Novel hyaluronidase variant

Abstract

Provided is a novel hyaluronidase variant. Specifically, the present invention relates to a novel PH20 variant, a coding nucleic acid thereof, a cell comprising the coding nucleic acid, a method for producing the PH20 variant, and a pharmaceutical composition comprising the PH20 variant.

Description

A novel hyaluronidase variant

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. CN202410854024.7, filed on June 27, 2024, Chinese Patent Application No. CN202411090079.1, filed on August 8, 2024, Chinese Patent Application No. CN202411620515.1, filed on November 13, 2024, and Chinese Patent Application No. CN202510421567.4, filed on April 3, 2025. TECHNICAL FIELD

[0003] The present application belongs to the field of medicine, which relates to a novel hyaluronidase variant. Specifically, the present application relates to a novel PH20 variant, its encoding nucleic acid, a vector comprising the encoding nucleic acid, a cell comprising the encoding nucleic acid, a method for producing the PH20 variant, and a pharmaceutical composition comprising the PH20 variant. BACKGROUND

[0004] Hyaluronidase (HAase) is an enzyme that degrades hyaluronic acid (HA) in the extracellular matrix. Five homologous hyaluronidases are encoded in the human genome: PH20 / SPAM1 (Sperm Adhesion Molecule 1), Hyal1, Hyal2, Hyal3, and Hyal4. PH20 is expressed on the sperm cell membrane and acrosomal membrane, and Hyal1 and Hyal2 are expressed in many tissues. Hyal3 is expressed in many tissues including the brain, but has not shown enzymatic activity on any known substrate. Hyal4 has specificity for chondroitin and chondroitin sulfate substrates and little activity on hyaluronic acid. Among them, only the PH20 gene product has activity under neutral pH conditions and can degrade hyaluronic acid under physiological conditions.

[0005] Although animal testis-derived hyaluronidase has been widely used in clinical practice, it usually faces the limitations of immunogenicity and impurity characteristics. To solve these problems, Halozyme Therapeutic, Inc. developed a recombinant human hyaluronidase (rHuPH20) consisting of 447 amino acids, which lacks a GPI anchor attachment motif at the C-terminus (see International Publication No. WO2004078140A2, which is incorporated herein by reference). When rHuPH20 is produced in Chinese hamster ovary (CHO) cells, the protein can be purified to a high specific activity. In the medical field, rHuPH20 is used as a carrier to facilitate the delivery of drugs under the skin. However, the current rHuPH20 still has deficiencies in enzyme activity, thermal stability, and expression efficiency. Therefore, there is a great demand in the clinic for recombinant hyaluronidase with improved biological and physicochemical properties.

[0006] Currently, Halozyme Therapeutic, Inc. has found rHuPH20 variants with high thermal stability or high enzyme activity, a high thermal stability variant (Halozyme II-F204P) constructed by substituting the amino acid residue F204P, and a high enzyme activity variant (Halozyme II-I70A) constructed by substituting the amino acid residue I70A (see International Publication No. WO2013102144A2, which is incorporated herein by reference). Alteogen Co., Ltd. has found a variant (also known as Alteogen-HP46) with high expression efficiency, high enzyme activity, and significantly improved protein aggregation temperature (T agg ) constructed by deleting 2 residues at the N-terminus, deleting 14 residues at the C-terminus, and substituting the amino acid residues corresponding to Hyal1 at a portion corresponding to the amino acid positions T306 to I326 (see International Publication No. WO2020022791A1, which is incorporated herein by reference).

[0007] In order to design rHuPH20 variants with high enzymatic activity or high stability, the present application deeply understands and analyzes the structural characteristics of rHuPH20. For this purpose, the present application first performs homology modeling of rHuPH20 using P38568.1.A as a template, obtains and analyzes the full-length three-dimensional (3-dimensional, 3D) structural characteristics of rHuPH20 (Figure 1). rHuPH20 consists of two domains, namely a catalytic domain and an epidermal growth factor-like domain (EGF-like domain), wherein the catalytic domain is in the form of TIM barrel (β / α)8. In this domain, the β-sheet is the main binding and catalytic center of hyaluronan, and the α-helix cooperates with the β-sheet to maintain the rigidity of the enzyme active center, which is very important for the function and stability of the enzyme. Among rHuPH20 and its homologous proteins Hyal1-4, only Hyal1 has a crystal structure (PDB ID: 2PE4), so each α-helix will be described by referring to the naming rules of Hyal1 (Figure 1).

[0008] Since the kinetic process can reveal the details of the stability and fluctuation of different components, it can be a key factor for promoting the enzyme activity and stability. Therefore, the present application performs 100 ns of molecular dynamics (MD) simulation on rHuPH20 and its variants to obtain the dynamic motion trajectory of hyaluronidase in solution. Subsequently, conformational analysis is performed on rHuPH20 and the variants based on these simulation trajectories. The simulation results show that more residues in α-helix 5 form a more stable α-helix after modification by F204P. The replacement of the connecting domain between α-helix 7 and α-helix 8 and part of α-helix 8 causes more hydrogen bonds to be formed within this domain. The above results show that enhancing the stability of the α-helix is conducive to the improvement of enzyme activity and thermal stability.

[0009] In enzyme engineering, in addition to active site residues, distal residues are also crucial for enzyme activity. Distal sites are residues that are not within the van der Waals distance of any part of the substrate, and amino acids within the van der Waals distance of the substrate are called "first shell" residues. Distal residues can affect the secondary structure of the enzyme by changing the enzyme, thereby affecting the structure and activity of the enzyme. Some distal sites, although not directly contacting the substrate, can still cause a change in the conformation of the active center by adjusting the position of the key residues in the catalytic center. In addition, distal sites also affect the physicochemical properties of the enzyme surface, such as hydrophobicity, charge distribution, and energy landscape, which not only affect the folding of the enzyme, but also affect the binding of the enzyme to the substrate, thereby affecting the activity.

[0010] The present application first identifies the first shell residues in rHuPH20 that bind HA from two dimensions of hydrogen bond and van der Waals interaction. Among the first shell residues, R45, I46, Y57, D59, R60, D111, E113, Y184, Y229, Y268, R270, T274, W304, R311 form hydrogen bonds with HA, which are specific and strong forces, and modification of these sites will have a significant impact on enzyme activity.

[0011] Subsequently, the present application identifies the distal residues that can affect enzyme activity by "fluctuation difference analysis" and "motion correlation coefficient". "Fluctuation difference analysis" is a difference analysis of the root mean square fluctuation (RMSF) of the same region under different conditions. After binding to HA, the RMSF of a certain region is significantly different from that when HA is not bound, indicating that this region is not in direct contact with HA, but is closely related to the binding of HA. Based on this, the identified amino acid residues are W123, K195, K196, P197, G198, L307, I309, M310, R311, L374, K376, G377, G378, F380, A425, D426. "Motion correlation coefficient" can identify distal residues that can cause a change in the conformation of the active site by adjusting the position of key residues in the catalytic center. Based on the identified first shell residues, the motion correlation coefficient is used to identify multiple distal residues that move with the first shell residues.

[0012] Excluding the identified first shell residues and distal residues that affect enzyme activity, sites that do not affect the enzyme active center and have modification potential can be obtained. These sites are 1-14 of the N-terminal, S69-T70, V141-L143, 1-14 of the C-terminal, and their secondary structures are all Loops. This indicates that further deletion of the N-terminal or C-terminal can improve the overall stability of the molecular structure. At the same time, for the intramolecular Loop, it can be mutated to make the enzyme activity or stability higher.

[0013] Therefore, the present application intends to modify and design rHuPH20 through multiple dimensions such as N-terminal deletion, C-terminal deletion, modification of alpha helix, and unit point mutation. And the designed rHuPH20 variants are evaluated in multiple dimensions such as enzyme activity, stability, and producibility, to obtain a hyaluronidase that meets the clinical needs.

[0014] SUMMARY

[0015] The present application relates to a novel PH20 variant, a nucleic acid encoding the same, a cell comprising the encoding nucleic acid, a method of producing the PH20 variant and a pharmaceutical composition comprising the PH20 variant. In particular, the novel PH20 variant exhibits at least one improved property compared to a wild-type PH20 polypeptide. More particularly, the improved property is selected from the group consisting of increased thermostability (e.g. increased resistance to denaturation at temperatures above 30°C), increased enzymatic activity, increased resistance to aggregation, increased solubility, increased resistance to cleavage and / or oxidation, and / or better post-administration skin barrier reconstitution properties.

[0016] A first aspect of the present application relates to a PH20 variant comprising at least one amino acid substitution compared to a wild-type PH20 polypeptide, wherein the PH20 variant exhibits at least one improved property compared to the wild-type PH20 polypeptide not containing the amino acid substitution (SEQ ID NO: 1).

[0017] In a further aspect, the PH20 variant comprises one or more amino acid substitutions compared to the wild-type PH20 polypeptide as set forth in SEQ ID NO: 1 at a position selected from the group consisting of P21, S22, E23, F24, G27, K28, F29, D30, E31, P32, L33, M35, A48, V58, S69, I70, V75, K82, I83, S84, Q86, D87, L89, D90, K91, A92, K93, D95, I96, T97, F98, M100, A120, K124, P125, K130, N131, R132, I134, E135, Q138, Q139, Q140, N141, V142, L165, V166, F204, N205, I208, K209, R210, D213, S215, I271, L278, T306, L307, S308, I309, M310, R311, S312, M313, K314, S315, L317, L318, D320, N321, M323, E324, T325, I326, N328, I331, I332, T335, L336, A338, K339, M340, Q343, V344, and L345, wherein the respective amino acid position is identified by alignment of the PH20 variant with the polypeptide as set forth in SEQ ID NO: 1.

[0018] In a still further aspect, the PH20 variant comprises one or more amino acid substitutions compared to the wild-type PH20 polypeptide set forth in SEQ ID NO: 1 selected from the group consisting of P21N, S22T, E23Q, F24W, G27E, K28R, F29H, D30G, E31V, P32D, L33V, M35V, A48N, V58Q, S69A, I70A, V75L, K82N, I83V, S84G, Q86W, D87A, L89R, D90K, K91M, A92L, K93Q, D95R, I96V, T97E, F98H, M100I, A120V, K124Q, P125D, K130R, N131R, R132L, I134R, E135Q, Q138A, Q139S, Q140R, N141H, V142R, L165D, V166R, F204P, N205S, N205D, I208V, K209A, R210Q, D213Q, S215A, I271L, L278R, T306D, T306S, L307W, S308E, I309N, M310T, R311T, S312N, S312T, M313T, M313K, K314E, S315T, L317Q, L317K, L318A, L318Y, D320K, N321D, M323L, E324D, E324T, T325R, I326L, I326T, N328V, I331V, I332V, T335S, L336W, A338T, K339Q, M340Y, Q343R, V344A, and L345Q, wherein the corresponding amino acid position is identified by alignment of the PH20 variant with the polypeptide set forth in SEQ ID NO: 1.

[0019] In a further aspect, the PH20 variant comprises one or more amino acid substitutions compared to the wild-type PH20 polypeptide set forth in SEQ ID NO: 1 selected from the group consisting of I70, M100, F204, T306, L307, S308, I309, M310, S312, M313, K314, L317, L318, D320, N321, E324, and I326, wherein the corresponding amino acid position is identified by alignment of the PH20 variant with the polypeptide set forth in SEQ ID NO: 1. Preferably, the PH20 variant comprises one or more amino acid substitutions compared to the wild-type PH20 polypeptide set forth in SEQ ID NO: 1 selected from the group consisting of I70, M100, F204, T306, L307, S308, I309, M310, S312, M313, K314, L317, L318, D320, N321, E324, and I326, and further comprises one or more amino acid substitutions at a position selected from the group consisting of P21, S22, E23, F24, G27, K28, F29, D30, E31, P32, L33, and M35, wherein the corresponding amino acid position is identified by alignment of the PH20 variant with the polypeptide set forth in SEQ ID NO: 1.

[0020] In a further aspect, the PH20 variant comprises one or more amino acid substitutions compared to the wild-type PH20 polypeptide set forth in SEQ ID NO: 1 selected from the group consisting of I70A, M100I, F204P, T306S, L307W, S308E, I309N, M310T, S312T, M313K, K314E, L317Q, L318A, D320K, N321D, E324D, and I326T, wherein the corresponding amino acid position is identified by alignment of the PH20 variant with the polypeptide set forth in SEQ ID NO: 1. Preferably, the PH20 variant comprises one or more amino acid substitutions compared to the wild-type PH20 polypeptide set forth in SEQ ID NO: 1 selected from the group consisting of I70A, M100I, F204P, T306S, L307W, S308E, I309N, M310T, S312T, M313K, K314E, L317Q, L318A, D320K, N321D, E324D, and I326T, and further comprises one or more amino acid substitutions selected from the group consisting of P21N, S22T, E23Q, F24W, G27E, K28R, F29H, D30G, E31V, P32D, L33V, and M35V, wherein the corresponding amino acid position is identified by alignment of the PH20 variant with the polypeptide set forth in SEQ ID NO: 1.

[0021] Preferably, the PH20 variant has at least 85%, at least 90%, at least 92%, at least 95%, at least 97% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 5-24.

[0022] More preferably, the PH20 variant comprises the amino acid sequence selected from any one of SEQ ID NOs: 5-24.

[0023] Also preferably, the PH20 variant further comprises a secretion signal peptide at the N-terminus.

[0024] A second aspect of the present application relates to a nucleic acid encoding the PH20 variant of the first aspect above.

[0025] A third aspect of the present application relates to a vector comprising the nucleic acid molecule of the second aspect above.

[0026] A fourth aspect of the present application relates to a cell comprising the nucleic acid of the second aspect above or the vector of the third aspect above.

[0027] A fifth aspect of the present application relates to a method of producing a PH20 variant, comprising: culturing the cell of the fourth aspect above under conditions wherein the PH20 variant is produced and secreted by the cell; and recovering the expressed PH20 variant.

[0028] A sixth aspect of the present application relates to a pharmaceutical composition comprising the PH20 variant of the first aspect above.

[0029] The particulars of the application are set forth in the accompanying description, which is best taken in conjunction with the appended drawings. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, illustrative methods and materials are now described. Other features, objects, and advantages of the application will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All patents and publications cited in this specification are incorporated herein by reference in their entirety.

[0030] The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. BRIEF DESCRIPTION OF DRAWINGS

[0031] FIG. 1 shows the three-dimensional structure of rHuPH20.

[0032] Figure 2 shows the plot of RMSD values of various hyaluronidases and variants as a function of simulation time.

[0033] Figure 3 shows the plot of Rg values of various hyaluronidases and variants as a function of simulation time.

[0034] Figure 4 shows the plot of enzyme activity of hyaluronidase variants as a function of time under incubation conditions at 40°C.

[0035] Figures 5-8 show the plots of hydrous particle size, SEC-monomer purity, ME-SDS purity and RP-HPLC purity of hyaluronidase variants as a function of time under incubation conditions at 40°C, respectively.

[0036] Figure 9 shows the plot of enzyme activity of hyaluronidase variants in Daratumumab co-formulation as a function of time under incubation conditions at 40°C.

[0037] Figures 10 and 11 show the plots of enzyme activity of rHuPH20, rHuPH20 variants and Alteogen-HP46 as a function of time under incubation conditions at 40°C in co-formulation with Trastuzumab Deruxtecan (Figure 10) and Trastuzumab-VC-MMAE (Figure 11) ADCs, respectively.

[0038] Figures 12 and 13 show the plots of enzyme activity of rHuPH20 (Figure 12) and hyaluronidase variants (Figure 13) as a function of time under incubation conditions at 40°C in co-formulation with different antibodies (Daratumumab and Pembrolizumab), respectively.

[0039] Figure 14 shows the plot of enzyme activity of hyaluronidase variants as a function of time under incubation conditions at 40°C in histidine and acetic acid co-formulation systems at the same pH.

[0040] Figure 15 shows the plot of enzyme activity of hyaluronidase variants as a function of time under incubation conditions at 40°C in histidine co-formulation in the range of pH 5.0-6.5.

[0041] Figure 16 shows the plot of enzyme activity of hyaluronidase variants as a function of time under incubation conditions at 40°C in acetic acid co-formulation in the range of pH 5.0-6.0.

[0042] Figure 17 shows the area of trypan blue diffusion caused by hyaluronidase variants after 1 minute under step injection and co-injection.

[0043] Figure 18 shows the comparison of the area of trypan blue diffusion after 30 minutes of injection of different hyaluronidases followed by injection of trypan blue solution 5 minutes later.

[0044] DETAILED DESCRIPTION

[0045] The present disclosure provides a novel PH20 variant. The novel PH20 variant exhibits at least one improved property as compared to a wild-type PH20 polypeptide. More particularly, the improved property is selected from the group consisting of increased thermal stability (e.g., increased resistance to denaturation at temperatures above 30°C), increased enzymatic activity, increased resistance to aggregation, increased solubility, increased resistance to cleavage and / or oxidation, and / or better post-application skin barrier reconstruction properties.

[0046] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections:

[0047] 1. Definitions;

[0048] 2. PH20 polypeptides;

[0049] 3. Pharmaceutical compositions; and

[0050] 4. Articles of manufacture and kits.

[0051] 1. Definitions

[0052] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the terms and techniques employed herein relating to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and immunological procedures are those commonly used by those skilled in the art. In addition, the definitions and explanations of the relevant terms are provided below for better understanding of the present application.

[0053] As used herein, hyaluronidase refers to a class of enzymes that degrade hyaluronic acid. Hyaluronidases include, but are not limited to, bacterial hyaluronidases (EC 4.2.2.1 or EC 4.2.99.1), leech, other parasites, and crustacean-derived hyaluronidases (EC 3.2.1.36), and mammalian-type hyaluronidases (EC 3.2.1.35). Hyaluronidases include any non-human derived enzymes, including but not limited to enzymes derived from murine, canine, feline, leporine, avian, bovine, ovine, porcine, equine, piscine, amphibian, bacterial, as well as any enzymes from leeches, other parasites, and crustaceans. Exemplary human hyaluronidases include HYAL1, HYAL2, HYAL3, HYAL4, and PH20. Hyaluronidases also include soluble hyaluronidases, including ovine and bovine PH20, and soluble PH20. Exemplary hyaluronidases include forms thereof having signal peptide sequences, mature forms thereof (lacking signal sequences), or allelic or species variants thereof. Hyaluronidases also include truncated forms thereof that exhibit hyaluronidase activity, including soluble N-terminal or C-terminal truncation variants.

[0054] As used herein, PH20 refers to a type of hyaluronidase that occurs in sperm and is neutral active. PH-20 occurs on the surface of sperm and in the lysosome-derived acrosome, where it binds to the inner face of the acrosomal membrane. PH20 includes those of any origin, including but not limited to those derived from human, chimpanzee, cynomolgus monkey, rhesus monkey, mouse, bovine, ovine, guinea pig, rabbit, and rat. Exemplary PH20 polypeptides include both precursor and mature forms, including those from human, chimpanzee, rhesus monkey, cynomolgus monkey, bovine; mouse; rat; rabbit; ovine, guinea pig; fox; gibbon, marmoset, and orangutan. Reference to PH20 includes both precursor PH20 polypeptides and mature PH20 polypeptides (such as those in which the signal sequence has been removed), truncated forms thereof that are active, and variants including allelic and species variants, variants encoded by splice variants, and other variants. PH20 polypeptides also include those containing chemical or post-translational modifications, and those not containing chemical or post-translational modifications. Such modifications include but are not limited to PEGylation, albuminization, glycosylation, farnylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art. Exemplary commercially available bovine or ovine soluble hyaluronidases are Hyaluronidase (Ovine Hyaluronidase) and Hyaluronidase (Bovine Hyaluronidase).

[0055] As used herein, the term "wild-type PH20 polypeptide (also referred to herein as rHuPH20)" refers to a PH20 polypeptide that lacks the N-terminal signal peptide sequence and is 447 amino acids in length, having the following amino acid sequence:

[0056] As used herein, a modification is a modification to the amino acid sequence of a polypeptide or the nucleotide sequence of a nucleic acid molecule, including deletions, insertions, and substitutions of amino acids and nucleotides, respectively. A modification can also include a post-translational modification or other alteration of the molecule that occurs as a result of direct or indirect conjugation or linkage to another moiety. Methods of modifying polypeptides are well known to those of skill in the art, for example, by using recombinant DNA methods.

[0057] As used herein, the term "denature" as used with respect to a protein refers to a biochemical change in the protein whereby a property or activity of the protein is reduced or eliminated. The biochemical change can be a change in the tertiary structure of the protein to unfolded. The property or activity can be completely eliminated or can be reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0058] As used herein, a property refers to a physical or structural property, such as three-dimensional structure, pi, half-life, conformation, solubility, and other such physical characteristics. For example, a change in a property can be exhibited by solubility, aggregation, or crystallization of the protein.

[0059] As used herein, activity refers to the functional activity associated with a full-length (intact) protein of a polypeptide or portion thereof. Functional activity includes, but is not limited to, biological activity, catalytic or enzymatic activity, antigenicity (the ability to bind or compete with a polypeptide for binding to an anti-polypeptide antibody), immunogenicity, the ability to form multimers, and the ability to specifically bind to a receptor or ligand for the polypeptide.

[0060] As used herein, hyaluronidase activity refers to the ability to catalytically cleave hyaluronic acid. Hyaluronidase activity is determined indirectly by the United States Pharmacopeia (USP) XXII assay for hyaluronidase, which measures the amount of higher molecular weight hyaluronic acid or acetylhyaluronic acid (HA) substrate remaining after the enzyme has been reacted with HA at 37°C for 30 minutes (USP XXII-NF XVII (1990) 644-645 United States Pharmacopeia Convention, Inc, Rockville, MD). Reference standard solutions can be used in the assay to determine the relative activity units of any hyaluronidase. In vitro assays to determine hyaluronidase activity of hyaluronidases, including modified PH20 polypeptides, are known in the art and described herein. An exemplary assay includes the microturbidity assay described herein, which measures the indirect cleavage of hyaluronic acid by a hyaluronidase by detecting the insoluble precipitate formed when un-cleaved hyaluronic acid binds to serum albumin. Reference standards can be used, for example, to generate a standard curve to determine the unit activity of a hyaluronidase being tested.

[0061] As used herein, "increased activity" refers to a modified PH20 hyaluronidase that exhibits higher hyaluronidase activity when tested under the same conditions as compared to an unmodified PH20 hyaluronidase that does not contain the amino acid substitution. For example, a modified PH20 hyaluronidase exhibits at least or about at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more of the unmodified or reference PH20 hyaluronidase activity.

[0062] As used herein, "solubility" of a protein refers to the protein being homogeneous in an aqueous solution, where the protein molecules are diffused and do not spontaneously precipitate. Thus, a soluble protein solution is one in which there are no visible or discrete particles in the solution containing the protein, whereby the particles cannot be readily filtered. Generally, a protein is soluble if there are no visible or discrete particles in the solution. For example, a protein is soluble if it contains no or very few particles that can be removed by filtration with a filter having a pore size of 0.22 μm.

[0063] As used herein, aggregation or crystallization of a protein refers to the presence of visible or discrete particles in a solution containing the protein. Typically, the particles are greater than 10 μm in size, such as greater than 15 μm, 20 μm, 25 μm, 30 μm, 40 μm, 50 μm, or more. Aggregation or crystallization can result from decreased solubility, increased protein denaturation, or covalent bond formation.

[0064] Thus, "stability" or "stable" of a formulation or co-formulation provided herein refers to a modified hyaluronan-degrading enzyme, such as a modified PH20 hyaluronidase, that is stable at or about 2°C to 8°C for at least 1 month or at or about 30°C to 42°C for at least 3 days when exposed to one or more denaturing conditions or denaturing agents (e.g., in the presence of a denaturing excipient such as a preservative).

[0065] As used herein, "increased stability" of a modified PH20 hyaluronidase refers to a modified PH20 hyaluronidase exhibiting a higher hyaluronidase activity in the presence of the same denaturing conditions (e.g., at an elevated temperature) as compared to an unmodified PH20 hyaluronidase that does not contain the amino acid substitution. For example, a modified PH20 hyaluronidase exhibits increased stability if it exhibits at least or about at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more of the activity of an unmodified or reference PH20 hyaluronidase in the presence of denaturing conditions (e.g., in the presence of a denaturing excipient such as a preservative).

[0066] As used herein, "elevated temperature" refers to a temperature that is higher than room temperature or ambient temperature. Generally, an elevated temperature is at least about or greater than 30°C, such as 30°C to 42°C, typically 32°C to 37°C or 35°C to 37°C (inclusive of the endpoints).

[0067] As used herein, room temperature refers to a temperature that is generally in the range of about or at 18°C to about or at 32°C. Those skilled in the art recognize that room temperature varies with locale and prevailing conditions. For example, room temperature can be higher in a warm climate such as Italy or Texas.

[0068] As used herein, "comparing" proteins under the same conditions refers to treating the different proteins the same or substantially the same, whereby one or more conditions that can affect the activity or property of the protein or substance do not change or do not substantially change between or among the substances being compared. For example, when comparing the hyaluronidase activity of a modified PH20 polypeptide to an unmodified PH20 polypeptide, any one or more conditions such as the amount or concentration of the polypeptide, the presence of excipients, carriers or other ingredients in the formulation in addition to the active substance (e.g., the modified PH20 hyaluronidase), the temperature, the storage time, the storage container, the storage conditions (e.g., agitation), and / or other conditions related to exposure or use are the same or substantially the same between and among the polypeptides being compared.

[0069] As used herein, "predetermined time" refers to a time that is determined or decided in advance. For example, the predetermined time can be a time that is preselected in accordance with the desired application or use of the protein in relation to the desired duration of activity of the hyaluronan-degrading enzyme. The predetermined time can be hours, days, months, or years. For example, the predetermined time can be at least about or about 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 6 months, 1 year, or more.

[0070] As used herein, "storage" refers to the maintenance of a formulation for a period of time under certain conditions (e.g., a certain temperature, time, and / or form (e.g., liquid or lyophilized form)) prior to administration to a subject after the formulation has been prepared. For example, a liquid formulation can be maintained at different temperatures for days, weeks, months, or years prior to administration to a subject, such as refrigerated (0°C-10°C, such as 2-8°C), room temperature (e.g., up to 32°C, such as 18°C to about or to 32°C), or elevated temperatures (e.g., 30°C-42°C, such as 32°C-37°C or 35°C-37°C).

[0071] As used herein, "excipient" refers to a compound in a formulation of an active substance that does not provide the biological effect of the active substance when the active substance is not present. Exemplary excipients include, but are not limited to, salts, buffers, stabilizers, tonicity modifiers, metals, polymers, surfactants, preservatives, amino acids, and sugars.

[0072] As used herein, "thermostable" refers to a protein, such as a modified PH20 polypeptide, that exhibits stability at elevated temperatures above or about 30°C, such as 30°C-42°C, and typically 32°C-37°C or 35°C-37°C. For example, a thermostable modified PH20 hyaluronidase typically exhibits increased stability when assayed under identical elevated temperature denaturing conditions as compared to an unmodified PH20 hyaluronidase that does not contain the amino acid substitution. For example, a modified PH20 hyaluronidase exhibits at least or about at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more activity of an unmodified or reference PH20 hyaluronidase at elevated temperatures.

[0073] As used herein, nucleic acid includes DNA, RNA and analogs thereof, including peptide nucleic acid (PNA) and mixtures thereof. Nucleic acids can be single-stranded or double-stranded.

[0074] As used herein, "amino acid" is an organic compound that contains both an amino group and a carboxylic acid group. Polypeptides contain two or more amino acids. For purposes of the present application, amino acids include the 20 naturally occurring amino acids, unnatural amino acids, and amino acid analogs (i.e., amino acids in which the alpha-carbon has a side chain).

[0075] As used herein, suitable conservative substitutions are known to those of skill in the art, and can typically be made without altering the biological activity of the resulting molecule. Those of skill in the art recognize that a single amino acid substitution in a non-essential region of a polypeptide will typically not alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. co., p. 224).

[0076] Other substitutions are also permissible, and can be determined empirically or according to known conservative substitutions.

[0077] As used herein, "corresponding to" a disclosed sequence, such as the sequence set forth in the sequence listing, refers to the nucleotide or amino acid position in the nucleotide or amino acid position in the sequence alignment that maximizes identity when the disclosed sequence is aligned with the sequence using a standard alignment algorithm, such as the GAP algorithm. For purposes of the present application, the alignment of PH20 sequences is with the amino acid sequence set forth in SEQ ID NO: 1. Thus, reference herein to a position or amino acid substitution corresponding to a position of SEQ ID NO: 1 also refers to the position or amino acid substitution corresponding to a position of SEQ ID NO: 1. By sequence alignment, one of skill in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides. Typically, to identify corresponding positions, the amino acid sequences are aligned so as to obtain the highest order match.

[0078] As used herein, "sequence identity" refers to the number of identical or similar amino acid or nucleotide bases detected in a comparison of a polypeptide or polynucleotide to a reference polypeptide or polynucleotide. Sequence identity can be determined by sequence alignment of nucleic acid or protein sequences to identify regions of similarity or identity. For purposes of the present application, sequence identity is typically determined by alignment to identify identical residues. The alignment can be local or global, but for purposes of the present application, the alignment is typically a global alignment in which the entire length of each sequence is compared. Between the sequences being compared, matches, mismatches, and gaps can be identified. Gaps are null amino acids or nucleotides inserted between sequence residues being aligned so as to align identical or similar properties. Typically, the gaps can be internal or terminal. Sequence identity can be determined by considering gaps as identical residue number / length of the shortest sequence x 100. When using a gap penalty, sequence identity can be determined with terminal gaps unpenalized (e.g., terminal gaps are not penalized).

[0079] As used herein, produced by recombinant means or using recombinant DNA methods refers to the expression of a protein encoded by cloned DNA using well known molecular biology methods.

[0080] As used herein, vector (or plasmid) refers to a discrete element used to introduce heterologous nucleic acid into a cell for expression or replication. The vector is typically maintained episomally, but can be designed to effect integration of the gene or a portion thereof into a chromosome of the genome. The term also encompasses vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. The selection and use of such vectors are well known to those skilled in the art.

[0081] As used herein, an expression vector includes a vector that is capable of expressing DNA operably linked to regulatory sequences such as a promoter region that effect the expression of the DNA segment. Such additional segments can include promoters and terminators, and optionally can include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are typically derived from plasmid or viral DNA, or can contain both elements. Accordingly, the expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector that, when introduced into an appropriate host cell, causes the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art, including those that can replicate in eukaryotic and / or prokaryotic cells, and those that remain episomal or integrate into the host cell genome.

[0082] As used herein, conjugation refers to the direct or indirect attachment of a modified PH20 polypeptide to one or more other polypeptides or chemical moieties. Such conjugates include fusion proteins, those produced by chemical conjugation, and those produced by any other method whereby at least one modified PH20 polypeptide is directly or indirectly attached to another polypeptide or chemical moiety, so long as the conjugate retains hyaluronidase activity. Exemplary conjugates provided by the application include PH20 polypeptides directly or indirectly attached to a multimerization domain (e.g., an Fc portion), a toxin, a label, or a drug.

[0083] As used herein, "composition" refers to any mixture of two or more products or compounds. It can be a solution, suspension, liquid, powder, paste, aqueous solution, non-aqueous solution, or any combination thereof.

[0084] As used herein, formulation refers to a composition containing at least one active pharmaceutical or therapeutic agent and one or more excipients.

[0085] As used herein, the singular form "a" or "an" includes plural referents unless specifically stated otherwise. Thus, for example, "a" or "an" agent includes one or more agents.

[0086] As used herein, ranges and amounts can be expressed in a specific term such as "about" and "approximately." About also includes exact amounts. Thus "about 5 amino acids" means "about 5 amino acids" and "5 amino acids."

[0087] As used herein, the term "about" when used in reference to a quantity is intended to refer to a value that can vary by 10% (±10%), preferably by 5% (±5%), of the stated value.

[0088] It will be understood by those within the art that, when a numerical range is recited herein, the end points of the range are included in the range, unless the context clearly dictates otherwise. Furthermore, when a numerical range is recited, it is intended to include all sub-ranges of the same width, as well as individual numbers that fall within the range, unless the context clearly indicates otherwise. For example, a range of 1 to 10 is intended to include all sub-ranges between (and including) 1 and 10, e.g., 1 to 3.5, 4.1 to 7.6, etc., as well as individual numbers within the range, e.g., 1, 4, 10, etc.

[0089] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0090] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are in accordance with their common usage, recognized abbreviations, or IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. 11 : 1726), unless otherwise indicated.

[0091] 2. PH20 variants

[0092] In one embodiment, the present application relates to a PH20 variant comprising at least one amino acid substitution as compared to a wild-type PH20 polypeptide, wherein the PH20 variant exhibits at least one improved property as compared to the wild-type PH20 polypeptide (SEQ ID NO: 1) that does not contain the amino acid substitution.

[0093] In a further embodiment, the improved property of the PH20 variant is increased thermal stability.

[0094] In yet further embodiments, the improved property is increased resistance to denaturation at temperatures above 30°C, for example, increased resistance to denaturation at temperatures of 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, or 42°C. Preferably, under the same conditions, the activity of the PH20 variant is at least or about at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more of the activity of the wild-type PH20 polypeptide after storage at the temperature for a certain time. In a further embodiment, the PH20 variants of the disclosure have enhanced stability under conditions of, for example, 40°C. In a further embodiment, the PH20 variants of the disclosure have enhanced stability under conditions of, for example, 40°C in co-formulation with an antibody.

[0095] In further embodiments, the improved property of the PH20 variant is increased enzyme activity. Preferably, under the same conditions, the enzyme activity of the PH20 variant produced by expression is at least or about at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more of the enzyme activity of the wild-type PH20 polypeptide.

[0096] In further embodiments, the improved property of the PH20 variant is increased resistance to aggregation. Preferably, under the same conditions, the amount of polymeric polypeptide produced in a formulation comprising the PH20 variant is significantly less than the amount of polymeric polypeptide produced in a formulation comprising the wild-type PH20 polypeptide, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100% or even more less.

[0097] In a further embodiment, the improved property of the PH20 variant is increased solubility. Preferably, the solubility of the PH20 variant is at least or about at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more of the solubility of the wild-type PH20 polypeptide under the same conditions.

[0098] In a further embodiment, the improved property of the PH20 variant is increased resistance to fragmentation and / or oxidation. Preferably, the rate of fragmentation and / or oxidation of the PH20 variant is significantly less than that of the wild-type PH20 polypeptide, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100% or even more less under the same conditions.

[0099] In a further embodiment, the improved property of the PH20 variant is less degradation. Preferably, the rate of degradation of the PH20 variant is significantly less than that of the wild-type PH20 polypeptide, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100% or even more less under the same conditions.

[0100] In yet another embodiment, the PH20 variant has at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. Preferably, the PH20 variant has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. Preferably, the PH20 variant has at least 92% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. Preferably, the PH20 variant has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. Preferably, the PH20 variant has at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0101] In another embodiment, the PH20 variant has one or more amino acid substitutions at positions selected from P21, S22, E23, F24, G27, K28, F29, D30, E31, P32, L33, M35, A48, V58, S69, I70, V75, K82, I83, S84, Q86, D87, L89, D90, K91, A92, K93, D95, I96, T97, F98, M100, A120, K124, P125, K130, N131, R132, I134, E135, Q138, Q139, Q140, N141, V142, L165, V166, F204, N205, I208, K209, R210, D213, S215, I271, L278, T306, L307, S308, I309, M310, R311, S312, M313, K314, S315, L317, L318, D320, N321, M323, E324, T325, I326, N328, I331, I332, T335, L336, A338, K339, M340, Q343, V344, and L345, compared to the wild-type PH20 polypeptide set forth in SEQ ID NO: 1, wherein the corresponding amino acid positions are identified by alignment of the PH20 variant and the polypeptide set forth in SEQ ID NO: 1.

[0102] In yet another embodiment, the PH20 variant comprises one or more amino acid substitutions compared to the wild-type PH20 polypeptide set forth in SEQ ID NO: 1 selected from the group consisting of P21N, S22T, E23Q, F24W, G27E, K28R, F29H, D30G, E31V, P32D, L33V, M35V, A48N, V58Q, S69A, I70A, V75L, K82N, I83V, S84G, Q86W, D87A, L89R, D90K, K91M, A92L, K93Q, D95R, I96V, T97E, F98H, M100I, A120V, K124Q, P125D, K130R, N131R, R132L, I134R, E135Q, Q138A, Q139S, Q140R, N141H, V142R, L165D, V166R, F204P, N205S, N205D, I208V, K209A, R210Q, D213Q, S215A, I271L, L278R, T306D, T306S, L307W, S308E, I309N, M310T, R311T, S312N, S312T, M313T, M313K, K314E, S315T, L317Q, L317K, L318A, L318Y, D320K, N321D, M323L, E324D, E324T, T325R, I326L, I326T, N328V, I331V, I332V, T335S, L336W, A338T, K339Q, M340Y, Q343R, V344A, and L345Q, wherein the corresponding amino acid position is identified by alignment of the PH20 variant with the polypeptide set forth in SEQ ID NO: 1.

[0103] In a further aspect, the PH20 variant comprises one or more amino acid substitutions compared to the wild-type PH20 polypeptide set forth in SEQ ID NO: 1 selected from the group consisting of I70, M100, F204, T306, L307, S308, I309, M310, S312, M313, K314, L317, L318, D320, N321, E324, and I326, wherein the corresponding amino acid position is identified by alignment of the PH20 variant with the polypeptide set forth in SEQ ID NO: 1. Preferably, the PH20 variant comprises one or more amino acid substitutions compared to the wild-type PH20 polypeptide set forth in SEQ ID NO: 1 selected from the group consisting of I70, M100, F204, T306, L307, S308, I309, M310, S312, M313, K314, L317, L318, D320, N321, E324, and I326, and further comprises one or more amino acid substitutions at a position selected from the group consisting of P21, S22, E23, F24, G27, K28, F29, D30, E31, P32, L33, and M35, wherein the corresponding amino acid position is identified by alignment of the PH20 variant with the polypeptide set forth in SEQ ID NO: 1.

[0104] In a further aspect, the PH20 variant comprises one or more amino acid substitutions compared to the wild-type PH20 polypeptide set forth in SEQ ID NO: 1 selected from the group consisting of I70A, M100I, F204P, T306S, L307W, S308E, I309N, M310T, S312T, M313K, K314E, L317Q, L318A, D320K, N321D, E324D, and I326T, wherein the corresponding amino acid position is identified by alignment of the PH20 variant with the polypeptide set forth in SEQ ID NO: 1. Further, the PH20 variant comprises one or more amino acid substitutions compared to the wild-type PH20 polypeptide set forth in SEQ ID NO: 1 selected from the group consisting of I70A, M100I, F204P, T306S, L307W, S308E, I309N, M310T, S312T, M313K, K314E, L317Q, L318A, D320K, N321D, E324D, and I326T, and further comprises one or more amino acid substitutions selected from the group consisting of P21N, S22T, E23Q, F24W, G27E, K28R, F29H, D30G, E31V, P32D, L33V, and M35V, wherein the corresponding amino acid position is identified by alignment of the PH20 variant with the polypeptide set forth in SEQ ID NO: 1.

[0105] In a further embodiment, the PH20 variant comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-24.

[0106] In a further embodiment, the PH20 variant comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-24.

[0107] In a further embodiment, the PH20 variant comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-24.

[0108] In a further embodiment, the PH20 variant comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-24.

[0109] In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 5. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 6. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 7. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 8. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 9. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 10. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 11. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 12. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 13. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 14. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 15. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 16. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 17. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 18. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 19. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 20. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 21. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 22. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 23. In one embodiment, the PH20 variant comprises the amino acid sequence of SEQ ID NO: 24.

[0110] In further embodiments, the PH20 variant further comprises a secretion signal peptide at the N-terminus.

[0111] In further embodiments, the PH20 variant is secreted upon expression from a cell and is soluble in the supernatant.

[0112] In further embodiments, the PH20 variant is substantially purified or isolated.

[0113] In a further embodiment, the PH20 variant is modified by a modification selected from glycosylation, sialylation, albumination, farnesylation, carboxylation, hydroxylation, and phosphorylation.

[0114] In a further embodiment, the PH20 variant is glycosylated, wherein the polypeptide comprises at least N-acetylglucosamine moieties, linked to each of at least three (e.g., three, four, five, or six) asparagine (N) residues.

[0115] In a further embodiment, the PH20 variant is conjugated to a moiety selected from a multimerization domain, a toxin, a detectable label, or a drug.

[0116] In a further embodiment, the PH20 variant is conjugated to an Fc domain.

[0117] In another aspect, the present application relates to a nucleic acid encoding a PH20 variant of the present application.

[0118] In yet another aspect, the present application relates to a vector comprising a nucleic acid of the present application.

[0119] In still another aspect, the present application relates to a cell comprising a nucleic acid of the present application or a vector of the present application. Preferably, the cell is a Chinese hamster ovary (CHO) cell.

[0120] In a further aspect, the present application relates to a method of producing a PH20 variant, the method comprising: culturing a cell of the present application under conditions wherein the PH20 variant is produced and secreted by the cell; and recovering the expressed PH20 variant.

[0121] 3. Pharmaceutical composition

[0122] While the PH20 variants described herein can be used (e.g., administered) alone, it is generally preferred that they be presented in the form of a composition or formulation.

[0123] In one aspect, the composition is a pharmaceutical composition (e.g., formulation, preparation, medicament) comprising a PH20 variant described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0124] In one aspect, the composition is a pharmaceutical composition comprising at least one PH20 variant described herein and one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including but not limited to pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, coloring agents, flavoring agents, and sweetening agents.

[0125] In one aspect, the composition further comprises other active agents, e.g., other therapeutic or prophylactic agents.

[0126] Suitable carriers, diluents, excipients and the like can be found in the standard pharmaceutical literature. See, e.g., Handbook of Pharmaceutical Additives, 2ndEdition (M. Ash and I. Ash, eds.), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20thEdition, Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2ndEdition, 1994.

[0127] When co-formulated with other therapeutic or prophylactic agents, the hyaluronidases of the present application can be present in the combined final formulation with the other therapeutic or prophylactic agents, or can be present separately, for co-administration at the time of use. For example, the components comprising the therapeutic or prophylactic agent and the hyaluronidase of the present application can be prepared in a single combined formulation, or in two separate formulations, mixed just prior to injection (use), or injected separately into different sites in the body, or used sequentially in successive injections.

[0128] Another aspect of the application relates to methods of preparing a pharmaceutical composition comprising mixing at least one PH20 variant as defined herein with one or more other pharmaceutically acceptable ingredients (e.g., carriers, diluents, excipients, etc.) well-known to those of ordinary skill in the art. If formulated as discrete units, such as tablets, each unit contains a predetermined amount (dosage) of the active compound.

[0129] As used herein, the term "pharmaceutically acceptable" in reference to a compound, ingredient, material, composition, dosage form, etc., refers to something that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0130] The formulations can be prepared by any method known or hereafter developed in the art of pharmacy. Such methods include the step of bringing into association the active compound with the carrier (s) constituting one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0131] The formulations can be prepared to provide rapid or slow release; immediate, delayed, timed, or sustained release; or combinations thereof.

[0132] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticulates). Such liquids can additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulations isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, e.g., water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers that can be employed include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Generally, the concentration of the active ingredient in the liquid can be from about 1 ng / ml to about 10 μg / ml, such as from about 10 ng / ml to about 1 μg / ml. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, such as water, for injection immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0133] In one embodiment, the present application relates to a pharmaceutical composition comprising a PH20 variant of the present application.

[0134] 4. Articles and kits

[0135] In another aspect, an article or kit is provided, comprising a PH20 variant described herein. The article or kit can further comprise instructions for use of a PH20 variant described herein in a method of the present application. Thus, in certain embodiments, the article or kit comprises instructions for use of a PH20 variant described herein in a method of treating a disease in a subject. In some embodiments, the subject is a human.

[0136] The article or kit can further comprise a container. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (e.g., single or dual-chamber syringes), and test tubes. In some embodiments, the container is a vial. The containers can be formed from a variety of materials such as glass or plastic. The container holds a formulation.

[0137] The article of manufacture or kit can further comprise a label or package insert on or associated with the container, which can indicate directions for reconstitution and / or use of the formulation. The label or package insert can further indicate that the formulation can be used or intended to be used for subcutaneous, intravenous (e.g., intravenous infusion), or other modes of administration to treat a disease in a subject. The container holding the formulation can be a single-use vial or a multi-use vial that allows repeated administration of the reconstituted formulation. The article of manufacture or kit can further comprise a second container comprising a suitable diluent. The article of manufacture or kit can also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0138] The article of manufacture or kit herein also optionally comprises a container comprising a second agent, wherein the PH20 variant is a first agent, and the article of manufacture or kit further comprises instructions on the label or package insert for treating a subject with an effective amount of the second agent.

[0139] In some embodiments, the PH20 variant is present in the container as a lyophilized powder. In some embodiments, the lyophilized powder is in a gas-sealed container (such as a vial, ampule, or pouch) that indicates the amount of active agent. In cases where the medicament is to be administered by injection, an ampule of sterile water for injection or saline can optionally be provided as part of the kit, such that the components can be mixed just prior to administration. Such kits can also include, if desired, one or more of various conventional pharmaceutical components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, and the like, as will be apparent to those skilled in the art. Printed instructions can also be included in the kit, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and / or instructions for mixing the components. Examples

[0140] The following examples are merely illustrative of the subject disclosure and should not be construed as limiting the subject disclosure in any way.

[0141] Example 1. rHuPH20 variant design considerations

[0142] Hyaluronidase (HAase) is an enzyme that degrades hyaluronic acid (HA) in the extracellular matrix. Five isoforms of hyaluronidase are encoded in the human genome: PH20 / SPAM1 (Sperm Adhesion Molecule 1), Hyal1, Hyal2, Hyal3, and Hyal4. PH20 is expressed on the sperm cell membrane and acrosomal membrane, and Hyal1 and Hyal2 are expressed in many tissues. Hyal3 is expressed in many tissues including the brain, but has not shown enzymatic activity toward any known substrate. Hyal4 is specific for chondroitin and chondroitin sulfate substrates, and has little activity toward hyaluronic acid. Of these, only the PH20 gene product is active at neutral pH and can degrade hyaluronic acid under physiological conditions.

[0143] Although animal testis-derived hyaluronidase has been widely used in clinical applications, it generally faces limitations of immunogenicity and impurity characteristics. To address these issues, Halozyme Therapeutic, Inc. developed a recombinant human hyaluronidase (rHuPH20) consisting of 447 amino acids, which lacks a GPI anchor attachment motif at the C-terminus (see International Publication No. WO2004078140A2, which is incorporated herein by reference). When rHuPH20 is produced in Chinese hamster ovary (CHO) cells, the protein can be purified to a high specific activity. In the medical field, rHuPH20 is used as a carrier to facilitate the delivery of drugs under the skin. However, the current rHuPH20 still has deficiencies in thermal stability and expression efficiency. Therefore, there is a great need in the clinical field for a recombinant hyaluronidase having improved biological and physicochemical properties.

[0144] Currently, Halozyme Therapeutic, Inc. has discovered rHuPH20 variants having high thermal stability or high enzyme activity, a high thermal stability variant (Halozyme II-F204P) constructed by substituting the amino acid residue F204P, and a high enzyme activity variant (Halozyme II-I70A) constructed by substituting the amino acid residue I70A (see International Publication No. WO2013102144A2, which is incorporated herein by reference). Alteogen has discovered rHuPH20 variants having high expression efficiency, high enzyme activity, and significantly improved protein aggregation temperature (T agg) variant (also known as Alteogen-HP46), which is constructed by deleting 2 residues at the N-terminus, deleting 14 residues at the C-terminus, and substituting a portion corresponding to amino acid positions T306 to I326 with amino acid residues corresponding to Hyal1 (see International Publication No. WO2020022791A1, which is incorporated herein by reference).

[0145] To design rHuPH20 variants with high enzyme activity or high stability, the present application deeply understands and analyzes the structural characteristics of rHuPH20. To this end, the present application first performs homology modeling of rHuPH20 using P38568.1.A as a template, obtains and analyzes the full-length three-dimensional (3-dimensional, 3D) structural characteristics of rHuPH20 (Figure 1). rHuPH20 is composed of two domains, namely a catalytic domain and an epidermal growth factor-like domain (EGF-like domain), wherein the catalytic domain is in the form of TIM barrel (β / α)8. In this domain, the β-sheet is the main binding and catalytic center of hyaluronan, and the α-helix cooperates with the β-sheet to maintain the rigidity of the enzyme active center, which is very important for the function and stability of the enzyme. Among rHuPH20 and its homologous proteins Hyal1-4, only Hyal1 has a crystal structure (PDB ID: 2PE4), so each α-helix will be described by referring to the naming rules of Hyal1 (Figure 1).

[0146] Since the kinetic process can reveal the details of the stability and fluctuation of different components, which can be a key factor for promoting enzyme activity and stability. Therefore, the present application performs 100 ns of molecular dynamics (MD) simulation on rHuPH20 and its variants to obtain the dynamic motion trajectory of hyaluronidase in solution. Subsequently, conformational analysis is performed on rHuPH20 and variants based on these simulation trajectories. The simulation results show that more residues in α-helix 5 form a more stable α-helix after modification of F204P. The replacement of the connecting domain between α-helix 7 and α-helix 8 and part of α-helix 8 causes more hydrogen bonds to form within this domain. The above results show that enhancing the stability of α-helix is conducive to the improvement of enzyme activity and thermal stability.

[0147] In enzyme engineering, besides active site residues, distal residues are also crucial for enzyme activity. Distal residues are residues not within the van der Waals distance of any part of the substrate, and the amino acids within the van der Waals distance of the substrate are called "first shell" residues. Distal residues can affect the secondary structure of the enzyme, thereby affecting the structure and activity of the enzyme. Some distal sites, although not in direct contact with the substrate, can still cause a change in the conformation of the active center by adjusting the position of key residues in the catalytic center. In addition, distal sites can also affect the physicochemical properties of the enzyme surface, such as hydrophobicity, charge distribution, and energy landscape, which not only affect the folding of the enzyme, but also affect the binding of the enzyme to the substrate, thereby affecting the activity.

[0148] The present application first identifies the first shell residues of rHuPH20 that bind to HA from the two dimensions of hydrogen bonding and van der Waals interaction. Among the first shell residues, R45, I46, Y57, D59, R60, D111, E113, Y184, Y229, Y268, R270, T274, W304, and R311 form hydrogen bonds with HA, which are specific and strong forces, and modification of these sites will have a significant impact on enzyme activity.

[0149] Subsequently, the present application identifies distal residues that can affect enzyme activity through "fluctuation difference analysis" and "motion correlation coefficient". "Fluctuation difference analysis" is a difference analysis of the root mean square fluctuation (RMSF) of the same region under different conditions. After binding to HA, the RMSF of a certain region is significantly different from that when HA is not bound, indicating that this region may not be in direct contact with HA, but is closely related to the binding of HA. Based on this, the identified amino acid residues are W123, K195, K196, P197, G198, L307, I309, M310, R311, L374, K376, G377, G378, F380, A425, and D426. "Motion correlation coefficient" can identify distal residues that can cause a change in the conformation of the active center by adjusting the position of key residues in the catalytic center. Based on the identified first shell residues, the motion correlation coefficient is used to identify multiple distal residues that move with the first shell residues.

[0150] Excluding the identified first shell residues and distal residues that affect enzyme activity, sites that do not affect the active center of the enzyme and have modification potential can be obtained. These sites are positions 1-14 of the N-terminus, S69-T70, V141-L143, and positions 1-14 of the C-terminus, and their secondary structures are all Loops. This indicates that further deletion of the N-terminus or C-terminus can improve the overall stability of the molecular structure. At the same time, for the intramolecular Loop, it can be mutated to make the enzyme more active or stable.

[0151] Therefore, the present application intends to design and modify rHuPH20 through multiple dimensions such as N-terminal deletion, C-terminal deletion, modification of alpha helix, and modification of single-point mutation. And the designed rHuPH20 variants are evaluated in multiple dimensions such as enzyme activity, stability, and producibility, so as to obtain a hyaluronidase that meets the clinical needs.

[0152] Example 2. Development and optimization of the calculation method

[0153] 2.1 Molecular dynamics simulation

[0154] The present application first performs homology modeling of hyaluronidase (HAase) using P38568.1.A as a template, and constructs a HAase-HA complex structure using the 3D structure of the disaccharide HA in the crystal structure 1FCV and the position relative to rHuPH20. Subsequently, the Gromacs-2022 is used to perform molecular dynamics simulation on the complex system to obtain its dynamic process. In order to simulate the conditions in the experiment, the side chains of Asp, Glu, Arg, His and Lys are charged (Asp -1 , Glu -1 , Arg +1 , His +1 , Lys +1 ), and the N- and C-termini are also charged (NH 3+ , COO - ). All MD simulations were performed in the isothermal-isobaric (NPT) ensemble using the GROMACS-2022 software package. The CHARMM36 force field was used. The temperature was maintained at about 300K by weak coupling with an external temperature bath with a coupling constant of 0.1 ps, and the pressure was maintained at 1 bar with a coupling time of 100 ps.

[0155] For the alpha helix 1 replacement variant, the alpha helix 1 part is modeled with the crystal structure of Hyal1 (PDB ID: 2PE4), and the other parts are modeled in the same way as rHuPH20.

[0156] 2.2 RMSD value calculation

[0157] The root-mean-square deviation (RMSD) changing with the simulation time was calculated using the rms module to evaluate the stability of the simulation trajectory. The reference structure used for calculation is the initial conformation of the simulation. The calculation formula of RMSD is:

[0158] In formula 1 and formula 2, N is the number of atoms, m i is the mass of the i-th atom, and r i(t) is the position of atom i at time t, M is the total mass of N atoms. To characterize the trajectory fluctuation of each variant during the simulation, the standard deviation of RMSD of all frames during the simulation was used as a characterization index.

[0159] 2.3 Rg calculation

[0160] The gyrate module was used to calculate the radius of gyration (Rg) as a function of simulation time to evaluate the spatial distribution uniformity and expansion of the molecule during the simulation. The standard deviation of Rg of all frames during the simulation was used as a characterization parameter of the conformational fluctuation of each variant.

[0161] 2.4 Hydrogen bond calculation

[0162] Hydrogen bonding was analyzed by the hbond module. If the distance between N and O atoms is ≤ 3.5 and the angle of N-H…O is ≥ 150°, it is considered that a hydrogen bond is formed.

[0163] 2.5 Secondary structure calculation

[0164] The secondary structure in the enzyme was identified by the do_dssp tool. do_dssp converts the protein structure at each time step into a format that can be processed by the DSSP program. Then, the DSSP program is called to classify the structure at each time step into secondary structure, including alpha helix, beta sheet, turn and coil, etc.

[0165] 2.6 Rosetta ΔG calculation

[0166] Rosetta energy function was used to evaluate the energy state and stability of the protein and its mutants. Rosetta energy function is an important tool for evaluating and predicting the stability of proteins and their complexes. Based on physical and statistical principles, the energy function calculates the energy of protein structures to provide guidance for structure prediction, design and optimization. The core of the Rosetta energy function is a combined scoring system. Through the Rosetta energy function, the free energy of the protein and its mutants can be effectively calculated to predict their stability and functional changes.

[0167] 2.7 Hyaluronidase-hyaluronic acid enzyme activity calculation

[0168] To reasonably and quantitatively predict the enzyme activity of each rHuPH20 variant, the structure is associated with the enzyme activity. Based on the literature (Sci Rep 6, 27808 (2016)), it is known that the binding energy of the enzyme and the substrate is positively correlated with the enzyme activity. The binding free energy of HAase and HA is calculated using the AutoDock semi-empirical free energy field. HAase is the receptor molecule (R), and the HA molecule is the ligand molecule (L). First, the energy change in the molecule when the HAase and HA molecules are converted from the unbound state to the bound state is calculated, and then the free energy difference between the HAase and HA molecules before and after the bound state is calculated, and the conformational entropy lost by the HAase and HA molecules due to the formation of the complex. According to the sum of the three pairs of pairing potential functions (V) and the lost conformational entropy (ΔS conf ), the binding free energy of each frame of HAase-HA molecule complex in the simulation can be obtained. The parameters of HAase and HA molecules are calculated by Python program in AutoDockTools according to the standard process.

[0169] To verify the correlation of average energy representing enzyme activity, the present application establishes a correlation equation using the rHuPH20 variants and their enzyme activities in the Alteogen patent (CN 113840921 A). The names of the three rHuPH20 variants are Alteogen-HP46, HM261 and HM268, and the enzyme activities (relative enzyme activity) of rHuPH20 are 2.4, 1.6 and 2.6, respectively. Then, the complex structures of the three variants and HA are constructed and MD simulation is performed for 10 ns. Subsequently, the average binding energy of rHuPH20 and the three variants during MD simulation is calculated, which is -7.873, -10.383, -7.976 and -10.676 kcal / mol, respectively. Based on the known relative enzyme activity and average energy, the present application constructs a correlation equation of "relative enzyme activity-average energy" (Formula 3). The R 2 of this correlation equation is 0.91, indicating that the accuracy of the equation is high. Average energy = -1.94 x relative enzyme activity - 5.54 (Formula 3)

[0170] Finally, the relative enzyme activity of each variant is calculated according to the "average energy-relative enzyme activity" formula. The lower the average energy, the higher the relative enzyme activity, and the higher the enzyme activity of the variant.

[0171] 2.8 Solubility calculation

[0172] Hyaluronidase as an important enzyme for clinical use, its solubility not only directly affects its activity and use efficiency, but also affects its effective absorption and distribution in the body, predicting its solubility can help to select the appropriate rHuPH20 variant. In order to evaluate the solubility of each variant, the present application predicts the CamSol score of each variant. This algorithm is based on the physical and statistical properties of protein sequences, and predicts the solubility of proteins through a specific mathematical model. First, a linear combination of biophysical properties is used to calculate the solubility score of each residue, and then the score is adjusted according to the position of each residue in the three-dimensional structure and the properties of the surrounding residues. Finally, based on the physical and statistical properties of protein sequences, the solubility of proteins is predicted through a specific mathematical model, and the prediction result is represented by CamSol score. The higher the CamSol score, the higher the solubility, and the better the enzyme activity in the production process and the effect in the body.

[0173] 2.9 pI, Net charge calculation

[0174] The isoelectric point (pI) and net charge of each variant are predicted using the Molecular Operating Environment (MOE) software. In the prediction process, first, the three-dimensional structure file of the protein is imported into MOE. MOE estimates the pKa value of the amino acid residues of the protein through the built-in pKa database and the extended Henderson-Hasselbalch equation. Then, MOE simulates the ionization state of amino acids under different pH conditions, and calculates the total net charge of the protein under each pH. The pH value when the net charge is zero is the isoelectric point (pI) of the protein. And get the net charge under the corresponding pH.

[0175] 2.10 Positive and negative patch area, hydrophobic patch area calculation

[0176] MOE software is used to divide the surface by recognizing the charge distribution and hydrophobic characteristics of the molecule. MOE uses charge distribution data to calculate the areas on the surface of the protein molecule with positive and negative charges. By integrating the surface area of these charged regions, the positive and negative charge patch areas are obtained, respectively. And according to the hydrophobicity index of amino acids, hydrophobic regions are identified, and by integrating the surface area of these hydrophobic regions, the hydrophobic patch area can be obtained.

[0177] 2.11 Oxidation risk site prediction

[0178] Residue Exposure in the process of molecular dynamics (MD) simulation reflects the degree of exposure of each amino acid residue to solvent in the solution environment, which is an important indicator for predicting the region prone to oxidation. Oxidation usually occurs at highly exposed amino acid residues, so the exposure of methionine (Met, M) and tryptophan (Trp, W) is dynamically evaluated by MD simulation. When the average exposure is greater than 30%, it is considered to have an oxidation risk, and the oxidation risk of each variant is evaluated according to the number of these risk sites.

[0179] 2.12 Deamidation risk site prediction

[0180] Using MOE software, the deamidation risk of each site is evaluated by identifying the solvent accessible surface area of each asparagine site in the protein, the secondary structure of the position, the dihedral angle inside the amino acid, etc. The deamidation risk of each variant is evaluated according to the number of deamidation risk sites of each variant.

[0181] 2.13 Isomerization risk site prediction

[0182] Using MOE software, the isomerization risk of each site is evaluated by identifying the solvent accessible surface area of each aspartic acid site in the protein, the secondary structure of the position, the dihedral angle inside the amino acid, etc. The isomerization risk of each variant is evaluated according to the number of aspartic acid risk sites of each variant.

[0183] Example 3. Design and screening of rHuPH20 variants

[0184] 3.1 First round of design

[0185] 3.1.1 Design scheme

[0186] Because the enhancement of α-helix stability can promote the improvement of enzyme activity, the first round of design mainly considers α-helix replacement and single-point mutation. The present application first compares the structure of rHuPH20 and the crystal structure of Hyal1, and the most different region is that the secondary structure of α-helix 1 of rHuPH20 is Loop, while that of Hyal1 is α-helix (α-helix). This indicates that α-helix 1 has the potential for modification, and replacing the amino acid positions corresponding to P21 to M35 in rHuPH20 with the amino acid residues of Hyal1 can make its secondary structure be α-helix, thereby improving the structural stability, so as to design the variant HAase-1 of rHuPH20.

[0187] Meanwhile, based on Hyal1 and Hyal2, according to the strength of each amino acid stabilizing alpha-helix, an alpha helix 5 with stronger stability was designed. The amino acid positions corresponding to F204P to Q220 in rHuPH20 were replaced with the designed alpha helix 5, and by enhancing its stability, the goal of enhancing its thermal stability and enzyme activity was achieved, thereby designing the variant HAase-2 of rHuPH20. The present application further superimposes the modification of alpha helix 1 and alpha helix 5, and designs the variant HAase-3.

[0188] Therefore, the two fragments S69-T71 and V1410-L143 are subjected to mutation modification on the basis of replacing the alpha helix 5, and the variants HAase-4 and HAase-5 are obtained. At the same time, the oxidation site and the breaking site are subjected to mutation modification, and the variant HAase-6 is obtained.

[0189] Table 1 rHuPH20 variant first round candidate molecules

[0190] 3.1.2 Evaluation results

[0191] The present application models the designed rHuPH20 variant, and constructs the complex structure of rHuPH20 variant and hyaluronic acid (HA), and then performs 10 ns MD simulation. The simulation results show that the relative enzyme activity of HAase-1 is higher than that of rHuPH20, indicating that the replacement of alpha helix 1 helps to improve the enzyme activity; and the relative enzyme activities of HAase-2 and HAase-3 are both less than 1, indicating that the replacement of alpha helix 5 is not conducive to the improvement of enzyme activity. The HAase-4 to HAase-6 based on the single-point mutation of alpha helix 5, compared with HAase-2, have different degrees of improvement in enzyme activity, indicating that these single-point mutations are conducive to the improvement of enzyme activity.

[0192] However, in terms of conformational stability, the characterization results by RMSD and Rosetta ΔG show that all the rHuPH20 variants are significantly inferior to Alteogen-HP46, indicating that the deletion of the N-terminus and C-terminus of Alteogen-HP46 is conducive to the conformational stability. Therefore, the next round of design will not only revolve around the replacement and single-point mutation of alpha helix 1, but also plans to superimpose the deletion of the N-terminus and C-terminus to further improve the performance of the variant.

[0193] Table 2 Calculation results of rHuPH20 variant first round candidate molecules

[0194] 3.2 Second round design

[0195] 3.2.1 Design scheme

[0196] In order to improve the overall stability of the rHuPH20 variants, the present application designs a new round of rHuPH20 variants by adding deletions at the N-terminus or C-terminus. Considering the proximity to the enzyme active center, the number of amino acids deleted at the N-terminus should be appropriately small.

[0197] Based on the evaluation results of the first round of designed variants, this round of design adds single-point mutations, N-terminal and C-terminal deletions to the replacement of alpha helix 1. Finally, 9 rHuPH20 variants (HAase-7 to HAase-15) are designed and obtained. The specific sequences of these variants are shown in Table 3 below.

[0198] Table 3 rHuPH20 variants of the second round of candidate molecules

[0199] 3.2.2 Evaluation results

[0200] The rHuPH20 variants designed in this round are modeled, the complex structure of rHuPH20 variants and HA is constructed, and 10 ns of MD simulation is performed. The simulation results show that, on the basis of the replacement of alpha helix 1, single-point mutations (N141H, V142R, S69A and I70A) can increase the enzyme activity. At the same time, the deletion of the C-terminus can also improve the enzyme activity. Specifically, the effect of S69A and I70A in single-point mutation is better than that of N141H and V142R.

[0201] The replacement of alpha helix 1 and the deletion of N-terminus and C-terminus significantly improve the conformational stability of the protein. For the aggregation tendency of the protein, the deletion of the C-terminus can effectively inhibit the aggregation of the protein. However, the effect of the replacement of part of alpha helix 1 with single-point mutation in improving the enzyme activity is weaker than that of the replacement of alpha helix 5 with single-point mutation.

[0202] Based on the evaluation results of the first round and this round, the next round of design will integrate these findings and make more detailed optimization of the variants. Specifically, for the N-terminus, 1 to 2 residues will be considered for deletion; for the alpha helix, replacement attempts of other alpha helices will be added; for the C-terminus, 3 to 14 residues will be considered for deletion; and single-point mutations will cover more combinations of sites. This optimization design aims to further improve the enzyme activity, conformational stability and anti-aggregation of the rHuPH20 variants.

[0203] Table 4 Calculation results of rHuPH20 variants of the second round of candidate molecules

[0204] 3.3 Third round of design

[0205] 3.3.1 Design Scheme

[0206] Based on the design and evaluation of the first and second rounds, N-terminal or C-terminal deletion, a-helix replacement, and amino acid mutation all have a positive impact on enzyme activity, conformational stability, inhibition of aggregation, and chemical degradation. Therefore, this round of design will further optimize the previous two rounds.

[0207] This round of design not only continues to focus on a-helix 1 and a-helix 5, but also evaluates other a-helices of the catalytic center of rHuPH20 (a-helix 3, a-helix η' and 4', a-helix 4, a-helix 6, a-helix 7, and a-helix 8 in Figure 1) to determine the a-helices with modification potential and select their modification direction.

[0208] a-helix 3 (P80 to Y99): There is a Coil inside this helix. After replacing the amino acid residues from P80 to Y99 with those of Hyal2, the number of internal hydrogen bonds increased from 22 to 27, improving the structural stability.

[0209] a-helix η' and 4' (W119 to Q140): These two a-helices are connected by a Loop, and the number of residues forming a-helix inside this part fluctuates greatly. After replacing the amino acid residues from W119 to Q140 with those of Hyal2, the number of hydrogen bonds increased from 22 to 24, enhancing the structural stability and having modification potential.

[0210] a-helix 4 (S145 to R176) and a-helix 6 (P236 to V253): The secondary structure of these two a-helices is very stable, so it is less likely to further improve its stability through modification, and it has no modification potential.

[0211] a-helix 7 (P281 to V298): Half of the amino acids inside the a-helix are distal residues that affect the enzyme active center residues, and modifying this helix will significantly affect the position of the enzyme active center residues. For this reason, a-helix 7 is not modified.

[0212] a-helix 8 (T306 to C346): There is a Loop inside the secondary structure of this part, and MD simulation shows that the number of a-helix amino acids decreases, having modification potential. In addition, replacing T306 to C346 with some or all of the amino acid residues of Hyal1 or Hyal2 to increase the number of hydrogen bonds and improve the structural stability.

[0213] Point mutation: Select and combine multiple mutation sites that improve the activity and stability of rHuPH20 to obtain a variant with high expression efficiency, enzyme activity, and stability.

[0214] Based on the above considerations, 20 rHuPH20 variants (HAase II-1 to HAase II-20) were designed in this round, and the specific sequences are shown in Table 5.

[0215] Table 5 rHuPH20 variants third round candidate molecules

[0216] Note: rHuPH20, Halozyme II-F204P, Alteogen-HP46 and Halozyme II-I70A serve as control molecules.

[0217] 3.3.2 Evaluation results

[0218] Through the above calculation evaluation method, all rHuPH20 variants in Table 5 were comprehensively evaluated from five aspects of enzyme activity, solubility, conformational stability, aggregation tendency and chemical degradation. Table 6 shows the detailed evaluation results of these variants. The evaluation results show that these variants have significant improvements in different evaluation dimensions, indicating that these variants have significant improvements in these different dimensions.

[0219] Table 6 rHuPH20 variants third round candidate molecules

[0220] 3.4 Conclusion

[0221] The present application improves rHuPH20 from the following three dimensions through iterative design: N-terminal and C-terminal deletion, α-helix replacement, and amino acid mutation, in order to obtain variants with high expression efficiency, enzyme activity and stability.

[0222] In the first, second and third rounds of design, the present application comprehensively considers these modification strategies, designs, evaluates and obtains a plurality of rHuPH20 variants. The rHuPH20 variants exhibit excellent enzyme activity, solubility, conformational stability, anti-aggregation tendency and anti-chemical degradation.

[0223] Example 4. Construction, expression and enzyme activity determination of rHuPH20 variant expression vector

[0224] 4.1 Construction of rHuPH20 variant plasmid

[0225] After codon optimization of the coding sequence of rHuPH20 or its variants as shown in Table 5, synthesis of rHuPH20 variant sequences was performed. Restriction enzyme cleavage sites BamHI (New England Biolabs, Cat. No. R3136S) and NgoMIV (New England Biolabs, Cat. No. R0564S) were designed at both ends of the rHuPH20 variant sequence in advance, and the synthesized rHuPH20 variant sequence was double-digested, and then the rHuPH20 variant sequence fragment obtained by enzyme digestion was inserted into the pCGS3 vector (Merck) using T4 DNA ligase (New England Biolabs, Cat. No. M0202L), thereby obtaining a final plasmid containing the rHuPH20 or its variant sequence. Subsequently, the plasmid was amplified in competent E. coli DH5α for subsequent cell transfection.

[0226] 4.2 Transfection of rHuPH20 variant plasmid and cell recovery

[0227] CHOZN cells were selected as host cells for transfection, and the constructed plasmid carrying the rHuPH20 variant sequence was transfected into the host cells by electroporation. Before electroporation, it was confirmed that the host cells were in the logarithmic growth phase (cell density 2-4 x 10 6 After electroporation, the cells were incubated at 37°C in an incubator, and the cell density and viability were detected by regular counting. When the cell viability was >70%, the cells were transferred to a shake flask for subculture.

[0228] 4.3 Fed-batch culture of rHuPH20 variant expression cells in a shake flask

[0229] When the shake flask subculture cell viability was >95%, the cells were inoculated at a density of 0.3 x 10 6 The cells were incubated in a 37°C, 200 rpm shaking incubator, and the culture period was eight days. The cell density, viability, and glucose content were detected regularly to maintain the glucose concentration above 1 g / L, and a certain volume of feed medium was added on the fourth and sixth days. Samples were taken on the fourth and eighth days, and the supernatant was collected after centrifugation for enzyme activity detection.

[0230] In order to confirm the industrial applicability of hyaluronidase, the activity of the enzyme needs to be analyzed. The detection method of enzyme activity is turbidimetry (transmittance % at 600 nm, optical path = 1 cm), which measures the absorbance of the precipitate produced when hyaluronic acid is mixed with bovine serum albumin. The principle of detecting enzyme activity is that when hyaluronic acid is hydrolyzed by hyaluronidase, the absorbance of the precipitate produced when mixed with albumin decreases.

[0231] Turbidity test was performed as follows: Hyaluronidase standard (European Standard) was diluted to 2, 5, 10, 20, 25 units / mL and prepared in each test tube. rHuPH20 variant supernatant samples were dissolved in enzyme dilution buffer (20 mM sodium phosphate with 77 mM sodium chloride and 0.01% (w / v) bovine serum albumin, pH 7.0, 37°C) to a certain concentration, then diluted to 6 concentration gradients in test tubes: 1x, 2x, 4x, 8x, 16x, 32x. After preheating at 37°C for 25 min, 20 μL of diluted sample was placed in a 96-well plate. Then 20 μL of 1 mg / mL hyaluronic acid solution preheated at 37°C was added to each well. After mixing, incubate at 37°C for 55 min, then add 160 μL of acid albumin solution preheated at 37°C to each well and incubate at room temperature for 5-10 min. Finally, read the absorbance value of each well at 600 nm.

[0232] After the sample detection was completed, first, the standard curve was drawn by linear fitting with the final concentration of the standard as the abscissa and the absorbance value as the ordinate. Then the enzyme activity corresponding to the absorbance value of the sample in the linear region was calculated, and the average enzyme activity of the dilution samples in the linear region was taken as the final result. In the enzyme activity detection on the 4th day, some samples had enzyme activity lower or higher than the linear region, so the results were summarized with enzyme activity <40 U / mL or >400 U / mL. The test results of enzyme activity are shown in Table 7.

[0233] Table 7 rHuPH20 and its variants of shake flask fed-batch enzyme activity data

[0234] From the data in Table 7, HAase II-14, HAase II-17 and HAase II-20 all showed certain enzyme activity on the 8th day after transfection, and HAase II-20 showed much higher enzyme activity than the control molecule. These molecules will be used for stability investigation, and subsequent monoclonal cell strain screening may be carried out.

[0235] 4.4 rHuPH20 variant expression cell reactor fed-batch culture

[0236] The rHuPH20 variant expression cells with high enzyme activity in the shake flask fed-batch culture were recovered, and after two amplification passages in shake flasks, they were inoculated into a 2L bioreactor at (0.8±0.2) x 10 6 The reactor was set to: pH 6.95±0.1, dissolved oxygen 50%, stirring speed 250 rpm, initial culture temperature 37.0°C, when VCD≥8 x 10 6When the cell density is 1.5 x 106cells / ml, the temperature will be 33.0°C. The culture cycle is 13 days, and after the culture ends, the cell culture fluid of rHuPH20 variants is purified.

[0237] During the culture process, a certain volume of feed medium is added every day from the third day to the twelfth day, and an appropriate amount of glucose solution is added to maintain the glucose concentration in the culture fluid greater than 1 g / L. The viable cell density, viability, metabolic parameters, and osmotic pressure are detected every day, and the supernatant of the eighth and thirteenth day samples is collected after centrifugation for enzyme activity detection. The test results of enzyme activity are shown in Table 8.

[0238] Table 8. Reactor fed-batch enzyme activity data of rHuPH20 and its variants

[0239] 4.5 Purification of rHuPH20 variants

[0240] After completing the reactor fed-batch culture, an appropriate amount of 10% glacial acetic acid is added to adjust the pH of the untreated cell harvest fluid to be acidic, and then clarified by deep filtration membrane.

[0241] The rHuPH20 variant protein is purified by two-step column chromatography, Capto S Impact (Cytiva) and Capto Phenyl (High sub) (Cytiva).

[0242] For protein purification using Capto S Impact filler, buffer A (20 mM sodium acetate, pH 5.3) and buffer B (20 mM sodium phosphate, pH 7.9) are prepared, the protein is bound to the Capto S Impact column, and then washed with 5 CV of buffer A to remove non-specifically bound proteins, and then eluted with 30 CV of buffer B with a concentration gradient of 0 to 100% to elute the protein.

[0243] For protein purification using Capto Phenyl (High sub), buffer A (20 mM sodium phosphate, pH 7.0, 1.2 M (NH4)2SO4) and buffer B (20 mM sodium phosphate, pH 7.0) are prepared, the protein is bound to the Capto Phenyl (High sub) column, and then washed with 5 CV of buffer A to remove non-specifically bound proteins, and then eluted with 30 CV of buffer B with a concentration gradient of 0 to 100% to elute the protein.

[0244] 4.6 Stability analysis of rHuPH20 variants

[0245] After obtaining the purified rHuPH20 variants, the melting temperature of the rHuPH20 variants was measured and compared with wild type rHuPH20 to assess the stability of the variants. The melting temperature was measured by a differential scanning fluorimeter. The instrument used was UNcle, and the module used was T m &T agg with optional DLS. The instrument settings and data collection procedures are described in Table 9.

[0246] Table 9 UNcle parameter settings

[0247] A 1 mg / mL protein sample in histidine buffer, pH 6.0, 150 mM NaCl, 30 mg / mL sucrose, 10 mM methionine, and 0.2 mg / mL Tween 80 was prepared.

[0248] The UNcle results showed that the T m and T m onset were the highest, 65°C and 61°C, respectively, which were about 10°C higher than other molecules. This indicated that the HAase II-20 variant was more stable against heat stress. Also, the aggregation temperature of HAase II-20 measured by UNcle was 67°C, which was 4°C higher than the aggregation temperature of rHuPH20, 63°C, also indicating an increase in thermal stability (Table 10).

[0249] Table 10 T m and T agg of rHuPH20 and its variants

[0250] The prepared solutions were each aliquoted in 0.3 mL into 2 mL vials. The samples were incubated at 40°C. At the prescribed time points, samples were withdrawn from the incubator and subjected to enzyme activity, visual inspection, hydrated particle size, and SEC-HPLC analysis. The results of the enzyme activity are shown in Table 11. At 40°C, the enzyme activity of Halozyme II-F204P and Halozyme II-I70A was almost completely lost within 5 days of incubation, the enzyme activity of HAase II-14 was reduced to 67%, and the enzyme activity of HAase II-17 was reduced to 31%. The enzyme activity of rHuPH20, HAase II-20, and Alteogen-HP46 did not change significantly over 5 days of incubation at 40°C, indicating that these three molecules have high stability. The results of the visual inspection showed that rHuPH20, Alteogen-HP46, Halozyme II-F204P, Halozyme II-I70A, HAase II-14, and HAase II-17 had visible particles after being stored at 40°C for 3 days, while HAase II-20 did not have visible particles, indicating that HAase II-20 has low aggregation. The hydrated particle sizes are shown in Table 12. The hydrated particle sizes of Halozyme II-F204P, Halozyme II-I70A, HAase II-14, and HAase II-17 increased significantly within 3 days of incubation, and were all > 100 nm. The increase in the hydrated particle sizes of rHuPH20 and Alteogen-HP46 was slightly lower, and the hydrated particle sizes increased to 16.6 and 33.2 nm, respectively, after 5 days of incubation. The hydrated particle size of HAase II-20 did not increase significantly within 5 days of incubation, and the hydrated particle size was still < 10.0 nm. The results of the SEC-monomer are shown in Table 13. The SEC-monomer of rHuPH20 increased significantly within 5 days of incubation, from 0.9% to 11.4%. The SEC-monomer of the rHuPH20 variants did not increase significantly.

[0251] Table 11 Relative enzyme activity of rHuPH20 and its variants at 40°C m Table 11 Relative enzyme activity of rHuPH20 and its variants at 40°C agg Based on the stability data at 25°C, 37°C, and 40°C, HAase II-20 has the best stability.

[0252] Table 11 Relative enzyme activity of rHuPH20 and its variants at 40°C

[0253] Table 12 Hydrated particle size of rHuPH20 and its variants at 40°C

[0254] Table 13 SEC-monomer content of rHuPH20 and its variants at 40°C

[0255] Example 5 Stability of rHuPH20 variants at high temperature

[0256] The stability of rHuPH20 variants from Example 4 under heat conditions was tested. HAase II-20 was tested for aggregation, degradation, and enzyme activity to evaluate its stability. It was prepared as 3 mg / mL protein samples in histidine buffer, pH adjusted to 6.0, NaCl concentration of 300 mM, sucrose concentration of 30 mg / mL, methionine concentration of 10 mM, and Tween 80 concentration of 0.2 mg / mL.

[0257] The prepared solutions were each aliquoted into 0.8 mL in 2 mL vials, and the samples were incubated at 40 °C. At the specified time points, samples were withdrawn from the incubator and tested for enzyme activity, visual inspection, sub-visible particles, hydrated particle size, SEC-HPLC, ME-SDS, and RP-UPLC. The enzyme activity results are shown in Figure 4. HAase II-20 did not show significant change in enzyme activity after 28 days of continuous incubation at 40 °C, indicating that this molecule is highly stable. The visual inspection results showed that HAase II-20 did not show visible particles after 28 days of continuous incubation at 40 °C. The hydrated particle size is shown in Figure 5. HAase II-20 did not show significant increase in hydrated particle size after 28 days of incubation. The SEC-monomer, ME-SDS, and RP-HPLC purity results are shown in Figures 6-8. There was no significant increase in purity after 28 days of incubation. In summary, HAase II-20 is highly stable in terms of enzyme activity, aggregation, and degradation.

[0258] Example 6. Stability of rHuPH20 variants in co-formulation under high temperature conditions

[0259] To demonstrate the stability results of HAase II-20, the purified HAase II-20 protein was formulated in 120 mg / mL Daratumumab, 8 mM Histidine, 269 mM Sorbitol, 0.4 mg / mL Tween 20, 0.9 mg / mL Met (methionine), pH 5.6, with enzyme activity in the range of 1000-3000 U / mL. The prepared solution was aliquoted into 0.3 mL and divided into 2 mL vials, and the Darzalex The drug solution was aliquoted into 0.3 mL in 2 mL vials as a control. The vials were incubated at 40 °C. At different time points, samples were withdrawn from the incubator, and enzyme activity was measured as described in Example 4.

[0260] The enzyme activity test results are shown in Figure 9. As can be seen from Figure 9, the Darzalex The enzyme activity in the enzyme solution was rapidly reduced at 40°C, and only 34% of the enzyme activity remained after 48 hours of incubation. In contrast, the activity of HAase II-20 did not significantly decrease during the entire study, and 88% of the enzyme activity remained after 48 hours of incubation. This indicates that the stability of HAase II-20 is excellent in the co-formulation.

[0261] Example 7. Stability of rHuPH20 variants in ADC co-formulation at high temperature

[0262] To demonstrate the stability of HAase II-20 in the ADC co-formulation, the purified HAase II-20 protein (with Alteogen-HP46 or rHuPH20 as a control) was formulated in 20 mg / mL Trastuzumab Deruxtecan (in-house preparation) or trastuzumab-VC-MMAE, 25 mM histidine, 85 mg / mL sucrose, 0.2 mg / mL Tween 20, pH 5.5 (co-formulation solution) at an enzyme activity in the range of 1000-3000 U / mL. Trastuzumab Deruxtecan and trastuzumab-VC-MMAE employed trastuzumab produced using a CHO cell line and purified by PA-CEX-AEX three-step purification.

[0263] The ADC conjugation process for Trastuzumab Deruxtecan was as follows: 20 mL of trastuzumab solution (20 mg / mL) was placed in a reaction bottle. EDTA was added to a final concentration of 10 mM, followed by adjusting the pH to 7.5 with 0.5 M sodium phosphate dibasic solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 5 equivalents, 1.35 mL) was slowly added, mixed well, and placed at room temperature for 1 h. After the antibody interchain disulfide bonds were completely opened, MC-GGFG-DXD solution (10-fold equivalents, 27 μmol) dissolved in dimethyl sulfoxide was added to the above solution system, stirred well, and placed at room temperature for 2 h. The system buffer was replaced with 25 mM His-HCl, pH 5.5 using a Pellicon3 Ultracel membrane pack, and 85 g / L sucrose was added to a final concentration. Trastuzumab Deruxtecan was finally obtained. Mass spectrometry detected a DAR value of 8.0.

[0264] Structure of Trastuzumab Deruxtecan (here, Ab is trastuzumab, and k is the DAR value)

[0265] The conjugation process of Trastuzumab-VC-MMAE is as follows: 20 mL of Trastuzumab solution (20 mg / mL) was taken in a reaction flask. EDTA was added to a final concentration of 10 mM, followed by adjusting the pH to 7.5 with 0.5 M sodium phosphate dibasic solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 2.1 eq, 0.56 mL) was added slowly, mixed well and kept at room temperature for 1 h. After the interchain disulfide bonds of the antibody were completely opened, the above solution system was added with a solution of Mc-VC-PABC-MMAE (its structure is shown below) dissolved in dimethyl sulfoxide solution (6 eq, 16.2 pmol) in advance, stirred and mixed well and kept at room temperature for 2 h. The system buffer was replaced with 25 mM His-HCl, pH 5.5 using a Pellicon3 Ultracel membrane package, and sucrose was added to a final concentration of 85 g / L. Finally, the conjugation product of Trastuzumab and Mc-VC-MMAE, Trastuzumab-VC-MMAE ADC, was obtained. The DAR value detected by HIC was 4.0.

[0266] Structure of Trastuzumab-VC-MMAE ADC (herein Ab is Trastuzumab, and k is the DAR value)

[0267] The prepared co-formulation solutions were equally divided into 0.3 mL and were divided into 2 mL vials, and Alteogen-HP46 and rHuPH20 proteins were prepared in the same prescription as controls. The vials containing the co-formulation solutions were incubated at 40°C. Samples were taken from the incubator at different time points, and the enzyme activity was measured as described in Example 4.

[0268] The results of enzyme activity detection are shown in FIGS. 10 and 11. As can be seen from FIG. 10, in the co-formulation with Trastuzumab Deruxtecan ADC, the enzyme activities of Alteogen-HP46 and rHuPH20 rapidly decreased when incubated at 40°C, and after 10 hours of incubation, the enzyme activities were only 57% and 33%. However, the activity of HAase II-20 did not significantly decrease throughout the study, and after 10 hours of incubation, the enzyme activity was still 96%. As can be seen from FIG. 11, in the co-formulation with Trastuzumab-VC-MMAE ADC, the enzyme activities of Alteogen-HP46 and rHuPH20 rapidly decreased when incubated at 40°C, and after 10 hours of incubation, the enzyme activities were only 28% and 35%. However, the activity of HAase II-20 did not significantly decrease throughout the study, and after 10 hours of incubation, the enzyme activity was still 95%. This indicates that the stability of HAase II-20 is excellent in the co-formulation with ADC.

[0269] Example 8. Assay of the stability of rHuPH20 variants in co-formulation under high temperature and different co-formulation antibody conditions

[0270] To demonstrate the stability of HAase II-20 as a co-formulation with different antibodies, the purified HAase II-20 protein was formulated in 120 mg / mL of different antibodies (Daratumumab and Pembrolizumab), 8 mM histidine, 269 mM sorbitol, 0.4 mg / mL Tween 20, 0.9 mg / mL Met (methionine), pH 5.6, with enzyme activity in the range of 1000-3000 U / mL. The purified rHuPH20 protein was also formulated in the same solution environment as a control. The prepared solutions were aliquoted into 0.3 mL and divided into 2 mL vials. The vials were incubated at 40°C. Samples were taken from the incubator at different time points, and the enzyme activity was measured as described in Example 4.

[0271] The results of the enzyme activity detection are shown in Figure 12. As can be seen from Figure 12, the enzyme activity of rHuPH20 co-formulated with different antibodies decreased significantly after incubation at 40°C for 19 hours, and the rate of decrease was different. However, the enzyme activity of HAase II-20 in co-formulation with different antibodies did not decrease significantly after incubation for 19 hours, as shown in Figure 13. This indicates that the enzyme activity stability of HAase II-20 is excellent and is not affected by the type of antibody molecule.

[0272] Example 9. Stability of rHuPH20 variants in co-formulation under high temperature and different buffer system conditions

[0273] To demonstrate the stability of HAase II-20 in different co-formulation buffer systems, the purified HAase II-20 protein was formulated in 120 mg / mL of Daratumumab, 8 mM histidine or acetic acid, 269 mM sorbitol, 0.4 mg / mL Tween 20, 0.9 mg / mL Met (methionine), pH 5.0-6.5, with enzyme activity in the range of 1000-3000 U / mL. The prepared solutions were aliquoted into 0.3 mL and divided into 2 mL vials. The vials were incubated at 40°C. Samples were taken from the incubator at different time points, and the enzyme activity was measured as described in Example 4.

[0274] The results of the enzyme activity detection are shown in Figure 14. As can be seen from Figure 14, the enzyme activity of HAase II-20 did not decrease significantly in histidine and acetic acid co-formulation systems at the same pH after incubation at 40°C for 28 days. This indicates that the enzyme activity stability of HAase II-20 is excellent and has no preference for buffer systems.

[0275] In histidine system, the enzyme activity of HAase II-20 in co-formulation did not decrease significantly at 40°C for 28 days at pH 5.0-6.5, as shown in Figure 15. This indicates that the enzyme activity of HAase II-20 is excellent and has no preference for pH in histidine system.

[0276] In acetic acid system, the enzyme activity of HAase II-20 in co-formulation did not decrease significantly at 40°C for 28 days at pH 5.0-6.0, as shown in Figure 16. This indicates that the enzyme activity of HAase II-20 is excellent and has no preference for pH in acetic acid system.

[0277] Example 10. Measurement of rHuPH20 variants in the area of intradermal blue dye diffusion in nude mice

[0278] The diffusion activity of rHuPH20 variant HAase II-20 was evaluated by measuring the area of dye diffusion in vivo. The blue dye diffusion was divided into two groups of experiments: step injection and co-injection. HAase II-20 and Alteogen-HP46 were prepared in buffer (10 mM histidine, 150 mM NaCl, 10 mM Met (methionine), 30 mg / mL sucrose, 0.2 mg / mL Tween 80, pH 6.0). Wild-type rHuPH20 was purchased from Shanghai Pu Hongzhi Biological Technology Co., Ltd. This study used SPF male BALB / c-nu nude mice, 6 in each group, as shown in Table 14.

[0279] Table 14 Overview of treatment groups for dye diffusion study

[0280] Step injection was performed as follows: all nude mice received an intradermal injection of 40 μL of test article at study time 0. 1 min after injection of the test article, a volume of 0.04 mL of blue dye stain (0.2% trypan blue solution) was administered by intradermal injection at the same injection site. Co-injection was performed as follows: the hyaluronidase variant (HAase II-20) was diluted 1:1 with 0.4% trypan blue, and all nude mice received an intradermal injection of 40 μL of the mixed sample at study time 0. The area of dye diffusion was measured at 1, 2.5, 5, and 20 minutes after injection, and photographs were taken of the injection site using a camera. The camera was precisely positioned at a predetermined distance from the area of trypan blue dye using a stand. The area of dye was determined using Image j-1.54g software. The calculated area is expressed in cm 2 .

[0281] The results are shown in Table 15 and Figure 17. The results show that in the step injection group, the diffusion activity of rHuPH20 variant HAase II-20 is substantially equivalent to that of Alteogen-HP46 and rHuPH20, and is significantly different from the negative control. And HAase II-20 reaches the same diffusion effect as 200 U / mL at 50 U / mL, which indicates that HAase II-20 functions at 50 U / mL. In the co-injection group, 70 U / mL of HAase II-20 significantly increases the trypan blue diffusion area compared to the negative control at 1 min after injection, which indicates that HAase II-20 functions at 70 U / mL. And the diffusion effect of 600 U / mL of HAase II-20 in the co-injection group is the same as that of the step injection, which indicates that 600 U / mL of HAase II-20 can achieve the same effect as the step injection. In summary, the results show that HAase II-20 functions at a lower dose of enzyme activity.

[0282] Table 15 Trypan blue diffusion

[0283] Example 11. Skin barrier reconstruction after administration of rHuPH20 variants

[0284] Skin barrier reconstruction after hyaluronidase administration was determined by monitoring the area of diffusion of 0.2% trypan blue over time. The amount of time required for the skin barrier to reconstruct itself after intradermal administration of hyaluronidase (HAase II-20, rHuPH20 or Alteogen-HP46) was determined. The proteins used in this study were purified rHuPH20 variant HAase II-20 and Alteogen-HP46 (prepared as described in Example 4), and wild-type rHuPH20 HAase II-20 and Alteogen-HP46 were formulated in buffer (10 mM histidine, 150 mM NaCl, 10 mM Met (methionine), 30 mg / mL sucrose, 0.2 mg / mL PS80, pH 6.0). Wild-type rHuPH20 SPF grade male BALB / c-nu nude mice were purchased from Shanghai Primate Cloning and Transgenic Co., Ltd. The study used SPF grade male BALB / c-nu nude mice, 6 in each group, as shown in Table 16.

[0285] Table 16 Overview of treatment groups for skin reconstruction study of rHuPH20 and its variants

[0286] All mice received an intradermal injection of test article 40 μL at study time 0. At 0.5, 12, 24, and 48 hours after the injection of test article, a volume of 0.04 mL of trypan blue stain (0.2% trypan blue solution) was administered by intradermal injection at the same injection site. At 5 and 20 minutes after the injection of trypan blue stain, the dye area at the injection site was measured by digital imaging of the area as described in Example 9.

[0287] The results, as shown in Table 17 and Figure 18, show that the dye spread area of HAase II-20 was significantly less than rHuPH20 when the trypan blue dye was injected 30 minutes after the injection of hyaluronidase, 5 minutes later, indicating that the subcutaneous tissue recovered faster for HAase II-20 than for rHuPH20. The above results show that the subcutaneous tissue recovered faster after injection of rHuPH20 variant HAase II-20 than after injection of rHuPH20.

[0288] In addition, when the trypan blue dye was injected >12 hours after the injection of hyaluronidase, there was no significant difference in the trypan blue spread area for HAase II-20, Alteogen-HP46, and rHuPH20, i.e., the subcutaneous tissue was similar, because the subcutaneous tissue had fully recovered within 12 hours.

[0289] Table 17 Skin Reformation

[0290] In addition to the various embodiments depicted and claimed, the disclosed subject matter is also directed to other embodiments having other combinations of the features disclosed and claimed herein. As such, the particular features presented herein can be combined in other ways to achieve other embodiments of the disclosed subject matter. The above description of the specific embodiments of the disclosed subject matter has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to the precise forms disclosed.

[0291] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the spirit or scope of the disclosure. Thus, it is intended that the present disclosure cover the modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.

[0292] Various publications, patents and patent applications are cited herein, the contents of which are hereby incorporated by reference in their entirety.

Claims

1. A PH20 variant comprising at least one amino acid substitution compared to a wild type PH20 polypeptide, wherein the PH20 variant exhibits at least one improved property compared to the wild type PH20 polypeptide (SEQ ID NO: 1) not containing the amino acid substitution.

2. The PH20 variant of claim 1, wherein the improved property is increased thermostability.

3. The PH20 variant of claim 1 or 2, wherein the improved property is increased resistance to denaturation at temperatures above 30°C.

4. The PH20 variant of any one of claims 1 to 3, wherein the improved property is increased enzymatic activity.

5. The PH20 variant of any one of the preceding claims, wherein the improved property is increased resistance to aggregation.

6. The PH20 variant of any one of the preceding claims, wherein the improved property is increased solubility.

7. The PH20 variant of any one of the preceding claims, wherein the improved property is increased resistance to fragmentation and / or oxidation.

8. The PH20 variant of any one of the preceding claims, wherein the improved property is less degradation.

9. The PH20 variant of any one of the preceding claims, wherein the improved property is better post-application skin barrier rebuilding properties.

10. The PH20 variant of any one of the preceding claims, wherein the PH20 variant has at least 85%, at least 90%, at least 92%, at least 95%, at least 97% sequence identity to the amino acid sequence set forth in SEQ ID NO:

1.

11. The PH20 variant of any of the preceding claims, wherein the PH20 variant comprises one or more amino acid substitutions at a position selected from P21, S22, E23, F24, G27, K28, F29, D30, E31, P32, L33, M35, A48, V58, S69, I70, V75, K82, I83, S84, Q86, D87, L89, D90, K91, A92, K93, D95, I96, T97, F98, M100, A120, K124, P125, K130, N131, R132, I134, E135, Q138, Q139, Q140, N141, V142, L165, V166, F204, N205, I208, K209, R210, D213, S215, I271, L278, T306, L307, S308, I309, M310, R311, S312, M313, K314, S315, L317, L318, D320, N321, M323, E324, T325, I326, N328, I331, I332, T335, L336, A338, K339, M340, Q343, V344, and L345, as compared to the wild-type PH20 polypeptide set forth in SEQ ID NO: 1, wherein the corresponding amino acid position is identified by alignment of the PH20 variant and the polypeptide set forth in SEQ ID NO:

1.

12. The PH20 variant of claim 11, wherein the amino acid substitution(s) is selected from one or more of: P21N, S22T, E23Q, F24W, G27E, K28R, F29H, D30G, E31V, P32D, L33V, M35V, A48N, V58Q, S69A, I70A, V75L, K82N, I83V, S84G, Q86W, D87A, L89R, D90K, K91M, A92L, K93Q, D95R, I96V, T97E, F98H, M100I, A120V, K124Q, P125D, K130R, N131R, R132L, I134R, E135Q, Q138A, Q139S, Q140R, N141H, V142R, L165D, V166R, F204P, N205S, I208V, K209A, R210Q, D213Q, S215A, I271L, L278R, T306S, L307W, S308E, I309N, M310T, R311T, S312N, M313T, K314E, S315T, L317Q, L317K, L318Y, D320K, N321D, M323L, E324T, T325R, I326L, N328V, I331V, I332V, T335S, L336W, A338T, K339Q, M340Y, Q343R, V344A, and L345Q, wherein the corresponding amino acid position is identified by alignment of the PH20 variant with the polypeptide set forth in SEQ ID NO:

1.

13. The PH20 variant of any of the preceding claims, wherein the PH20 variant comprises one or more amino acid substitution(s) at a position selected from I70, M100, F204, T306, L307, S308, I309, M310, S312, M313, K314, L317, L318, D320, N321, E324, and I326, as compared to the wild-type PH20 polypeptide set forth in SEQ ID NO: 1, wherein the corresponding amino acid position is identified by alignment of the PH20 variant with the polypeptide set forth in SEQ ID NO:

1.

14. The PH20 variant of any of the preceding claims, wherein the PH20 variant comprises one or more amino acid substitution(s) selected from I70A, M100I, F204P, T306S, L307W, S308E, I309N, M310T, S312T, M313K, K314E, L317Q, L318A, D320K, N321D, E324D, and I326T, as compared to the wild-type PH20 polypeptide set forth in SEQ ID NO: 1, wherein the corresponding amino acid position is identified by alignment of the PH20 variant with the polypeptide set forth in SEQ ID NO:

1.

15. The PH20 variant of claim 13 or 14, wherein the PH20 variant further comprises one or more amino acid substitutions at a position selected from P21, S22, E23, F24, G27, K28, F29, D30, E31, P32, L33, and M35, as compared to the wild-type PH20 polypeptide set forth in SEQ ID NO: 1, wherein the corresponding amino acid position is identified by alignment of the PH20 variant with the polypeptide set forth in SEQ ID NO:

1.

16. The PH20 variant of any one of claims 13-15, wherein the PH20 variant further comprises one or more amino acid substitutions selected from P21N, S22T, E23Q, F24W, G27E, K28R, F29H, D30G, E31V, P32D, L33V, and M35V, as compared to the wild-type PH20 polypeptide set forth in SEQ ID NO: 1, wherein the corresponding amino acid position is identified by alignment of the PH20 variant with the polypeptide set forth in SEQ ID NO:

1.

17. The PH20 variant of any one of the preceding claims, wherein the PH20 variant has 1 or 2 amino acids deleted from the N-terminus relative to the polypeptide set forth in SEQ ID NO:

1.

18. The PH20 variant of any one of the preceding claims, wherein the PH20 variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids deleted from the C-terminus relative to the polypeptide set forth in SEQ ID NO:

1.

19. The PH20 variant of any one of the preceding claims, wherein the PH20 variant has at least 85%, at least 90%, at least 92%, at least 95%, at least 97% sequence identity to an amino acid sequence selected from any one of SEQ ID NOs: 5-24.

20. The PH20 variant of any one of the preceding claims, wherein the PH20 variant comprises an amino acid sequence selected from any one of SEQ ID NOs: 5-24.

21. The PH20 variant of any one of the preceding claims, wherein the PH20 variant further comprises a secretion signal peptide at the N-terminus.

22. The PH20 variant of any one of the preceding claims, wherein the PH20 variant is secreted upon expression from a cell and is soluble in the supernatant.

23. The PH20 variant of any one of the preceding claims, wherein the PH20 variant is substantially purified or isolated.

24. The PH20 variant of any one of the preceding claims, wherein the PH20 variant is modified by a modification selected from glycosylation, sialylation, albuminization, farnesylation, carboxylation, hydroxylation, and phosphorylation.

25. The PH20 variant of any one of the preceding claims, wherein the PH20 variant is glycosylated, wherein the polypeptide comprises at least N-acetylglucosamine moieties, attached to each of at least three, e.g., three, four, five, or six asparagine (N) residues.

26. The PH20 variant of any of the preceding claims, wherein the PH20 variant is conjugated to a moiety selected from a multimerization domain, a toxin, a detectable label, or a drug.

27. The PH20 variant of any of the preceding claims, wherein the PH20 variant is conjugated to an Fc domain.

28. A nucleic acid encoding the PH20 variant of any of claims 1-27.

29. A vector comprising the nucleic acid of claim 28.

30. A cell comprising the nucleic acid of claim 28 or the vector of claim 29.

31. The cell of claim 30, wherein the cell is a Chinese hamster ovary (CHO) cell.

32. A method of producing a PH20 variant, comprising: culturing the cell of claim 30 or 31 under conditions wherein the PH20 variant is produced and secreted by the cell; and recovering the expressed PH20 variant.

33. A pharmaceutical composition comprising the PH20 variant of any of claims 1-27.

34. A kit comprising the PH20 variant of any of claims 1-27 and instructions for use.

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