 and the shortest truncated sequence SEQ ID NO:3.

[0033] In this article, the term "vector" refers to a self-replicating DNA molecule used in genetic engineering and recombinant DNA technology to transfer a target gene into a recipient cell. These vectors not only replicate themselves, carrying the target gene into the host cell, but also enable it to be expressed or replicated within the host cell. Plasmids are small, circular DNA molecules found in bacterial cells that can replicate autonomously and are among the most commonly used vectors. Besides plasmid vectors, there are also viral vectors, bacteriophage vectors, etc.

[0034] The term "recombinant expression vector" in this article refers to a vector constructed using molecular cloning technology, in which foreign genes are inserted to enable their expression in host cells. This technology is not only crucial in basic biological research but also a key tool in the biotechnology and pharmaceutical industries.

[0035] The term "transfection" as used herein refers to a method of introducing plasmids into cells using chemical or physical means. The plasmids can be inserted into the genome or remain free in the cytoplasm, thereby enabling the cells to express the target protein. Transfection is classified into transient transfection and stable transfection. In this disclosure, a chemical method using liposomes is employed for transient transfection, in which the plasmid containing the target gene remains free in the cytoplasm.

[0036] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio. The terms "pharmaceutical excipient" or "pharmaceutical acceptable excipient" refer to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, or carriers administered with therapeutic agents.

[0037] As used herein, the terms “disease,” “symptom,” or “disorder,” etc., refer to any alteration or dysregulation that impairs or interferes with the normal function of cells, tissues, or organs. For example, “disease” includes, but is not limited to: tumors, pathogen infections, autoimmune diseases, T-cell dysfunction disorders, or deficiencies in immune tolerance (such as transplant rejection).

[0038] As used in this article, the term "treatment" refers to a clinical intervention in an attempt to alter an individual's or treat a disease caused by cells, which can be preventative or intervention in a clinicopathological process. Treatment effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and alleviating or improving prognosis.

[0039] As used herein, "acetylhyaluronic acid-related diseases, disorders, or conditions" refers to any such disease or condition in which elevated acetylhyaluronic acid levels are the cause, consequence, or phenomenon of the disease or condition. Acetylhyaluronic acid-related diseases and conditions are associated with elevated acetylhyaluronic acid expression in tissues or cells, increased interstitial hydraulic pressure, decreased vascular volume, and / or increased water content in tissues. Acetylhyaluronic acid-related diseases, disorders, or conditions can be treated by administering a composition containing an acetylhyaluronic acid-degrading enzyme such as hyaluronidase, for example, soluble hyaluronidase, either alone or in combination with or separately from another treatment and / or drug. Examples of diseases or conditions include, but are not limited to, cancers with acetylhyaluronic acid enrichment, such as tumors, including solid tumors such as advanced cancer, metastatic cancer, undifferentiated cancer, ovarian cancer, carcinoma in situ (ISC), squamous cell carcinoma (SCC), prostate cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, colon cancer, and other cancers. Examples of acetylated hyaluronic acid-related diseases and conditions also include those associated with increased interstitial fluid pressure (IFP), such as those related to intervertebral disc pressure and edema, for example, edema due to organ transplantation, stroke, traumatic brain injury, or other injuries. Examples of acetylated hyaluronic acid-related diseases and conditions include those associated with increased interstitial fluid pressure, decreased vascular volume, and / or increased water content in tissues, including cancer, intervertebral disc pressure, and edema. In one instance, treatment of acetylated hyaluronic acid-related conditions, diseases, or disorders includes improving, alleviating, or otherwise benefiting one or more of the increased interstitial fluid pressure (IFP), decreased vascular volume, and increased water content in tissues.

[0040] The annotation of amino acids contained in the molecules disclosed herein follows the conventions of the art, and the location of mutations is indicated by the single-character symbol and number of the wild-type amino acid, for example, Thr at position 417 is referred to as "T417". Mutations are indicated by the single-character symbol and number of the wild-type amino acid and the single-character symbol of the mutated amino acid, for example, a mutation that replaces Thr at position 417 with Val is referred to as "T417V", and if the mutant has multiple mutations, the multiple mutations are indicated by the separator " / ".

[0041] In this disclosure, the PH20 mutant T417 is a mutant consisting of a sequence in which Thr at position 417 of the wild-type PH20, as shown in SEQ ID NO:2, is replaced by other natural amino acids.

[0042] The mutants disclosed herein need not have the full-length sequence; as long as the regions important for PH20 activity are retained, they are within the scope of this disclosure. Furthermore, recombinant proteins containing the aforementioned fragment and appropriately adding, for example, signal peptide sequences, purification tags (e.g., His-tags), adapter sequences (e.g., GGGS), or other functional components to their N-terminus or C-terminus to form novel fusion proteins that do not affect PH20 activity are also within the scope of this disclosure. Additionally, truncated forms containing the aforementioned fragment and appropriately deleting from their N-terminus or C-terminus are also within the scope of this disclosure.

[0043] In the amino acid sequence of the mutants disclosed herein, one or more amino acids may be substituted, deleted, inserted, and / or added at positions other than the required mutation positions described below, without affecting enzyme activity. Any position may be chosen for such amino acid alterations, as long as they do not affect enzyme activity. In this disclosure, the term "a few" refers to 20 or fewer, preferably 10 or fewer, more preferably 5 or fewer, and most preferably 4, 3, 2, or 1.

[0044] When used in this disclosure, a designation without a specific quantity can mean one or more. When used in conjunction with the word "comprising," a designation without a specific quantity can mean one or more. When used in this disclosure, "another" can mean at least a second or more.

[0045] As used herein, "high purity" refers to the separation of the target protein from contaminants that accompany it in its native state or from contaminants generated or used during the preparation of the target protein. Typically, the target protein is considered high purity when the grayscale of the target protein band after gel electrophoresis accounts for at least 90% of the total grayscale of all bands. Preferably, in some embodiments, the grayscale of the target protein accounts for at least 95% of the total grayscale of all bands, and most preferably at least 98%.

[0046] This disclosure further provides a recombinant gene encoding the aforementioned PH20 mutant, a gene construct containing the recombinant gene such as a plasmid or expression vector, a host cell transformed with the gene construct, and a method for generating the mutant of this disclosure, which includes the step of collecting the mutant of this disclosure from a culture of the host cell. The recombinant gene, gene construct, host cell, etc., can be prepared based on the amino acid sequence of the mutant of this disclosure using known genetic engineering techniques.

[0047] Host cells transformed by introducing a gene encoding a mutant of this disclosure can be cultured under appropriate conditions according to cell type (cells commonly used for protein production, such as animal cells, plant cells, *E. coli*, yeast, etc., can be appropriately selected), and mutants of this disclosure can be collected from the culture. Collection of mutants is carried out by appropriately combining conventional purification techniques based on the physical properties of the protein. To facilitate collection, gene constructs can be designed to express the mutant in the form of a tagged peptide, such as GST, which is pre-linked to the mutant to make collection possible using the affinity for the tagged peptide. The tagged peptide can be removed after purification, but when it has no effect on the enzymatic activity of the mutant, the mutant with the tagged peptide linked thereto can be used for reactions such as hyaluronic acid hydrolysis. Mutants of this disclosure comprise amino acid sequences containing a tagged peptide linked thereto.

[0048] The features and advantages of this disclosure are more fully demonstrated by the following non-limiting embodiments.

[0049] Examples

[0050] Materials and Methods

[0051] In this disclosure, ExpiFectamine CHO reagent and ExpiCHO expression medium were purchased from Thermo Fisher Scientific, horse serum from Gibco, phosphate buffer solution from Adamas Life, hyaluronidase standard from the National Institutes for Food and Drug Control, and hyaluronic acid from TCI. Unless otherwise specified, all other reagents were conventional reagents produced by Merck, Sigma, Sinopharm, etc. The packing materials, chromatographic columns, and instruments used in this disclosure include: mPES hollow fiber column (10kDa, Riplekin (Shanghai) Biotechnology Co., Ltd.), Polar MC30-Ni Excel 1mL column, gel imaging system (Gel Doc EZ Imager, Bio-RAD), and PR.NT.Plex (Nano Temper) protein stability analyzer.

[0052] Construction of recombinant plasmids for site-directed mutagenesis of recombinant PH20 wild-type enzyme and mutant enzymes thereof

[0053] 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 inserted together between XbaI and AflII in the multiple cloning site region of the vector plasmid pCDNA3.4. The codon of the mutant amino acid was introduced using primers containing the mutation, and recombinant plasmids containing the nucleic acid sequences of wild-type PH20 and its mutant were produced using E. coli DH5α strain.

[0054] Expression and purification of recombinant PH20 wild-type enzyme and mutant enzymes thereof

[0055] PH20 wild-type and PH20 mutant proteins were expressed via transient transfection into ExpiCHO cells. One day prior to transfection, cells were passaged to a final viable cell density of 3E6–4E6 cells / mL and allowed to grow overnight. On the day of transfection, when the cell density reached 7E6–10E6 cells / mL and cell viability reached 95–99%, the recombinant plasmid of PH20 WT or the mutant was transfected into ExpiCHO cells using ExpiFectamine CHO reagent. Based on ExpiCHO expression medium, the cells were continuously cultured for 7 days at 37°C and 5% CO2 in a shaker at 130 rpm to achieve expression of the PH20-6×His fusion protein (hereinafter referred to as PH20 protein).

[0056] After culture, cell supernatant containing wild-type PH20 or PH20 mutant protein was obtained by centrifugation at 3000 rpm for 30 min. The supernatant was then replaced with equilibration buffer (20 mM PB, 500 mM NaCl, 10 mM imidazole, pH 7.5) using a 10 kDa mPES hollow fiber column, followed by purification of the PH20 protein using a 1 mL Polar MC30-Ni Excel column. The fraction containing the PH20 protein was concentrated using an Amicon centrifugal filter (10 kDa, Millipore) and stored in storage buffer (10 mM HEPES, 130 mM NaCl, pH 7.0). Protein purity was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel imaging (Gel Doc EZImager, Bio-RAD) using Image Lab scanning software. Protein concentration was quantified using a spectrophotometer (Nano-300).

[0057] Stability of wild-type PH20 and mutants thereof

[0058] T protein stability was detected using the PR.NT.Plex protein stability analyzer. agg The PH20 WT and mutant protein were replaced with 20 mM histidine, 130 mM NaCl, pH 6.5 buffer using an Amicon centrifuge filter (10 kDa, Millipore), and diluted to 0.5 mg / mL. After centrifugation, the solution was aspirated into a capillary chip and placed in the detection cell for detection. The temperature program was set to 30℃~95℃, 0.5℃ / min. After detection, the analysis software was used for analysis.

[0059] Determination of enzyme activity of wild-type PH20 and mutants thereof by turbidimetry

[0060] Prepare the following reagents: (1) Acetic acid buffer solution - add 14.0 g of potassium acetate and 25.0 mL of glacial acetic acid to water to prepare a 1000 mL solution. (2) Phosphate buffer solution - purchased from Adamas Life. (3) Enzyme dilution stock solution - add 500 mL of phosphate buffer solution to 500 mL of water. (4) Enzyme dilution working solution - add 33 mg of hydrolyzed gelatin to 50 mL of 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 enzyme dilution working solution and mix thoroughly. (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 hours. Store the solution at 4 °C and use within 30 days. (7) Serum working solution - Add 10 mL of serum stock solution to 30 L of acetate buffer solution, and bring to room temperature after preparation. (8) Hyaluronic acid stock solution - Prepare an aqueous solution of sodium hyaluronate with a concentration of 5.0 mg / mL. (9) Hyaluronic acid working solution - Add 0.75 mL of hyaluronic acid stock solution to 4.25 mL of phosphate buffer solution. (10) Standard stock solution - Prepare an aqueous solution of national standard hyaluronidase (STD) with a concentration of 1000 units / mL in a container, divide into 50 μL portions, and store at -20℃. (11) Standard working solution - Add 40 μL of 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 it for determination immediately after preparation.

[0061] Follow the instructions below to dilute all enzyme samples in a low protein binding 96-well plate.

[0062] a) The maximum sensitivity of this assay is in the range of 10–30 units / mL. To obtain results within this range, the assay needs to be repeated. To minimize the number of repetitions, first determine an approximate total number of units / mL of the sample, and then select a dilution factor (integer) so that the final concentration is approximately 20 units / mL.

[0063] 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.

[0064] c) For serially diluted samples, dilute 1:10 in triplicate by aspirating 360 μL of SSB solution and 40 μL of sample into each well on a low protein binding 96-well plate.

[0065] The enzyme-diluted working solution was used to prepare standard solutions (40, 32, 24, 16, 8, 4, 0 units / mL) with STD to plot the standard curve.

[0066] Reaction plate: Pipette 30 μL of hyaluronic acid working solution into a flat-bottomed 96-well microtiter plate.

[0067] Preheating stage: Place the low-protein binding 96-well plate containing diluted sample, standard sample, and control sample, and the flat-bottomed 96-well plate containing hyaluronic acid working solution on the heating block and preheat them at 37°C for 5 minutes.

[0068] The reaction was initiated by adding the enzyme to the substrate: 30 μL of preheated enzyme solution from the enzyme standard solution and 30 μL of the sample plate to be tested were added to the wells of a 96-well flat plate (containing substrate) and mixed thoroughly to carry out the hydrolysis reaction at 37°C for 6 min.

[0069] Termination of reaction: 240 μL of serum working solution was pipetted into each well and mixed thoroughly. The absorbance at 640 nM was measured. A linear curve was fitted from the standard curve, from which the results of the test sample could be inferred.

[0070] Example 1 Preparation of recombinant proteins of wild-type PH20 and PH20 mutants

[0071] Human hyaluronidase PH20 (EC 3.2.1.35) belongs to the GH56 family of glycosylhydrolases, consisting of 509 amino acid residues, as shown in SEQ ID NO:1. Positions 1-35 are the signal peptide, and positions 491-509 are the GPI-anchored sequence. It 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 definitively resolved. Based on structural simulations of its family members, human Hyal1 (PDB ID: 2PE4) and bee venom hyaluronidase (PDB ID: 1FCV), the catalytic domain of human PH20 is presumably a (β / α)8 barrel configuration, consisting of eight repeating sequences of a β chain and an α helix. D146 and E148 (relative to the sequence positions in SEQ ID NO:1) are the catalytic residues.

[0072] Referring to the binding mode of bee venom hyaluronidase (PDB ID: 1FCV) to its substrate hyaluronic acid, to improve the stability of PH20 without affecting its activity, residues located far from the substrate, on the protein surface, and at flexible loop linkers or interacting with surface loops on the α-helix are selected as cleavage sites. In this disclosure, SEQ ID NO:2 is used as the basis for WT, and site-directed mutagenesis is 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 a His-tag protein to the C-terminus. High-purity PH20 protein can be obtained by purification using a Polar MC30-Ni Excel 1mL column. Figure 1 As shown.

[0073] Example 2 Aggregation temperature detection of wild-type PH20 and mutants thereof

[0074] Accumulation temperature (T) agg PH20 is an indicator of protein stability. In the co-formulation development of protein drugs and PH20, PH20 acts as a penetration enhancer, promoting drug dispersion and absorption after subcutaneous injection. Improved PH20 stability can reduce the risk of enzyme aggregation and inactivation during co-formulation storage, ensuring effective drug dispersion and absorption after injection. Protein aggregation is usually irreversible and tends to form larger aggregates. PH20 aggregation may affect drug protein activity through enzyme-protein drug interactions and increase the risk of immunogenicity. Furthermore, PH20 T... agg Improving the viscosity of high-concentration co-formulated drugs helps reduce the risk of increased viscosity, allowing for higher concentrations of drug to be administered, and can also alleviate injection site pain caused by high formulation viscosity. This disclosure measures the aggregation temperature of PH20 WT and its mutants, and the results are shown in Table 1. The Taggregation temperature of PH20 WT... agg The temperature was 46.7℃, which is similar to the PH20 WT T in patent WO2020022791A1. agg This matches 46.5℃. Compared to WT, E31M and I46K T... agg An increase to 48℃ or higher indicates a suitable improvement in stability; the aggregation temperature of mutant R248Q decreased by 8.7℃; surprisingly, the aggregation temperature of mutant T417V increased to 64.2℃, 17.5℃ higher than WT. Historical data shows that the HM4 mutant T in patent WO2020022791A1... agg The temperature was 56.5℃. The mutant HM280 mutant T in patent WO2021150079A1... aggThe temperature is 59℃. The T417V mutant in this disclosure is a known T mutant of human hyaluronidase PH20 and its mutants. agg The highest level of mutation.

[0075] Table 1. Hyaluronidase pH20 WT and aggregation temperature of various mutants

[0076]

[0077] Example 3 Enzyme activity detection of wild-type PH20 and mutants thereof

[0078] Hyaluronidase activity was detected using the turbidimetric method according to the Chinese Pharmacopoeia. The principle of the turbidimetric method is based on the fact that hyaluronic acid forms a precipitate with serum under acidic conditions. When hyaluronic acid is decomposed, the amount of precipitate in the serum-hyaluronic acid mixture decreases. Therefore, hyaluronic acid was used as a substrate to detect hyaluronidase activity, and the activity was calculated by measuring the absorbance of the hyaluronic acid-serum mixture under acidic conditions. The enzyme activity data of PH20 WT and its mutants are shown in Table 2. The enzyme activities of mutants R248Q and T417V were comparable to those of WT, while mutants E31M and I46K showed significantly increased enzyme activities, with activities of 165% and 178% of WT, respectively.

[0079] Table 2. Hyaluronidase and enzyme activities of various mutants

[0080]

[0081]

[0082] Based on the aggregation temperature detection results in Example 2, this disclosure yields an enzyme with activity comparable to WT, and T agg This is the known optimal level (64.2℃) of the T417V single-point mutant. The HM4 mutant T in patent WO2020022791A1... agg The temperature was 56.5℃, but only about 15% of the enzyme activity was retained. The HM280 mutant T in WO2021150079A1... agg The temperature was 59℃, and the enzyme activity was preserved. However, this mutant was obtained through multi-point amino acid combination mutations and truncation. Such significant alterations to the protein sequence may introduce potential immunogenicity issues. Furthermore, this disclosure also obtained T... agg Improved to 48.0 and 48.2 °C (WT 46.7 °C), and the activity of the E31M and I46K mutants was increased to 165% and 178% of WT, respectively.

[0083] This disclosure describes a mutant enzyme derived from human hyaluronidase PH20, comprising mutation sites E31M, I46K, and / or T417V. Compared to previously reported PH20 and its mutants, T417V exhibits the highest known aggregation temperature of 64.2°C, representing a 17.5°C increase over the maximum yield (WT), while maintaining enzyme activity comparable to WT. E31M and I46K, while possessing enhanced enzyme activity (165% and 178%, respectively), also improve aggregation temperature to some extent (increasing by 1.3°C and 1.5°C, respectively). Therefore, the mutant enzymes of this disclosure have better application potential in protein drug co-formulations.

[0084] Those skilled in the art to which this disclosure pertains should understand that the above embodiments are for illustrative purposes only, and various alterations and modifications can be made to the mutant protein without departing from the scope of this disclosure, including but not limited to the insertion and / or truncation of the N-terminus and / or C-terminus, glycosylation, sialylation, albuminization, farnesylation, carboxylation, hydroxylation, phosphorylation, and conjugation to polymers, etc. Importantly, the protein possesses hyaluronidase activity and contains mutations relative to T417V, E31M, I46K, and / or R248Q in the sequence of human hyaluronidase PH20 SEQ ID NO:2.

Claims

1. A PH20 mutant enzyme, wherein a single amino acid substitution exists in the unmutated PH20 polypeptide, wherein: The unmutated PH20 polypeptide is the polypeptide shown in SEQ ID NO:1 or a truncated polypeptide of its N-terminus or C-terminus, and the truncated polypeptide contains at least the polypeptide shown in SEQ ID NO:2; the PH20 mutant enzyme has hyaluronic acid hydrolytic activity and an increased aggregation temperature relative to wild-type PH20; the substitution position is based on the amino acid sequence shown in SEQ ID NO:2, and the substitution is T417V.

2. A nucleic acid encoding the PH20 mutant enzyme as described in claim 1.

3. A vector comprising the nucleic acid of claim 2.

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

5. A pharmaceutical composition comprising the PH20 mutant enzyme of claim 1.

6. The pharmaceutical composition of claim 5, further comprising other pharmaceutically active ingredients and / or pharmaceutically acceptable excipients.

7. The pharmaceutical composition of claim 6, wherein, The active pharmaceutical ingredient is an immunoglobulin, recombinant protein, synthetic polypeptide, RNA, DNA, or chemical drug.

8. The use of the PH20 mutant enzyme as described in claim 1 in the preparation of a pharmaceutical composition, wherein, The pharmaceutical composition is used to deliver therapeutic agents or to increase the penetration of chemotherapeutic agents into solid tumors.

9. The use as described in claim 8, wherein, The pharmaceutical composition is formulated for subcutaneous, intramuscular, or intradermal injection, and the amount of mutant enzyme in the pharmaceutical composition is sufficient to make the pharmaceutical composition therapeutically effective.

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