Human hyaluronidase 1 mutant

Abstract

Disclosed are human hyaluronidase 1 (HYAL1) mutants that highly express and exhibit hyaluronidase activity between pH 3.5 and pH 5.5.

Description

[0001] Reference to Electronically Submitted Sequence Listing

[0002] This application contains a sequence listing that has been electronically submitted in XML format and is hereby incorporated by reference in its entirety. The XML file was created on February 21, 2023, named 25596-WO-PCT_SL.XML and is 50,765 bytes in size. Background Art (1) Field of the Invention

[0003] The present invention relates to human hyaluronidase 1 (HYAL1) mutants that are highly expressed and exhibit hyaluronidase activity between pH 3.5 and pH 5.5.

[0004] (2) Description of Related Art

[0005] Subcutaneous (SC) delivery of biopharmaceuticals is a faster, less painful, and more convenient alternative to traditional intravenous injection or infusion 1-4 . In addition, many SC formulations are amenable to home administration via various devices, benefiting both patients and healthcare providers 1,4 . However, the acceptable maximum tolerated volume for SC injection is approximately 1.5 mL, which is incompatible with the typically large doses of most biopharmaceuticals 4 . Co-formulation with hyaluronidase (HAase) is a well-established method to significantly increase the tolerated volume for SC delivery of biopharmaceuticals and other molecules, up to 500 mL in some cases 2,5 . In fact, since the 1950s, bovine and ovine hyaluronidases have been used clinically as "spreading factors" to enhance tissue permeability 6-9 . In the past 20 years, recombinant human PH20 (rHuPH20) hyaluronidase, marketed under the trade name HYLENEX, has further broadened the availability of novel SC formulations for oncology and immunotherapy.

[0006] Hyaluronidase acts by degrading hyaluronic acid (HA) polymers, which are one of the major structural components of the extracellular matrix in the dermal layer of the skin 10 . These polymers release sequestered water upon cleavage by hyaluronidase, which allows for the diffusion of injection formulations within the dermal layer and absorption through underlying capillaries and lymphatics 11。In humans, there are five hyaluronidase isotypes: HYAL1-4 and HYAL5 (more commonly known as PH20 or SPAM1). PH20 is the only isotype with high enzymatic activity at neutral pH, severely limiting the clinical prospects of other human hyaluronidases. Efforts to increase the pH range of HYAL1 have met with some success, where activity can be observed up to pH 5.9 12 。 SUMMARY OF THE INVENTION

[0007] The present invention provides human hyaluronidase 1 (HYAL1) mutants that have been evolved by iterative protein engineering to have greatly improved hyaluronidase activity compared to wild-type HYAL1 and, more importantly, hyaluronidase activity over an extended pH range. Additionally, the HYAL1 mutants have an improved expression yield relative to wild-type HYAL1. These HYAL1 mutants can be used to increase the absorption of therapeutic agents into tissues and have the potential to reduce tissue damage in the case of extravasation of therapeutic agents. The HYAL1 mutants of the present invention provide HYAL1 mutants with a pH-controlled activity profile that reduces the effect of active hyaluronidase activity in the skin after the co-administered therapeutic agent has diffused through the skin, and thus reduces the undesirable side effects of hyaluronidase activity that persists after the therapeutic agent has diffused

[0008] The present invention provides HYAL1 mutants comprising an amino acid sequence having at least 95% (and in specific embodiments, 96%, 97%, 98%, 99% and 100%) identity to the amino acid sequence of amino acids 20 to 433 of the amino acid sequence shown in SEQ ID NO:2, wherein the hyaluronidase 1 mutant further comprises a combination of amino acid substitutions selected from: (a) the amino acid substitutions D68N, M246L, D323N, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M and W433E; (b) the amino acid substitutions D68N, M246L, D323N, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E and V412R; (c) the amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, V412R, R238C, I287P, F345T and L377S; (d) the amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, V412R, R238C, F345T and L377S; and (e) the amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, P23V, S304T, L379M, W433E, R194D, D299E, V412R, N235D, I287P, F345T and L377S; and wherein the hyaluronidase 1 mutant (i) includes at least one N-glycosylation site comprising the consensus sequence asparagine-X-serine / threonine, wherein X is any amino acid other than proline, the site is glycosylated, and (ii) exhibits hyaluronidase activity between pH 3.5 and pH 5.35 (or pH 5.5).

[0009] In certain embodiments, the HYAL1 mutant comprises the amino acid substitutions D68N, M246L, D323N, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, and W433E.

[0010] In certain embodiments, the HYAL1 mutant comprises the amino acid substitutions D68N, M246L, D323N, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, and V412R.

[0011] In certain embodiments, the HYAL1 mutant comprises the amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, V412R, R238C, I287P, F345T, and L377S.

[0012] In certain embodiments, the HYAL1 mutant comprises the amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, V412R, R238C, F345T, and L377S.

[0013] In certain embodiments, the HYAL1 mutant comprises the amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, P23V, S304T, L379M, W433E, R194D, D299E, V412R, N235D, I287P, F345T, and L377S.

[0014] In a further embodiment, at least one N-glycosylation site resulting from the D68N, D323N, G235N, or R326S substitution is glycosylated. In a further embodiment, the N-glycosylation sites resulting from the D68N, D323N, G235N, and R326S substitutions are each glycosylated. In a further embodiment, the native N-glycosylation site and the N-glycosylation sites resulting from the D68N, D323N, G235N, and R326S substitutions are each glycosylated.

[0015] The present invention further provides a HYAL1 mutant comprising an amino acid sequence having at least 95% (and in specific embodiments, 96%, 97%, 98%, 99%, and 100%) identity to the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24, wherein the hyaluronidase 1 mutant (i) includes at least one N-glycosylation site comprising the sequence asparagine-X-serine / threonine, wherein X is any amino acid other than proline, and the site is glycosylated, and (ii) exhibits hyaluronidase activity between pH 3.5 and pH 5.35 (or pH 5.5).

[0016] The present invention further provides a HYAL1 mutant comprising an amino acid sequence having at least 95% (and in specific embodiments, 96%, 97%, 98%, 99%, and 100%) identity to the amino acid sequences shown in SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24, wherein the hyaluronidase 1 mutant (i) includes at least one N-glycosylation site comprising the sequence asparagine-X-serine / threonine, wherein X is any amino acid other than proline, and the site is glycosylated, and (ii) exhibits hyaluronidase activity between pH 3.5 and pH 5.5.

[0017] The present invention further provides a HYAL1 mutant as disclosed herein conjugated to a polymer. In a particular embodiment, the polymer is dextran or polyethylene glycol.

[0018] The present invention further provides the HYAL1 mutants disclosed herein and a pharmaceutically acceptable carrier. In a further embodiment, the composition further comprises a therapeutic agent, and thus in certain embodiments, the present invention provides a composition comprising the HYAL1 mutants disclosed herein and a therapeutic agent. Representative therapeutic agents include, but are not limited to, small molecules, peptides, macrocyclic peptides, proteins or protein complexes. In certain embodiments, the small molecule comprises an antibiotic or an anti-inflammatory agent. In certain embodiments, the peptide comprises an insulinotropic peptide, growth hormone, insulin or an insulin mutant. In certain embodiments, the therapeutic agent comprises an antibody, an antigen-binding protein, a scFv, a Fab or a fusion protein comprising the Fc domain of an antibody.

[0019] In a further embodiment, the present invention provides a composition comprising tumor-specific CAR-T cells or CAR-NK cells conjugated to the HYAL1 mutants disclosed herein. See, e.g., Zhao et al., Bioorthogonal Equipping CAR-T Cells with Hyaluronidase and Checkpoint Blocking Antibody for Enhanced Solid Tumor Immunotherapy, ACS Cent. Sci. 8:603-614 (2022).

[0020] The present invention further provides a method for increasing the diffusion of a therapeutic agent in a tissue of a subject, which comprises administering to the tissue of the subject a composition comprising the HYAL1 mutants disclosed herein in an amount sufficient to increase the diffusion of the therapeutic agent; and administering to the tissue a composition comprising the therapeutic agent. The composition comprising the HYAL1 mutants and the composition comprising the therapeutic agent may be administered to the subject sequentially or simultaneously.

[0021] The present invention further provides a method for increasing the diffusion of a therapeutic agent in a tissue of a subject, which comprises administering to the tissue of the subject a composition comprising the therapeutic agent and a certain amount of the HYAL1 mutants disclosed herein in an amount sufficient to increase the diffusion of the therapeutic agent in the tissue.

[0022] The present invention further provides the use of the HYAL1 mutants disclosed herein for the manufacture of a medicament for use in combination with a therapeutic agent for treating a disease or disorder.

[0023] The present invention further provides the use of the HYAL1 mutants disclosed herein for the manufacture of a medicament comprising hyaluronidase and a therapeutic agent for treating a disease or disorder.

[0024] The present invention further provides the use of the HYAL1 mutants disclosed herein for increasing the diffusion of a therapeutic agent in the tissue of a subject for treating a disease or disorder.

[0025] The present invention further provides the use of a composition comprising the HYAL1 mutants disclosed herein and a therapeutic agent for treating a disease or disorder.

[0026] The present invention further provides a nucleic acid molecule encoding the HYAL1 mutants disclosed herein. The present invention further provides a nucleic acid molecule encoding the HYAL1 mutants disclosed herein, which is fused with a signal sequence targeting the HYAL1 mutants to the endoplasmic reticulum.

[0027] The present invention further provides an expression vector comprising a nucleic acid molecule encoding the HYAL1 mutants disclosed herein. In a specific embodiment, the nucleic acid molecule is fused with a signal sequence targeting the HYAL1 mutants to the endoplasmic reticulum.

[0028] The present invention further provides a host cell comprising a nucleic acid molecule encoding the HYAL1 mutants disclosed herein. In a specific embodiment, the nucleic acid molecule is fused with a signal sequence targeting the HYAL1 mutants to the endoplasmic reticulum. The present invention further provides a host cell comprising an expression vector comprising a nucleic acid molecule encoding the HYAL1 mutants disclosed herein. In a specific embodiment, the nucleic acid molecule is fused with a signal sequence targeting the HYAL1 mutants to the endoplasmic reticulum. In a further embodiment, the host cell is a mammalian host cell or a recombinant yeast host cell modified to produce complex N-glycans.

[0029] In a further embodiment, the mammalian host cell is a Chinese hamster ovary cell, and the recombinant yeast host cell is Pichia pastoris.

[0030] The present invention further provides a method for producing the HYAL1 mutants disclosed herein, which comprises: (a) introducing a nucleic acid molecule encoding the HYAL1 mutants or an expression vector comprising a nucleic acid molecule encoding the HYAL1 mutants disclosed herein into a host cell to produce a recombinant host cell; (b) culturing the recombinant host cell in a culture medium under conditions sufficient for the recombinant host cell to express the HYAL1 mutants and secrete them into the culture medium; and (c) obtaining the HYAL1 mutants from the culture medium.

[0031] In a further embodiment, the host cell is a mammalian host cell, and in a further embodiment, it may be a Chinese hamster ovary cell. In a further embodiment, the host cell is a recombinant yeast host cell modified to produce complex N-glycans, and in a further embodiment, it may be the recombinant yeast host cell Pichia pastoris.

[0032] In a further embodiment of the method, the HYAL1 mutant comprises amino acids 1 to 433 of the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13. These sequences contain a signal sequence that targets the HYAL1 mutant to the endoplasmic reticulum. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 An overview of the design / test section of the workflow for evolving human hyaluronidase 1 (HYAL1) into a HYAL1 mutant with a broader pH activity range, which can be used for the delivery of therapeutics for subcutaneous administration.

[0034] Figure 2 The results of the first round of screening are shown. This is a library constructed mainly based on sequence homology and literature reports on the effects of mutations on HYAL1 and other hyaluronidase homologs. The figure is sorted by FIOP from highest to lowest activity. FIOP stands for (fold improvement over parent) and refers to the activity of each mutant relative to the starting point of each round (e.g., in the first round, the starting point is wild-type HYAL1). FIOP is simply calculated as (activity of the variant) / (activity of the parent). The points on the graph show similar FIOP values regarding the expression level. Activity was measured using turbidimetry, and expression data were collected using a commercial HTRF assay kit.

[0035] Figure 3 Shows data very similar to Figure 2 In this case, the library variants were screened at both pH 4 (top) and pH 5 (bottom).

[0036] Figure 4A - Figure 4IThe process of HYAL1 evolution is shown in a round-by-round format. Each box represents a separate library, and each column of boxes represents a round of evolution (matching the table at the top). The "Fold improvement" row in the table shows how the HYAL1 backbone in that round compares to the backbone of the previous round. Rosetta refers to the RosettaCommons.org software used to model mutations.

[0037] Figure 5 In vivo data are shown that confirm the activity of the engineered HYAL1 mutants in a mouse skin dispersion model.

[0038] Figure 6 A workflow for expressing, purifying, and crystallizing the HYAL1 mutants is shown.

[0039] Figure 7 Shows the resulting structure from the Figure 6 workflow. The term "ASU" means "asymmetric unit".

[0040] Figure 8 In the 8th round of HYAL1 evolution, the focus became reducing the immunogenicity of the mutations introduced so far. ExpiVax software (EpiVax Inc.) was used to scan the regions of HYAL1 in which the mutations had been prepared. The software provides epitope scores that can be used to evaluate the immunogenicity of each possible 9-mer peptide in the scanned regions. The peptide sequences from top to bottom are represented by SEQ ID NO:34 to SEQ ID NO:44.

[0041] Figure 9 Seven epitope regions are shown that have an increased immunogenicity risk due to the mutations introduced during the evolution process. Depending on the score, these regions are each classified as low risk, medium risk, or high risk. EpiVax software (see Figure 8 ) was used for the analysis.

[0042] Figure 10 A method for designing additional HYAL1 mutations that will reduce the immunogenicity risk at the sites identified by EpiVax analysis is outlined. The peptide sequences from top to bottom are represented by SEQ ID NO:27 to SEQ ID NO:33.

[0043] Figure 11 The results of the 8th round of point mutant library screening are shown. The graph at the top shows the number of epitopes removed by each mutation introduced. The graph is divided into individual regions (A, B, C, D, E, and FG) that match the regions mapped on the Figure 9 structure shown.

[0044] Figure 12Shows the results of library screening for point mutations where the combination reduces immunogenicity. The figure shows Figure 12 the activities and expressed FIOPs of the mutants shown in Detailed Description

[0045] Definitions

[0046] As used herein, the terms "N-glycan" and "glycoform" are used interchangeably and refer to N-linked oligosaccharides, e.g., oligosaccharides attached by asparagine-N-acetylglucosamine linkage to the asparagine residue of a polypeptide. N-linked glycoproteins contain N-acetylglucosamine residues linked to the amide nitrogen of asparagine residues in the protein. The major saccharides found on glycoproteins are glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), and sialic acid (Sia). Sialic acid is a class of α-ketoacid sugars with a nine-carbon backbone. The most common member of this group is N-acetylneuraminic acid (Neu5Ac or NANA) found in animals and some prokaryotes. Processing of the sugar groups occurs co-translationally in the lumen of the ER and, for N-linked glycoproteins, continues post-translationally in the Golgi apparatus.

[0047] N-glycans have a common pentasaccharide core of Man3GlcNAc2, which contains a mannose linked at its reducing end by a β1,4 bond to the non-reducing end of a chitobiose core (GlcNAβ1-4GlcNAc), and one mannose residue linked by an α1,3 bond to the β1,4-linked mannose and another mannose linked by an α1,6 bond to the β1,4-linked mannose, represented by the structure showing the Man3GlcNAc2 linked to the asparagine residue that constitutes the N-glycosylation site in the glycoprotein

[0048]

[0049] The GlcNAc residue at the reducing end may also be linked by an α1,6 bond to a fucose residue. Generally, the structure of N-glycans is presented with the non-reducing end on the left and the reducing end on the right, or the non-reducing end on top and the reducing end on the bottom. The reducing end of an N-glycan is the end attached to the Asn residue that constitutes the glycosylation site on the protein. N-glycans differ in terms of the number of branches (antennae) of peripheral sugars (e.g., GlcNAc, galactose, fucose, and sialic acid) that are added to the Man3GlcNAc2 ("Man3") core structure, which is also referred to as the "trimannose core", "pentasaccharide core", "trimannosyl core", or "high-mannose core".

[0050] N-glycans are classified according to their branching composition (e.g., high-mannose, complex, or hybrid). "High-mannose" type N-glycans contain five or more mannose residues. "Complex" type N-glycans typically have at least one GlcNAc residue at the non-reducing end of a mannose residue attached by a β1,2 bond to the non-reducing end of a 1,3 mannose arm, and at least one GlcNAc residue at the non-reducing end of a mannose residue attached by a β1,2 bond to the non-reducing end of a 1,6 mannose arm of the trimannose core. Complex N-glycans may further include galactose ("Gal") or N-acetylgalactosamine ("GalNAc") residues, which may optionally be further linked to sialic acid ("Sia") or Sia derivatives (e.g., "NANA" or "NeuAc", where "Neu" refers to neuraminic acid and "Ac" refers to acetyl). Complex N-glycans may also have in-chain substitutions containing a "bisecting" GlcNAc residue and a core fucose ("Fuc") residue. Complex N-glycans may also have multiple antennae on the "trimannose core", often referred to as "multi-antennary N-glycans". "Hybrid" N-glycans contain at least one GlcNAc attached by a β1,2 bond to the non-reducing end of a mannose residue at the non-reducing end of a 1,3 mannose arm attached to the trimannose core, and zero or more mannose residues at the non-reducing end of a mannose attached to the non-reducing end of a 1,6 mannose arm of the trimannose core. The GlcNAc residue may then be attached to a galactose residue, and the galactose residue may be attached to a sialic acid residue. The various N-glycans are also referred to as "glycoforms".

[0051] Regarding complex N-glycans, the terms "G-2", "G-1", "G0", "G1", "G2", "A1", and "A2" mean the following. "G-2" refers to an N-glycan structure that can be characterized as Man3GlcNAc2; the term "G-1" refers to an N-glycan structure that can be characterized as GlcNAcMan3GlcNAc2; the term "G0" refers to an N-glycan structure that can be characterized as GlcNAc2Man3GlcNAc2; the term "G1" refers to an N-glycan structure that can be characterized as GalGlcNAc2Man3GlcNAc2; the term "G2" refers to an N-glycan structure that can be characterized as Gal2GlcNAc2Man3GlcNAc2; the term "A1" refers to an N-glycan structure that can be characterized as SiaGal2GlcNAc2Man3GlcNAc2; and, the term "A2" refers to an N-glycan structure that can be characterized as Sia2Gal2GlcNAc2Man3GlcNAc2. Unless otherwise specified, the terms “G-2", "G-1", "G0", "G1", "G2", "A1", and "A2" refer to N-glycan species that lack fucose attached to the GlcNAc residue at the reducing end of the N-glycan. When the term includes "F", "F" indicates that the N-glycan species contains a fucose residue on the GlcNAc residue at the reducing end of the N-glycan. For example, G0F, G1F, G2F, A1F, and A2F all indicate that the N-glycan further includes a fucose residue attached to the GlcNAc residue at the reducing end of the N-glycan. Lower eukaryotes such as yeast and filamentous fungi generally do not produce N-glycans that produce fucose.

[0052] Complex N-glycans include biantennary N-glycans, bisected N-glycans, and multiantennary N-glycans. Regarding biantennary N-glycans, the N-glycan contains a 1,6 mannose arm and a 1,3 mannose arm, wherein each of the 1,6 mannose arm and the 1,3 mannose arm contains one GlcNAc residue that is linked by a β1,2 glycosidic bond to a mannose residue at the non-reducing end of the arm. Each GlcNAc residue may independently be further attached to a galactose residue, and each galactose residue may independently be further attached to a sialic acid residue. Thus, biantennary N-glycans may include N-glycans having the shorthand formula GlcNAc2Man3GlcNAc2, Gal (1-2) GlcNAc2Man3GlcNAc2 or Sia (1-2) Gal (1-2) GlcNAc2Man3GlcNAc2. The term “1-2” means 1 or 2 sugar residues.

[0053] Regarding multiantennary N-glycans, the term "multiantennary N-glycan" refers to a biantennary N-glycan that further comprises: (i) a GlcNAc residue at the non-reducing end of a mannose residue that is attached by a β1,4 linkage to the non-reducing end of the 1,6 or 1,3 arm of the N-glycan; or (ii) a GlcNAc residue at the non-reducing end of a mannose residue that is attached by a β1,4 linkage to the non-reducing end of the 1,6 arm and a GlcNAc residue at the non-reducing end of a mannose residue that is attached by a β1,4 linkage to the non-reducing end of the 1,3 arm of the N-glycan. Each GlcNAc residue may independently be further attached to a galactose residue, and the galactose residue may independently be further attached to a sialic acid residue. Thus, multiantennary N-glycans can be characterized by the shorthand formula GlcNAc (3-4) Man3GlcNAc2, Gal (1-4) GlcNAc (3-4) Man3GlcNAc2 or Sia (1-4) Gal (1-4) GlcNAc (3-4) Man3GlcNAc2. The term "1-4" refers to 1, 2, 3, or 4 residues, and the term "3-4" refers to 3 or 4 sugar residues.

[0054] Regarding bisecting N-glycans, the term "bisecting N-glycan" refers to an N-glycan in which the reducing end of a GlcNAc residue is attached by a β1,4 linkage to the non-reducing end of the central mannose residue at the non-reducing end of the trimannose core. Bisecting N-glycans may be characterized by the formula GlcNAc3Man3GlcNAc2, in which each mannose residue is linked to a GlcNAc residue at its non-reducing end. In contrast, when a multiantennary N-glycan is characterized as GlcNAc3Man3GlcNAc2, the shorthand formula indicates that two GlcNAc residues are linked to mannose residues at the non-reducing ends of one of the two arms of the N-glycan and one GlcNAc residue is linked to a mannose residue at the non-reducing end of the other arm of the N-glycan. Although the GlcNAc residues at the ends of the 1,3 and 1,6 arms of bisecting N-glycans may each be further linked to a galactose residue, which may be further linked to a sialic acid residue, the bisecting GlcNAc residue does not extend further.

[0055] Table 1 provides a list of representative biantennary and bisecting N-glycans, which includes the linkage to the Asn residue in an N-glycosylation site containing the consensus sequence Asn-X-Ser / Thr, where X may be any amino acid other than proline.

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] The abbreviations used herein have their ordinary usage in the art. See, for example, the abbreviation for sugar above. Other common abbreviations include "PNGase", or "glycosidase" or "glucosidase", all of which refer to peptide N-glycosidase F (EC 3.2.2.18).

[0064] As used herein, the term "glycoprotein" refers to any protein having one or more N-glycans and / or one or more O-glycans attached thereto. Thus, the term refers both to proteins generally recognized in the art as glycoproteins and to proteins that have been genetically engineered to contain one or more N-linked glycosylation sites and / or O-linked glycosylation sites.

[0065] As used herein, "humanized glycoprotein" or "human-like glycoprotein" alternatively refers to a protein having an N-glycan with fewer than four mannose residues attached thereto, and a synthetic glycoprotein intermediate having at least five mannose residues (which is also useful and can be further manipulated in vitro or in vivo). In a particular embodiment, the glycoprotein produced according to the invention contains at least transiently at least 30 mol%, preferably at least 40 mol% and more preferably 50, 60, 70, 80, 90 or even 100 mol% of the Man5GlcNAc2 intermediate. This can be achieved, for example, by engineering the host cell of the invention to express a "better", i.e., more efficient, glycosylation enzyme. For example, a mannosidase is selected such that it has optimal activity under the conditions at the site where the protein is glycosylated in the host cell, and it is introduced into the host cell, preferably by targeting the enzyme to the host cell organelle where its activity is required.

[0066] As used herein, the term "recombinant host cell" is intended to mean a cell into which a recombinant vector has been introduced. It should be understood that such term is intended to refer not only to the particular subject cell but also to the progeny of such cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell located in a living tissue or organism. Preferred host cells are yeast and fungi.

[0067] As used herein, the term "hyaluronidase" or "HYAL" refers to hyaluronoglucosaminidase (EC 3.2.1.35), which catalyzes the hydrolysis of glycosaminoglycans including hyaluronic acid. Hyaluronidase 1 (HYAL1) is the hyaluronidase species found in sperm. Other species of hyaluronidase include HYAL2, HYAL3, HYAL4, and HYAL5 (also known as SPAM1 or PH20).

[0068] Four different purified hyaluronidases have been approved for use in the United States, three of animal origin and one recombinant. They are used as adjuvants in subcutaneous fluid administration to achieve hydration, to increase the dispersion and absorption of other injected drugs, or to improve the resorption of contrast agents in subcutaneous urography. The three hyaluronidases of natural origin are orthologs of human HYAL5 (PH20) obtained from testicular preparations. They are sold under the following trade names: VITRASE (ovine, approved by the FDA in May 2004), AMPHADASE (bovine, October 2004), and HYDASE (bovine, October 2005). Human recombinant hyaluronidase (HYLENEX) was approved for use in the United States in December 2005 and corresponds to a soluble fragment of human HYAL5 (PH20) produced in genetically engineered Chinese hamster ovary (CHO) cells containing a DNA plasmid encoding the enzyme.

[0069] As used herein, the term "antibody" or "immunoglobulin" as used herein refers to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) that are interconnected by disulfide bonds. Each HC is composed of a heavy chain variable region or heavy chain variable domain (V H ) and a heavy chain constant region or heavy chain constant domain. Each LC is composed of an LC variable region or LC variable domain (V L) and an LC constant domain. In some naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region consists of three domains: CH1, CH2, and CH3. Generally speaking, the basic antibody structural unit of an antibody is a Y-shaped tetramer containing two HC / LC pairs (2H). Each tetramer consists of two pairs of identical polypeptide chains, each pair having one LC (about 25 kDa) and one HC chain (about 50 - 70 kDa) (H+L). Each HC:LC pair contains one V H: One V L pair. One V H: One V L pair may be referred to by the term "Fab". Thus, each antibody tetramer contains two Fabs, one per arm of the Y-shaped antibody.

[0070] The LC constant domain consists of one domain CL. Human V H includes seven family members: V H 1, V H 2, V H 3, V H 4, V H 5, V H 6, and V H 7; and human V L includes 16 family members: V κ 1, V κ 2, V κ 3, V κ 4, V κ 5, V κ 6, V λ 1, V λ 2, V λ 3, V λ 4, V λ 5, V λ 6, V λ 7, V λ 8, V λ 9, and V λ 10. These family members can each be further divided into specific subtypes. V H and V L can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each V H and V LIt consists of three CDR regions and four FR regions arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The amino acid numbering in VH may be determined using the Kabat numbering scheme. See Béranger, et al., eds. Ginetoux, Correspondence between the IMGT unique numbering for C-DOMAIN, the IMGT exon numbering, the Eu and Kabat numberings: Human IGHG, Created: 17 / 05 / 2001, Version: 08 / 06 / 2016, which is accessible at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html.

[0071] The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). Generally, the amino acid numbering in the heavy chain constant domain starts from number 118, which is consistent with the EU numbering scheme. The EU numbering scheme is based on the amino acid sequence of human IgG1 (Eu), which has a constant domain starting at amino acid position 118 of the amino acid sequence of IgG1 described in Edelman et al., Proc. Natl. Acad. Sci. USA. 63:78-85 (1969), and is shown for the IgG1, IgG2, IgG3, and IgG4 constant domains in Béranger et al., supra.

[0072] As used herein, production by recombinant means using recombinant DNA methods means using well-known molecular biology methods for expressing a protein encoded by cloned DNA.

[0073] As used herein, the term "Fc domain" or "Fc" as used herein is the crystallizable fragment domain or region obtained from an antibody, which contains the CH2 and CH3 domains of the antibody. In an antibody, the two Fc domains are bound together by two or more disulfide bonds and hydrophobic interactions of the CH3 domains. The Fc domain may be obtained by digesting the antibody with the protease papain. Generally, the amino acids in the Fc domain are numbered according to the EU numbering convention (see Edelmann et al., Biochem. 63:78-85 (1969)).

[0074] As used herein, the term "antigen" as used herein refers to any substance or part thereof that induces an immune response in vivo.

[0075] As used herein, the term "antigen-binding fragment" refers to one or more polypeptides comprising a fragment of a full-length antibody, said fragment retaining the ability to specifically bind to an antigen bound by the full-length antibody, and / or the ability to compete with the full-length antibody for specific binding to an antigen. Examples of antigen-binding fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv regions, and scFv.

[0076] As used herein, the term "Fab fragment" refers to an antigen-binding agent comprising one antibody light chain and the CH1 and V of one antibody heavy chain H The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab fragment" can be the product of papain cleavage of an antibody.

[0077] As used herein, the term "Fab' fragment" refers to an antigen-binding agent comprising one antibody light chain and a portion or fragment of one antibody heavy chain, said portion or fragment containing V H and the CH1 domain up to the region between the CH1 and CH2 domains such that an interchain disulfide bond can form between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0078] As used herein, the term "F(ab')2 fragment" refers to an antigen-binding agent comprising two antibody light chains and two heavy chains, said two heavy chains containing V H and the CH1 domain up to the region between the CH1 and CH2 domains such that an interchain disulfide bond forms between the two heavy chains. An F(ab')2 fragment thus consists of two Fab' fragments held together by a disulfide bond between the two heavy chains. An "F(ab')2 fragment" can be the product of pepsin cleavage of an antibody.

[0079] As used herein, the term "Fv region" refers to an antigen-binding agent comprising the variable regions from both the heavy and light chains of an antibody, but lacking the constant regions.

[0080] As used herein, the term "ScFv" or "single-chain variable fragment" refers to a fusion protein comprising V fused or linked together by a short linker peptide of 10 to about 25 amino acids H and V L The linker is typically flexible and glycine-rich, and serine- or threonine-rich for solubility, and can link the N-terminus of V H to the C-terminus of V L or vice versa. Despite the removal of the constant regions and the introduction of the linker, this protein retains the specificity of the original immunoglobulin.

[0081] As used herein, the term "diabody" refers to an antigen-binding agent comprising a small antibody fragment having two antigen-binding domains, the fragment comprising a heavy-chain variable domain (V L ) linked to a light-chain variable domain (V H ) in the same polypeptide chain (V H -V L or V L -V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domain on the other chain and form two antigen-binding domains. Diabodies are described more fully, for example, in EP 404,097; WO 93 / 11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. For a review of engineered antibody variants, see generally Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0082] These and other potential constructs are described in Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0083] These and other potential constructs are described in Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0084] As used herein, the term "chimeric antigen receptor" (CAR) refers to a recombinant polypeptide that comprises at least an extracellular domain that specifically binds to an antigen or target, a transmembrane domain, and an intracellular T cell receptor activation signaling domain. Engagement of the extracellular domain of the CAR with a target antigen on the surface of a target cell results in clustering of the CAR and delivery of an activating stimulus to the cell containing the CAR. The CAR redirects the specificity of immune effector cells and triggers proliferation, cytokine production, phagocytosis, and / or the production of molecules that can mediate the death of cells expressing the target antigen in a major histocompatibility (MHC)-independent manner.

[0085] As used herein, when referring to CAR use, the terms "extracellular antigen-binding domain", "extracellular domain", or "extracellular ligand-binding domain" refer to the portion of the CAR that is located outside the cell membrane and is capable of binding to an antigen, target, or ligand.

[0086] As used herein, when referring to CAR use, the term "hinge region" refers to the portion of the CAR that connects two adjacent domains of the CAR protein (e.g., the extracellular domain and the transmembrane domain).

[0087] As used herein, the term "transmembrane domain" refers to the portion of the CAR that extends through the cell membrane and anchors the CAR to the cell membrane.

[0088] As used herein, the terms "intracellular T cell receptor activation signaling domain", "cytoplasmic signaling domain", or "intracellular signaling domain" refer to the portion of the CAR that is located inside the cell membrane and is capable of transducing effector signals.

[0089] As used herein, the term "engineered immune cell" refers to an immune cell, also referred to as an immune effector cell, that has been genetically modified by adding additional genetic material in the form of DNA or RNA to the cell's total genetic material. According to the embodiments herein, the engineered immune cells have been genetically modified to express a human tumor-targeting CAR and are further conjugated to a HYAL1 mutant.

[0090] As used herein, the term "stimulatory molecule" refers to a molecule expressed by a T cell that provides a major cytoplasmic signaling sequence that regulates, in a stimulatory manner, at least some aspects of the T cell signaling pathway for the primary activation of the T cell receptor (TCR) complex. Stimulatory molecules contain two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation (referred to as "primary signaling domains"), and those that act in an antigen-independent manner to provide secondary co-stimulatory signals (referred to as "co-stimulatory signaling domains").

[0091] As used herein, the terms "immune cell" or "immune effector cell" refer to cells involved in an immune response, such as promoting an immune effector response. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid phagocytes. According to certain embodiments, the engineered immune cells are T cells and are referred to as CAR-T cells because they have been engineered to express a CAR containing a ScFv targeting a human tumor; or the engineered immune cells are NK cells and are referred to as CAR-NK cells because they have been engineered to express a CAR containing a ScFv targeting a human tumor disclosed herein. In the present invention, the CAR-T cells and CAR-NK cells are conjugated to the HYAL1 mutant disclosed herein.

[0092] As used herein, the term "isolated" antibody or antigen-binding fragment thereof is at least partially free of other biomolecules from the cells or cell culture in which they are produced. Such biomolecules include nucleic acids, proteins, lipids, carbohydrates, or other materials such as cell debris and growth medium. The isolated antibody or antigen-binding fragment may further be at least partially free of recombinant host cell components, such as biomolecules from the host cell or its growth medium. In general, the term "isolated" is not intended to mean the complete absence of such biomolecules, or the absence of water, buffer, or salts, or components of a pharmaceutical formulation that includes the antibody or fragment.

[0093] As used herein, the term "monoclonal antibody" refers to a population of antibodies that are substantially homogeneous, i.e., the antibody molecules that make up the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a variety of different antibodies that have different amino acid sequences in their variable domains, which are often specific for different epitopes. The modifier "monoclonal" indicates the characteristic of the antibody as being derived from a substantially homogeneous population of antibodies and should not be construed as requiring antibody production by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention may be prepared by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991). See also Presta, J. Allergy Clin. Immunol. 116:731 (2005).

[0094] As used herein, the term "gene" is used broadly to refer to any nucleic acid segment associated with a biological function. Thus, a gene includes coding sequences and / or regulatory sequences required for its expression. For example, a "gene" refers to a nucleic acid fragment that expresses mRNA, functional RNA, or a specific protein, including regulatory sequences. A "gene" also includes non-expressed DNA segments, which form recognition sequences for other proteins, for example. A "gene" can be obtained from a variety of sources, including cloning from a source of interest or synthesis from known or predicted sequence information, and may include sequences designed to have desired parameters. A gene includes both naturally occurring nucleotide sequences encoding a molecule of interest and synthetically derived nucleotide sequences encoding a molecule of interest, such as complementary DNA (cDNA) obtained from a messenger RNA (mRNA) nucleotide sequence.

[0095] As used herein, the term "germline" or "germline sequence" refers to the sequence of an unrearranged immunoglobulin DNA sequence. Any suitable source of unrearranged immunoglobulin sequences may be used. Human germline sequences may be obtained, for example, from the website of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the United States National Institutes of Health germline database. Mouse germline sequences may be obtained, for example, as described in Giudicelli et al., Nucleic Acids Res. 33:D256-D261 (2005).

[0096] As used herein, the term "library" generally refers to a collection of related but different polynucleotides, which are generally in a common vector backbone. For example, a light chain or heavy chain immunoglobulin library may contain polynucleotides in a common vector backbone that encode light chains and / or heavy chain immunoglobulins that are different but related in their nucleotide sequences; for example, the immunoglobulins are functionally different in their ability to form complexes with other immunoglobulins and bind specific antigens.

[0097] As used herein, the term "polynucleotide" discussed herein forms part of the present invention. "Polynucleotide", "nucleic acid", or "nucleic acid molecule" includes single-stranded or double-stranded DNA and RNA. In one embodiment of the present invention, for example, the polynucleotide encoding the HYAL1 mutant of the present invention may be flanked by native regulatory (expression control) sequences, or may be associated with heterologous sequences, said heterologous sequences including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'-non-coding regions, and the like.

[0098] For example, a polynucleotide encoding a HYAL1 mutant of the present invention may be operably linked to a promoter. In one embodiment of the present invention, a "promoter" or "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell (e.g., directly or through other proteins or substances that bind to the promoter) and initiating transcription of a coding sequence. Generally, a promoter sequence is bounded at its 3' end by a transcription start site and extends upstream (in the 5' direction) to include the minimum number of bases or elements required to initiate transcription at any level. A transcription start site (e.g., conveniently defined by nuclease S1 mapping) and protein-binding domains (consensus sequences) responsible for binding RNA polymerase may be found within the promoter sequence. A promoter may be operably linked to other expression control sequences including enhancer and silencer sequences or to the nucleic acids of the present invention. Promoters that may be used to control gene expression include, but are not limited to, the cytomegalovirus (CMV) promoter (U.S. Patent Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist et al., Nature 290:304-310 (1981)), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Komaroff et al., Proc. Natl. Acad. Sci. USA 75:3727-3731 (1978)), or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)); see also "Useful Proteins from Recombinant Bacteria" in Scientific American 242:74-94 (1980); and promoter elements from yeast or other fungi, such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerate kinase) promoter, or the alkaline phosphatase promoter.

[0099] As used herein, the terms "vector", "cloning vector", and "expression vector" include such media (e.g., plasmids) into which a DNA or RNA sequence may be introduced into a host cell so as to transform the host and optionally promote expression and / or replication of the introduced sequence. In one embodiment of the present invention, a polynucleotide encoding a HYAL1 mutant of the present invention may be in a vector.

[0100] As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It should also be understood that not all progeny may have exactly the same DNA content due to either intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as screened in the original transformed cell are included. When different designations are intended, it will be clear from the context.

[0101] As used herein, the term "control sequence" or "regulatory sequence" means a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for expression in eukaryotes include, for example, promoter, operator or enhancer sequences, transcription termination sequences, and polyadenylation sequences for expressing messenger RNA encoding a protein and ribosome binding sites for facilitating translation of the messenger RNA.

[0102] As used herein, a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence such as a regulatory sequence. For example, DNA encoding a presequence or secretory leader is operably linked to DNA encoding a polypeptide if the presequence or secretory leader is expressed as a preprotein participating in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the DNA sequences to be linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide linkers or adaptors are used in accordance with conventional practice.

[0103] As used herein, the term "encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide such as a gene, cDNA, or mRNA to serve as a template for the synthesis of other polymers and macromolecules having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence in a biological process, and the biological properties that result therefrom. Thus, if transcription and translation of mRNA corresponding to a gene produce a protein in a cell or other biological system, the gene encodes that protein. Both the coding strand (the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand (which serves as a template for gene or cDNA transcription) can be said to encode the protein or other product of the gene or cDNA. Unless otherwise indicated, the "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of one another and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may include introns.

[0104] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence.

[0105] As used herein, the term "treat" or "treating" means the administration, locally, subcutaneously, intramuscularly, intradermally, or systemically, of a therapeutic agent, such as a composition comprising any human HYAL1 mutant of the invention, to an individual in need thereof. The amount of a therapeutic agent effective to treat a disease or disorder (including, in specific embodiments, cancer or a proliferative disease) in an individual may vary depending on factors such as the state of the disease or disorder, age, and / or weight of the individual, and the ability of the therapeutic agent to elicit the desired response in the individual. Whether a treatment goal has been achieved can be evaluated by the individual and / or by any clinical measurements commonly used by a physician or other skilled healthcare provider to evaluate the severity or progression of the treatment. Thus, the term denotes a beneficial result that has been or will be conferred on a human or animal individual in need thereof.

[0106] As used herein, when the term "treat" is applied to a human or veterinary individual, it refers to therapeutic treatment as well as diagnostic applications. When "treat" is applied to a human or veterinary individual, it encompasses the contact of the HYAL1 mutants or compositions of the invention with a human or animal subject.

[0107] As used herein, the term "therapeutically effective amount" refers to the quantity of a specific substance sufficient to achieve a desired effect in an individual to be treated. For example, this may be the amount required to inhibit or reduce the severity of a disease or disorder in an individual.

[0108] As used herein, the term "disease" or "disorder" refers to a pathological condition in an organism that results from, for example, infection, dysfunction, cancer, or genetic defect, and is characterized by identifiable symptoms.

[0109] Human hyaluronidase 1 (HYAL1) mutants

[0110] The present invention provides human hyaluronidase 1 (HYAL1) mutants that have been evolved by iterative protein engineering to have significantly improved hyaluronidase activity compared to wild-type HYAL1 and activity over an extended pH range. Wild-type HYAL1 has maximal activity at pH 3.5 and little to undetectable activity at pH 5.5. The HYAL1 mutants of the present invention have high activity in the pH 5-6 range, with little to no activity above pH 7, which differentiates the HYAL1 mutants from other commercial hyaluronidases. Additionally, the HYAL1 mutants have improved expression yields relative to wild-type HYAL1.

[0111] The HYAL1 mutants of the present invention can be used to increase the absorption of therapeutic agents into tissues and to reduce tissue damage in the case of therapeutic agent extravasation. The HYAL1 mutants of the present invention are highly active at pH 5.5 (formulation pH) and inactivated at pH 7.4 (skin pH), thus providing a hyaluronidase with switchable activity. The HYAL1 mutants have a narrower diffusion range compared to the FDA-approved hyaluronidase product HYLENEX. After introducing the HYAL1 mutants in a composition containing a pH 5.3 or 5.5 buffer into the skin, the enzyme dissipates in the skin and the local environment increases to the normal skin pH of pH 7.0 or 7.2, under which the enzyme activity decreases. The HYAL1 mutants of the present invention provide a hyaluronidase with a pH-controlled activity profile in the skin, which reduces the duration of active hyaluronidase in the skin after the co-administered therapeutic agent diffuses through the skin, thus reducing the undesirable side effects of hyaluronidase activity that persists after the therapeutic agent has diffused.

[0112] Accordingly, the present invention provides a HYAL1 mutant comprising an amino acid sequence having at least 95% (and in specific embodiments, 96%, 97%, 98%, 99% and 100%) identity to the amino acid sequence of amino acids 20 to 433 of the amino acid sequence shown in SEQ ID NO:2, wherein the hyaluronidase 1 mutant further comprises a combination of amino acid substitutions selected from the following: (a) the amino acid substitutions D68N, M246L, D323N, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M and W433E; (b) the amino acid substitutions D68N, M246L, D323N, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E and V412R; (c) the amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, V412R, R238C, I287P, F345T and L377S; (d) the amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, V412R, R238C, F345T and L377S; and (e) the amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, P23V, S304T, L379M, W433E, R194D, D299E, V412R, N235D, I287P, F345T and L377S; and wherein the hyaluronidase 1 mutant (i) comprises at least one N-glycosylation site comprising the sequence asparagine-X-serine / threonine, wherein X is any amino acid other than proline, the site is glycosylated, and (ii) exhibits hyaluronidase activity between pH 3.5 and pH 5.5.

[0113] In certain embodiments, the HYAL1 mutant comprises the amino acid substitutions D68N, M246L, D323N, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, and W433E.

[0114] In certain embodiments, the HYAL1 mutant comprises the amino acid substitutions D68N, M246L, D323N, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, and V412R.

[0115] In certain embodiments, the HYAL1 mutant comprises the amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, V412R, R238C, I287P, F345T, and L377S.

[0116] In certain embodiments, the HYAL1 mutant comprises the amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, V412R, R238C, F345T, and L377S.

[0117] In certain embodiments, the HYAL1 mutant comprises the amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, P23V, S304T, L379M, W433E, R194D, D299E, V412R, N235D, I287P, F345T, and L377S.

[0118] In a further embodiment, at least one N-glycosylation site generated by D68N, D323N, G235N or R326S substitution is glycosylated. In a further embodiment, the N-glycosylation sites generated by D68N, D323N, G235N and R326S substitutions are each glycosylated. In a further embodiment, the native N-glycosylation site and the N-glycosylation sites generated by D68N, D323N, G235N and R326S substitutions are each glycosylated.

[0119] The present invention further provides a HYAL1 mutant comprising an amino acid sequence having at least 95% (and in specific embodiments, 96%, 97%, 98%, 99% and 100%) identity to the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, wherein the hyaluronidase 1 mutant (i) includes at least one N-glycosylation site comprising the sequence asparagine-X-serine / threonine, wherein X is any amino acid other than proline, the site is glycosylated, and (ii) exhibits hyaluronidase activity between pH 3.5 and pH 5.5.

[0120] The present invention further provides a HYAL1 mutant comprising an amino acid sequence having at least 95% (and in specific embodiments, 96%, 97%, 98%, 99% and 100%) identity to the amino acid sequences shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, wherein the hyaluronidase 1 mutant (i) includes at least one N-glycosylation site comprising the sequence asparagine-X-serine / threonine, wherein X is any amino acid other than proline, the site is glycosylated, and (ii) exhibits hyaluronidase activity between pH 3.5 and pH 5.5.

[0121] The present invention further provides a HYAL1 mutant disclosed herein conjugated to a polymer. In a particular embodiment, the polymer is dextran or polyethylene glycol.

[0122] The present invention further provides the HYAL1 mutants disclosed herein and a pharmaceutically acceptable carrier. In a further embodiment, the composition further comprises a therapeutic agent. Thus, in certain embodiments, the present invention provides a composition comprising a HYAL1 mutant disclosed herein and a therapeutic agent. Representative therapeutic agents include, but are not limited to, small molecules, peptides, macrocyclic peptides, proteins or protein complexes. In certain embodiments, the small molecule comprises an antibiotic or an anti-inflammatory agent. In certain embodiments, the peptide comprises an insulinotropic peptide, growth hormone, insulin or an insulin mutant. In certain embodiments, the therapeutic agent comprises an antibody, an antigen-binding protein, a scFv, a Fab or a fusion protein comprising the Fc domain of an antibody.

[0123] The present invention further provides a method for increasing the diffusion of a therapeutic agent in a tissue of a subject, which comprises administering to the tissue of the subject a composition comprising a HYAL1 mutant disclosed herein in an amount sufficient to increase the diffusion of the therapeutic agent; and administering to the tissue a composition comprising the therapeutic agent. The composition comprising the HYAL1 mutant and the composition comprising the therapeutic agent are administered to the subject either consecutively or simultaneously.

[0124] The present invention further provides a method for increasing the diffusion of a therapeutic agent in a tissue of a subject, which comprises administering to the tissue of the subject a composition comprising the therapeutic agent and an amount of a HYAL1 mutant disclosed herein sufficient to increase the diffusion of the therapeutic agent in the tissue.

[0125] The present invention further provides the use of a HYAL1 mutant disclosed herein for the manufacture of a medicament for use in combination with a therapeutic agent for treating a disease or disorder.

[0126] The present invention further provides the use of a HYAL1 mutant disclosed herein for the manufacture of a medicament comprising hyaluronidase and a therapeutic agent for treating a disease or disorder.

[0127] The present invention further provides the use of a HYAL1 mutant disclosed herein for increasing the diffusion of a therapeutic agent in a tissue of a subject for treating a disease or disorder.

[0128] The present invention further provides the use of a composition comprising a HYAL1 mutant disclosed herein and a therapeutic agent for treating a disease or disorder.

[0129] The present invention further provides nucleic acid molecules encoding the HYAL1 mutants disclosed herein. In a particular embodiment, the HYAL1 mutant encoded by the nucleic acid molecule comprises an amino acid sequence having at least 95% (and in specific embodiments, 96%, 97%, 98%, 99% and 100%) identity to the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.

[0130] The present invention further provides nucleic acid molecules encoding the HYAL1 mutants disclosed herein, which are fused with a signal sequence targeting the HYAL1 mutant to the endoplasmic reticulum. In a particular embodiment, the nucleic acid molecule comprises amino acids 1 to 433 of the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13.

[0131] The present invention further provides an expression vector comprising a nucleic acid molecule encoding the HYAL1 mutants disclosed herein, and in a specific embodiment, the nucleic acid molecule is fused with a signal sequence targeting the HYAL1 mutant to the endoplasmic reticulum.

[0132] The present invention further provides a host cell comprising a nucleic acid molecule encoding the HYAL1 mutants disclosed herein, and in a specific embodiment, the nucleic acid molecule is fused with a signal sequence targeting the HYAL1 mutant to the endoplasmic reticulum. The present invention further provides a host cell comprising an expression vector comprising a nucleic acid molecule encoding the HYAL1 mutants disclosed herein, and in a specific embodiment, the nucleic acid molecule is fused with a signal sequence targeting the HYAL1 mutant to the endoplasmic reticulum. In a further embodiment, the host cell is a mammalian host cell or a recombinant yeast host cell modified to produce complex N-glycans.

[0133] In a further embodiment, the mammalian host cell is a Chinese hamster ovary cell, and the recombinant yeast host cell is Pichia pastoris.

[0134] The present invention further provides a method for producing the HYAL1 mutants disclosed herein, which comprises: (a) introducing a nucleic acid molecule encoding a HYAL1 mutant or an expression vector comprising a nucleic acid molecule encoding a HYAL1 mutant disclosed herein into a host cell to produce a recombinant host cell; (b) culturing the recombinant host cell in a culture medium under conditions sufficient for the recombinant host cell to express the HYAL1 mutant and secrete it into the culture medium; and (c) obtaining the HYAL1 mutant from the culture medium.

[0135] In a further embodiment, the host cell is a mammalian host cell, and in a further embodiment, it may be a Chinese hamster ovary cell. In a further embodiment, the host cell is a recombinant yeast host cell modified to produce complex N-glycans, and in a further embodiment, it may be the recombinant yeast host cell Pichia pastoris.

[0136] In a further embodiment of the method, the HYAL1 mutant comprises amino acids 1 to 433 of the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13. These amino acid sequences contain a signal sequence (amino acids 1-19) that targets the HYAL1 mutant to the endoplasmic reticulum.

[0137] N-linked glycosylation of hyaluronidase (e.g., the HYAL1 mutants disclosed herein) can be very important for its catalytic activity and stability. While altering the glycan type of modified mutants can have significant effects on the antigenicity, structural folding, solubility, and stability of proteins, most enzymes are thought not to require glycosylation for optimal enzyme activity. Hyaluronidase is thus unique in this regard, as removal of N-linked glycosylation can lead to almost complete inactivation of hyaluronidase activity. For such hyaluronidases, the presence of N-linked glycans is crucial for generating an active enzyme. In one aspect of the invention, complex glycosylation capped with sialic acid residues is described, with other glycosylations capped with galactose, GlcNAc, or mannose residues also contemplated. In certain embodiments, the HYAL1 mutants disclosed herein contain N-glycans selected from the N-glycans shown in Table 1. In some embodiments, the HYAL1 mutant includes an N-glycan containing a terminal sialic acid residue. The invention further provides compositions in which the major N-glycan is selected from the N-glycans shown in Table 1. In some embodiments, the major N-glycan is an N-glycan containing a terminal sialic acid residue. In certain embodiments, the HYAL1 mutant contains three wild-type N-glycosylation sites and mutations that generate at least one non-natural N-glycosylation site. In certain embodiments, the HYAL1 mutant contains three wild-type N-glycosylation sites and mutations that generate two or three non-natural N-glycosylation sites. In certain embodiments, the HYAL1 mutant contains at least five N-glycans. In certain embodiments, the HYAL1 mutant contains at least six N-glycans.

[0138] Preparations of hyaluronidase 1 mutants are also provided. For example, the HYAL1 mutants may be formulated in lyophilized form and stable solutions. Preparations containing specific metal ions such as calcium, magnesium, or sodium can be used for optimal activity at neutral pH. In addition to stable solution formulations, sustained-release formulations are also contemplated herein for prolonging the removal of glycosaminoglycans or for prolonging the facilitation of the dispersion or diffusion of agents such as pharmacologics. Kits are also provided that provide pre-packaged syringes of the HYAL1 mutant for administering small volumes of the HYAL1 mutant for intraocular surgical procedures and other small volume procedures. Balanced salt formulations for ex vivo use in assisted reproductive technology procedures are also provided.

[0139] Also provided are methods for using HYAL1 mutants in the removal of glycosaminoglycans. Hyaluronidases comprising HYAL1 mutants open channels in the interstitial space by degradation of glycosaminoglycans, which channels generally permit diffusion of molecules smaller than about 500 nm. Depending on the dose and formulation, these channels can remain relatively open for a period of 24 - 48 hours. Such channels can be used to facilitate the diffusion of exogenously added molecules such as fluids, small molecules, proteins, nucleic acids, and gene therapy vectors, as well as other molecules smaller than about 500 nm. Additionally, without being limited to a particular theory or mechanism of action, it is believed that the formation of such channels can promote substantial fluid flow within the interstitial space, which in turn can promote the dispersion or movement of solutes (such as detectable molecules or other diagnostic agents, anesthetics or other tissue modifiers, pharmacological or pharmaceutically active agents, or cosmetic or other beauty agents), in a process sometimes referred to herein as "convective transport" or simply convection, wherein the solutes are effectively carried by the fluid. Such convective transport can substantially exceed the rate and cumulative effect of molecular diffusion, and can thus cause more rapid and efficient perfusion of tissues by therapeutic or other administered molecules. Further, when molecules such as therapeutic agents or other agents (such as small molecule drugs or larger molecules or complexes) are co-formulated or co-administered with HYAL1 mutants, and both are injected into a relatively confined local site, such as a site of non-intravenous parenteral administration (e.g., intradermal, subcutaneous, intramuscular, or other internal tissues, organs, or other relatively confined spaces within or around the body), the fluid associated with the administered dose can both provide a local driving force (i.e., hydrostatic pressure) and a lower flow resistance (by opening channels within the interstitial matrix) - both of which tend to increase fluid flow and, concomitantly, the convective transport of the therapeutic agent or other molecules contained within the fluid. As described and illustrated in more detail herein, and as would be understood by one of ordinary skill in the art, these aspects of the use of HYAL1 mutants can have significant utility for improving bioavailability and manipulating other pharmacokinetic and / or pharmacodynamic properties of co-formulated or co-administered agents.

[0140] In a specific embodiment, a single short-acting dose is preferred. Transient removal of glycosaminoglycans can be used to enhance the delivery of solutions and drugs into and / or through the interstitial space. This can be used for the diffusion of anesthetics and the administration of therapeutic fluids, molecules, and proteins. Subcutaneous, intradermal, and intramuscular administration of molecules is also facilitated for more rapid systemic distribution in the presence of HYAL1 mutants (and / or other glycosaminoglycanases). Such methods are very useful when intravenous access is unavailable or when more rapid systemic delivery of molecules is desired. By way of example, the delivery of other macromolecules (such as factor VIII) with low bioavailability after subcutaneous administration may be injected together with HYAL1 mutants to increase their utilization.

[0141] In the context of non-intravenous parenteral injection (such as intradermal injection, subcutaneous injection, intramuscular injection, and other injections into spaces other than blood vessels), HYAL1 mutants and another agent (such as a co-formulation or mixture containing HYAL1 mutants and another agent such as a diagnostic agent, anesthetic, pharmacological agent, cosmetic agent, or a combination thereof) in a certain volume of liquid (such as a pharmaceutical excipient or other solution) can be introduced into one or more sites within the body by injection or infusion. Without being bound by theory, it is believed that several forces can be stimulated to enhance the delivery of pharmacological agents or other agents (the extent of which depends in part on, for example, the specific composition, volume, and administration site). These driving forces can include an increase in hydrostatic pressure when a certain volume of fluid is effectively forced into a closed space (such as the sub-Tenon's space, or the site of intradermal injection, subcutaneous injection, intramuscular injection, or other non-IV parenteral injection), a subsequent increase in convective transport (or convection) of solutes when fluid flow increases along its pressure gradient (and dissolved molecules or macromolecular complexes are carried along), and an increase in diffusion and / or osmosis mediated by the degradation of glycosaminoglycans and the concomitant formation of channels within the downstream extracellular matrix or intercellular spaces.

[0142] As would be understood by those skilled in the art, HYAL1 mutants can thus be used to effectively facilitate the delivery of a variety of anesthetic agents, diagnostic agents, pharmacological agents, and / or other agents to the posterior segment of the eye for treating conditions such as retinal detachment, retinal vein occlusion, proliferative retinopathy, diabetic retinopathy, inflammatory conditions (such as uveitis, choroiditis, retinitis, etc.), and degenerative diseases, vascular diseases, and various tumors. Again, as would be understood by those skilled in the art, various pharmacological agents or pharmaceutically effective agents can be used for treating such posterior segment conditions and diseases, by way of example, including anesthetic agents and pharmacological agents such as those described and illustrated below.

[0143] Co-formulations or co-administrations of HYAL1 mutants with other substances may also be envisioned for injectable pens for small-volume or rapid subcutaneous administration. Examples that can be formulated include Epipen TM 、insulin, and other fluids. The methods of the present invention include administering the HYAL1 mutant or a pharmaceutical composition containing the HYAL1 mutant before, simultaneously with, or after the administration of other therapeutic molecules. The HYAL1 mutant may be administered at a site different from the site of administration of the therapeutic molecule, or the HYAL1 mutant may be administered at the same site as the site of administration of the therapeutic molecule.

[0144] Without wishing to be bound by theory, the ability of the HYAL1 mutants to cause degradation of some of the glycosaminoglycans in the intercellular spaces between cells results in transient opening of channels within the stroma, which in turn tends to increase interstitial fluid flow and, concomitantly, promote diffusion and / or convective solute transport (convection) of dissolved components within the interstitial fluid (such as anesthetics, drugs and other pharmacological agents, markers and diagnostic agents, etc.). As would be appreciated by those skilled in the art, the HYAL1 mutants of the present invention can be applied to enhance the bioavailability of a variety of pharmacological and other agents (and potentially improve other pharmacokinetic and / or pharmacodynamic properties), and such other agents can be used to treat or diagnose various disease conditions or otherwise modify one or more tissues in the body. Illustrative classes of such therapeutic agents include: anti-cancer agents, anti-infective agents, anesthetics, anti-inflammatory agents, cytokines, antibodies and other proteins, nucleic acids, macromolecular complexes, and numerous other molecules and pharmacological agents that modify cells or other physiological activities, including various classes of agents (and their exemplary members) described herein and in the art.

[0145] Although diffusion and convective transport of even small molecules can be enhanced by opening interstitial channels and increasing fluid flow, in the case of larger pharmacological or other agents, such as many biotherapeutic drugs (including antibodies and other proteins, large nucleic acids, macromolecular complexes (such as liposomes and other macromolecular carriers), and gene therapy vectors, etc.), the size of the molecule and the presence of stromal components (such as glycosaminoglycans) substantially impede diffusion and / or convection of the agent. This can lead to several consequences that potentially limit the usefulness of the agent. For example, the pharmacokinetics of the agent can be effectively impaired due to slowed absorption and thus distribution of the agent. Additionally, retention of a portion of the agent at or near the site of administration not only limits its bioavailability but can also lead to toxicity due to potentially sustained and high local doses. In the latter aspect, local toxicity, which may be associated with pain or other side effects, is a problem for many large biomolecules administered by non-intravenous injection, such as subcutaneous injection, intradermal injection, or intramuscular injection. As a result, many pharmacological agents have a pharmacokinetic (PK) and / or pharmacodynamic (PD) profile that can be enhanced by co-formulating the agent with a HYAL1 mutant and / or co-administering the agent with a HYAL1 mutant, which may be provided before, simultaneously with, or after the agent and administered at the same or different sites, and the parameters will be the subject of optimization in a standard model (such as an animal model commonly used to evaluate the pharmacokinetics and pharmacodynamics of an agent). Any of a variety of therapeutic agents, pharmacological agents, and other agents (such as cosmetic or beauty preparations) that are typically administered parenterally can thus be enhanced using a HYAL1 mutant.

[0146] In non-IV parenteral situations, HYAL1 mutants can also be used to permit administration of a medicament by a more convenient route and / or with higher efficiency. By way of example (and without limitation), a medicament that is normally administered by subcutaneous injection can instead be administered by intradermal injection by re-formulating and / or co-administering the medicament with a HYAL1 mutant (locally or systemically and before, simultaneously with, or after administration of the medicament), and a medicament that is normally administered by intramuscular injection can instead be administered by subcutaneous or intradermal injection. Alternatively, the medicament can be administered by the same route but with improved pharmacokinetics and / or pharmacodynamics using a HYAL1 mutant.

[0147] The use of HYAL1 mutants to reformulate IV drugs and other medicaments for non-IV parenteral use also enables delivery of the drugs using any of a variety of new injection devices designed to simplify and / or accelerate delivery and facilitate self-administration. Such devices include, for example, ultra-sharp and microneedle devices (such as those being developed by Becton Dickinson and other companies) and needleless injection devices (such as the Biojector TM and other devices available from Bioject; the IntraJect TM and other devices available from Aradigm; the Medijector TM device, and the like). Many devices (such as the Biojector) can be particularly useful for facilitating intradermal injection, which when using a standard needle tends to require more experience (due to the possibility of penetrating the dermis during needle placement and delivering the medicament to a deeper tissue site such as the subcutaneous layer). For some pharmacological medicaments, such as vaccines (e.g., DNA-based vaccines or other vaccines), delivery of the medicament into the dermis can be particularly advantageous as compared to the sub-dermal layer because there tend to be relatively high concentrations of antigen-presenting cells (APCs) located within the dermis.

[0148] In the context of those agents that are typically delivered by intradermal injection (whether via a standard needle or other newer devices), or many other agents that are not currently but could potentially be delivered by intradermal injection, co - introduction of HYAL1 mutants locally can be used to enhance the delivery of the delivered pharmacological or other agents, and to facilitate their diffusion or dispersion within the dermis. In the case of vaccines where the dermis may be the primary target tissue, given the local abundance of APCs, the HYAL1 mutants can thus facilitate the diffusion of the vaccine within the target tissue, thereby increasing the likelihood and extent of the interaction between the vaccine and APCs (which can significantly enhance the generation of immunomodulatory responses). In the case of other agents, the dermis may not be the primary target tissue, but rather the tissue into which a dose of the agent is introduced, with the hope that the agent is absorbed from that tissue into another tissue, typically the bloodstream. In the latter case, the bloodstream itself may be the target tissue of interest (e.g., for blood - regulating factors as described herein and in the art), or the bloodstream itself may be the delivery tissue through which the agent is transported to distal target tissues (e.g., body tissues supplied by the bloodstream). In either of these latter cases (where delivery is intradermal, but the agent is desired to be delivered to the bloodstream), the HYAL1 mutants (and potentially the volume of fluid into which it is injected) can facilitate the dispersion of the agent first within the dermis and thus into the vascular system draining the dermis and ultimately into the larger blood supply), as described and illustrated herein.

[0149] The use of HYAL1 mutants to enhance the pharmacokinetics and / or pharmacodynamics of other therapeutic or pharmacological agents can also be applicable to agents delivered via routes other than non - IV parenteral administration. For example, without wishing to be bound by theory, the presence of HYAL1 mutants within the interstitial spaces of the body (which can be achieved by local and / or systemic administration of HYAL1 mutants) is thought to tend to promote an increase in fluid flow within gap junctions and interstitial spaces, which in turn promotes both the diffusion and convective transport of agents (such as pharmacological and other agents) dissolved in tissue fluid. By way of illustration, agents delivered directly into the bloodstream (e.g., via intravenous injection) or orally administered (and thus absorbed into the bloodstream, for example, after absorption from the gastrointestinal tract) can still be subject to the constraints of their pharmacokinetics during the post - absorption (i.e., distribution phase). Without wishing to be bound by theory, it is thought that HYAL1 mutants can enhance delivery to target cells by increasing interstitial permeability and fluid flow and thus increasing the diffusion and / or convection of agents within the interstitial spaces (which effectively form the medium between almost all pharmacological delivery routes and target cells).

[0150] In addition, it is considered that changes in colloid osmotic pressure caused by the administration of HYAL1 mutants can contribute to enhanced delivery of pharmacological agents. Again, without wishing to be bound by theory, the presence of HYAL1 mutants and the concomitant degradation along the interstitial side of vascularized tissues tend to reduce the colloid osmotic pressure within the intercellular space, which in turn enhances fluid filtration from the vascular system into the intercellular space.

[0151] In the context of many cancers where the interstitial pressure within the tumor (the tumor interstitial pressure of tumor interstitial fluid (TIF)) is elevated, the possibility of reducing the interstitial pressure is regarded as particularly important. High TIF can result in a relative resistance to fluid flow from the vascular system towards the tumor center, thereby limiting the amount of anti-cancer agents that can effectively reach the tumor, particularly deeper parts within the tumor mass. Introducing HYAL1 mutants into the tumor interstitium thus tends to enhance the delivery of locally and systemically available anti-cancer agents, which can more readily penetrate the tumor when the interstitial colloid osmotic pressure is reduced and diffusion and / or convective transport are increased. Measurements of TIF and hydraulic conductivity (K) within the tumor (e.g., using tagged molecules such as albumin labeled with Evans blue dye) can be used to quantitatively evaluate the effects of various concentrations of HYAL1 mutants on tumor hydrodynamics in vivo, as described herein and / or in the art.

[0152] Further exacerbating the reduced hydrodynamics within many tumors and highlighting additional potential benefits of applying HYAL1 mutants to tumors is the fact that many tumors exhibit an accumulation of glycosaminoglycans, particularly hyaluronic acid (which may be due to the fact that the lymphatic system is impaired or absent in many tumors, which is the major pathway for hyaluronic acid catabolism). Such excess hyaluronic acid can contribute to an impediment to hydraulic conductivity. The introduction of HYAL1 mutants can thus be used to counteract the accumulation of glycosaminoglycans in various tumors, improve the hydraulic conductivity within the tumor, and effectively render it more sensitive to anti-tumor agents (whether delivered locally or systemically).

[0153] As will be appreciated by those skilled in the art, the principles described herein can also be applied to numerous other pharmacological and other agents that are desired to be delivered to internal body sites, such as for the prevention, diagnosis, and / or treatment of diseases or otherwise for modulating physiological functions.

[0154] In addition to its use in potentially improving and / or reformulating various parenterally administered pharmacologies and / or medicaments, HYAL1 mutants can also be effectively employed in combination with non-parenteral medicaments (e.g., medicaments formulated as pills, liquids, or other forms for ingestion and general absorption through the gastrointestinal tract). For example, since most non-parenteral drugs must ultimately reach cells within the interstitium in order to exert their desired effects, the use of HYAL1 mutants to enhance interstitial diffusion and / or convective transport (systemic or local (e.g., via local or targeted administration of HYAL1 mutants)) can be applied to improve the extent and / or rate at which non-parenteral (as well as parenteral) drugs reach their desired target cells.

[0155] In addition, many medicaments that are typically administered non-parenterally (e.g., orally) may be reformulated, or their active ingredients may be reformulated, for parenteral administration to render a combination with HYAL1 mutants more effective and / or safe. To illustrate this widely applicable approach, the ability of HYAL1 mutants to promote targeted delivery to specific tissues (e.g., HYAL1 mutants provide the ability for transdermal delivery to tissues within the posterior segment of the eye) can be used to directly and preferentially deliver various medicaments (including medicaments previously administered systemically) to local sites of interest within the body. This can not only provide a more desired and / or more rapidly achieved concentration at the site of interest, but can also substantially reduce the potential problems and limitations associated with systemic administration, where concentrations at undesired sites may equal or even exceed concentrations at the desired target site (potentially triggering adverse side effects as well as wasting medicaments). In some cases, for example, medicaments that have not been widely used or are not used for certain indications or certain patients may be effectively applied to benefit additional patients by co-formulating or co-administering with HYAL1 mutants described and illustrated herein.

[0156] As would be appreciated by those skilled in the art, the ability to employ HYAL1 mutants to enhance, accelerate, and / or target the biodistribution of co-formulated and / or co-administered medicaments, and to manipulate other aspects of their pharmacokinetics or pharmacodynamics (e.g., in order to improve their risk:benefit profile or to facilitate their use by patients, families, or healthcare professionals), provides an important opportunity to improve drugs and other medicaments for the treatment, diagnosis, or prevention of disease.

[0157] Not limited to a particular set of applications, the HYAL1 mutants as described herein can be used to effectively effect bolus or bolus-like delivery of any number of pharmacological and other medicaments via non-intravenous parenteral routes as well as other routes of administration (e.g., by enhancing the delivery of medicaments into and / or through target tissues after they leave the bloodstream, whether they are introduced into the bloodstream directly (e.g., via IV administration) or indirectly (e.g., via oral or non-IV parenteral administration)).

[0158] Without being limited to the specific mechanism of action or aspects thereof, the ability of these enzymes to transiently degrade the interstitial matrix components between cells, which occupy a significant portion of the fluid space in the body and the correspondingly large space that pharmacological agents and other agents must cross in order to reach most target cells, can significantly facilitate the delivery of agents to target cells by one or more of several potentially synergistic means.

[0159] First, although a large amount of tissue fluid exhibits a certain degree of flow, this flow is often restricted or impeded by glycosaminoglycans such as hyaluronic acid and other interstitial components. As described and illustrated herein, the ability of HYAL1 mutants to open channels within such cell gaps can be used to reduce impedance and increase the degree and rate of "downstream" flow resulting from any given "upstream" pressure.

[0160] Second, in the case of non-IV parenteral injection of a HYAL1 mutant and another pharmacological agent or other agent, the volume of the non-IV parenteral injection can be used to increase the upstream driving pressure or head (e.g., by increasing the hydrostatic pressure within an enclosed space), which can further facilitate flow.

[0161] Third, since the fluid volume containing the introduced HYAL1 mutant and other therapeutic agents is effectively driven along an increasing pressure gradient (i.e., a gradient increase generated by raising the hydrostatic pressure within the injection "bolus" and simultaneously reducing the interstitial pressure within the surrounding tissue), the solutes within the bolus can be effectively carried (e.g., by convective transport) into adjacent tissues. The injected fluid or bolus can thus move efficiently and relatively rapidly into adjacent tissues and deliver any pharmacological agent or other agent introduced (e.g., by co-formulating or co-administering an agent in combination with the HYAL1 mutant) at or near the same site of introduction.

[0162] Methods for evolving HYAL1

[0163] In some embodiments, to prepare the HYAL1 mutants of the present disclosure, the HYAL1 mutants that catalyze the hyaluronidase reaction are obtained (or derived) from the ExpiCHO-S suspension cells of the Chinese hamster ovary (CHO) cell line. In some embodiments, the parental polynucleotide sequence is codon-optimized to enhance the expression of HYAL1 in the host cell. The parental polynucleotide sequence designated as SEQ ID NO:1 is codon-optimized for expression in CHO cells, and the codon-optimized polynucleotide is cloned into an expression vector suitable for heterologous protein expression in CHO cells. Clones expressing active HYAL1 mutants in CHO cells are identified, and the genes encoding them are sequenced to confirm their identity.

[0164] The HYAL1 mutants disclosed herein may be obtained by subjecting the polynucleotide encoding the parental sequence to mutagenesis and / or directed evolution methods. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling as described in Stemmer, 1994, Proc. Natl. Acad. Sci. USA 91:10747-10751; WO 95 / 22625; WO 97 / 20078; WO 97 / 35966; WO98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Patent No. 6,537,746. Other directed evolution procedures that may be used include, inter alia, the staggered extension process (StEP), in vitro recombination (Zhao et al., 1998, Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell et al., 1994, PCR Methods Appl. 3:S136-S140) and cassette mutagenesis (Black et al., 1996, Proc. Natl. Acad. Sci. USA 93:3525-3529).

[0165] HYAL1 mutants with the desired improved enzyme properties are screened among the clones obtained after mutagenesis treatment. Wild-type HYAL1 is only active at very acidic pHs and is inactive at pHs greater than 4.5, and the expression level of wild-type human HYAL1 is not high enough for large-scale manufacture. In addition, evolution of HYAL1 may introduce immunogenicity risks and the immunogenicity risks need to be measured for HYAL1 mutants. The desired improved properties of the HYAL1 mutants are: (i) being active at pH≥5.5, (ii) having a specific activity greater than 30,000 units / mg, (iii) having stable activity (greater than 80%) at 2-8 °C for 12-30 months, and (iv) having a low immunogenicity risk.

[0166] Measurement of the enzyme activity from the expression library may be performed over time at different pHs using a turbidimetric or ELISA activity assay. The relative expression of the HYAL1 mutants may be determined by expressing the HYAL1 mutants as a fusion protein with a C-terminal His-6 tail (SEQ ID NO:46) and measuring the expression level using a His-tag homogeneous time-resolved fluorescence (HTRF) expression assay. The immunogenicity risk may be determined by scanning the region containing the mutated HYAL1 mutants using a computer program such as ExpiVax software. The computer program provides epitope scores for the scanned regions, which can be used to evaluate the immunogenicity of each possible 9-mer peptide in the scanned regions. Clones containing the polynucleotide encoding the HYAL1 mutants are then isolated, sequenced to identify nucleotide sequence changes (if any), and used to express the enzyme in host cells.

[0167] When the sequence of a polypeptide is known, polynucleotides encoding the enzyme can be prepared by standard solid-phase methods according to known synthetic methods. In some embodiments, fragments up to about 100 bases can be synthesized individually and then ligated (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form any desired contiguous sequence. For example, the polynucleotides and oligonucleotides of the present invention can be prepared by chemical synthesis, using, for example, the classical phosphoramidite method described by Beaucage et al., 1981, Tet. Lett. 22:1859-69, or the method described by Matthes et al., 1984, EMBO J. 3:801-05, e.g., as it is typically practiced in automated synthesis methods. According to the phosphoramidite method, oligonucleotides are synthesized, for example, in an automated DNA synthesizer, purified, annealed, ligated and cloned into an appropriate vector. Additionally, substantially any nucleic acid can be obtained from any of a variety of commercial sources, such as The Midland Certified Reagent Company, Midland, Tex., The Great American Gene Company, Ramona, Calif., ExpressGen Inc., Chicago, Ill., Operon Technologies Inc., Alameda, Calif., and many other companies.

[0168] The HYAL1 mutants expressed in host cells can be recovered from the cells and / or the culture medium using one or more of the well-known techniques for protein purification, which include, inter alia, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation and chromatography.

[0169] Chromatographic techniques for separating HYAL1 mutants include, inter alia, reverse-phase chromatography, high performance liquid chromatography (RP-HPLC), ion-exchange chromatography, gel electrophoresis and affinity chromatography. The conditions for purifying a particular enzyme depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those skilled in the art.

[0170] In some embodiments, affinity techniques can be used to isolate improved HYAL1 mutants. For affinity chromatography purification, the protein sequence can be tagged with a recognition sequence to allow purification. Common tags include cellulose-binding domains, poly-His tags (e.g., His-6 (SEQ ID NO:46)), di-His chelates, FLAG tags, and many other tags that will be apparent to those skilled in the art. Antibodies can also be used as affinity purification reagents. Any antibody that specifically binds to the HYAL1 mutant can be used.

[0171] All experiments in this work were repeated at least 2 times (N = 3). Consistent results were obtained. Statistical analysis was performed using GraphPad Prism 8 for all included data points.

[0172] General methods

[0173] Transient transfection and expression of HYAL1

[0174] Expi-CHO-S cells (Gibco, catalog #A29127) were cultured according to the manufacturer's guidelines. Two days before transfection, cells were seeded at 1.5 x 106 cells / mL in Expi-CHO expression medium (Gibco, catalog #A2910003) in a non-baffled 250 mL shake flask (Thermo Scientific, catalog #4115-0125). On the day of transfection, cells were counted using a Countess II cell counter and then diluted to 6 x 106 cells / mL in fresh ExpiCHO expression medium. Cells were then transferred to the inner 60 wells of a 96-well culture plate (Corning, catalog #PDW20CS) at a volume of 800 μL / well. The outermost wells of each plate were filled with 800 μL of sterile phosphate-buffered saline (PBS).

[0175] Transfection was performed using the ExpiCHO-S transfection kit (Gibco, catalog #A29133) according to the manufacturer's protocol. A DNA vector containing an open reading frame encoding amino acids 23 - 433 of HYAL1 or its mutant, which is linked at the N-terminus to a non-native signal peptide containing the amino acid sequence of SEQ ID NO:25 and at the C-terminus to a TG-His6 peptide (SEQ ID NO:26), OptiMEM, and Expifectamine were combined in separate 96-well plates and then transferred to the cell culture plate. The cultures were sealed with a gas-permeable membrane (E&K Scientific, catalog #T896100-S) and incubated at 37 °C, 8% CO2 with shaking at 900 revolutions per minute (rpm). On the day after transfection, ExpiCHO-S enhancer-1 and enhancer-2 were added at a total volume of 200 μL per well according to the manufacturer's protocol. On the sixth day after transfection, cells were harvested by centrifugation at 2000 x g for 10 minutes. The supernatant was then transferred to a fresh plate and stored at 4 °C until Hyal-1 activity and expression levels were assayed.

[0176] HYAL1 activity screening

[0177] The relative activity of HYAL1 and its mutants was measured using turbidimetry. Cell culture supernatants containing HYAL1 variants were diluted between 2X–500X into reaction buffer (20 mM sodium phosphate, 77 mM NaCl, 0.01% BSA, pH 4.0, 4.5, 5.0, 5.4, 5.5, 6.0, 6.5, or 7.5). The reaction was then carried out in a 96-well plate (Biorad, catalog #HSP9601) as follows: 25 μL of each Hyal-1 sample was mixed with 25 μL of 0.3 mg / mL hyaluronic acid (Sigma Aldrich, catalog #H7630) (adjusted to match the reaction buffer pH), and the sealed plate was incubated at 37 °C for the duration of the reaction (15 minutes to 18 hours depending on pH and evolutionary round). The reaction was then quenched by heating the plate at 95 °C on a PCR thermocycler for 5 minutes. After quenching, 30 μL of each sample was mixed with 150 μL of acidic albumin solution (24 mM NaOAc, 79 mM HOAc, 0.1% BSA, pH 3.75) in a clear 96-well plate. The plate was incubated on the bench for 5 minutes, and then turbidity was measured at 600 nm on a Spectramax i3x.

[0178] HYAL1 His-tag HTRF expression assay

[0179] The relative expression level of HYAL1 was measured using a His-tag-based detection kit (Cisbio, catalog #64HISPEH). Cell culture supernatants containing Hyal-1 variants were diluted between 1X–200X in the kit assay buffer, and 10 μL of each sample was transferred to a white 1 / 2 area 96-well plate (Corning, catalog #3693). Following the kit manufacturer's protocol, 5 μL of XL665 was added, followed by 5 μL of Gold Eu conjugate (samples were mixed by pipetting for each reagent addition). The plate was then sealed with a foil seal and incubated at room temperature on an orbital shaker (250 rpm) for 2 hours. Then, HTRF measurements were collected using a PerkinElmer EnVision HRTF plate reader according to the vendor's protocol.

[0180] Recombinant expression of HYAL1 in ExpiCHO-S cells

[0181] HYAL1 mutants were expressed recombinantly in ExpiCHO-S based on the manufacturer's recommendations TMRecombinant expression was carried out in ExpiCHO-S cells (Thermo Fisher Scientific). Briefly, plasmid DNA encoding each HYAL1 mutant sterilized through a 0.2-μm filter was obtained at a maximum or gigaprep scale, and the sequences were verified by Sanger sequencing from Elim Biopharm (Hayward, CA). The DNA complex was first formed by mixing 160 μg of plasmid DNA with 8 mL of cold OptiPRO TM SFM (Thermo Fisher Scientific), and mixed by gentle inversion. Cold ExpiFectamine TM CHO Reagent (Thermo Fisher Scientific; 640 μL) was added to 7.4 mL of cold OptiPRO TM SFM and gently inverted. Next, the diluted ExpiFectamine TM CHO Reagent was added to the diluted plasmid DNA, and after 1 minute this mixture was added to 200 mL of ExpiCHO-S cell culture diluted to 6 x 10 6 cells / mL. The culture was placed in a shaking incubator (Infors HT Multitron) shaking at 120 rpm at 37 °C, 8% CO2, 80% humidity. The next day, 48 mL of ExpiCHO TM Feed and 1.2 mL of ExpiFectamine TM CHO Enhancer were added to each culture and incubated for an additional 6 days.

[0182] On day 7, the cultures were pelleted at 500 x g and the culture supernatant was filtered using a vacuum 0.2-μm bottle top filter (Thermo Fisher Scientific). The filtered ExpiCHO TMThe supernatant was buffered to a final concentration of 10 mM HEPES using 1 M HEPES [pH 7.0] (Teknova) and to a final concentration of 20 mM imidazole using 4 M imidazole [pH 8.0] (AdvancedBioReagents). These supernatants were applied by gravity to 3 - 4 mL of Ni Sepharose excel resin (Cytiva) pre - equilibrated with binding buffer (20 mM HEPES, 100 mM NaCl, 20 mM imidazole [pH 7.0]). Subsequently, the resin was washed with 3 column volumes (CV) of binding buffer, 3 CV of binding buffer with 50 mM imidazole, and 3 CV of binding buffer with 75 mM imidazole. Finally, the HYAL1 mutant sample was eluted with binding buffer with 250 mM imidazole. The HYAL1 mutant was mainly found in the 75 mM imidazole wash or elution fractions, and the pure protein fractions were combined after analysis by SDS - PAGE. The HYAL1 mutant sample was concentrated to ∼1 mL using an Ultra 15 mL centrifugal filter (10 kDa MWCO; Millipore Sigma) and applied to a HiLoad 16 / 600 Superdex 200 pg (Cytiva) size - exclusion chromatography column equilibrated in 20 mM HEPES, 100 mM NaCl [pH 7.0] using an avant25 FPLC system (Cytiva). The pure HYAL1 mutant fractions were combined after analysis by SDS - PAGE and used for activity and biophysical analysis.

[0183] Coating of biotinylated hyaluronic acid (BHA) ELISA plates

[0184] A 96-well Nunc microplate (Thermo Fisher) was coated with 100 μL / well of 1.2 μg / mL biotinylated hyaluronic acid (BHA) (CreativePEGWorks) in sodium phosphate buffer (100 mM phosphate buffer, 2 M NaCl, 50 mM MgSO4 [pH 5.8]) together with 2.2 μg / mL sulfo-N-hydroxysulfosuccinimide (Sulfo-NHS) (Thermo Fisher) and 1.5 μg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCA) (Thermo Fisher). The plate was incubated at 4 °C for 24 h. The plate was then washed 3x with PBST (PBS + 0.05% v / v Tween 20) on a BioTek plate washer. The plate was stored in storage solution (2 M NaCl, 50 mM MgSO4 in PBS [pH 7.4]) at -20 °C and used within one week of coating.

[0185] Hyaluronidase ELISA Activity Assay

[0186] The HYAL1 construct was analyzed together with control hyaluronidases: rHuPH20 Hylenex (Halozyme), rHuPH20 (AcroBioSystems), rHu-HYAL1 (R&D Systems), and bee venom hyaluronidase. Serial dilutions were prepared in different buffers depending on the pH being analyzed: histidine buffer (20 mM histidine, 130 mM NaCl, 1 mM CaCl2, and 0.04% ) for pH 5.0 - 7.5; sodium acetate buffer (20 mM sodium acetate, 130 mM NaCl, 1 mM CaCl2, and 0.04% BSA) for pH 4.0 and 4.5, and formate buffer (20 mM, 130 mM NaCl, 1 mM CaCl2, and 0.04% BSA) for pH 3.0 and 3.5.

[0187] Serial dilutions and transfers to ELISA plates were performed using an automated liquid handler (Agilent Bravo). Samples were further diluted 10x by adding 20 μL of diluted protein to 180 μL of buffer. Serial dilutions (100 μL each) were transferred to BHA-coated ELISA plates and incubated at 37 °C for 1 hour. The plates were then washed 3x with PBST buffer via a BioTek plate washer. Subsequently, 200 μL of 6 M guanidine hydrochloride was added as a stop solution to quench hyaluronidase activity, incubated for 5 minutes at room temperature (RT), and washed 3x with PBST. After that, a PBS solution of 0.1 μg / mL horseradish peroxidase (HRP)-conjugated streptavidin (Thermo Fisher) at 100 μL / well was added and placed on an oscillator at room temperature for 30 minutes. The plates were then washed 3x with PBST, and 100 μL of KLP peroxidase substrate (containing 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate); Sera Care) was added to each well and incubated in the dark for 20 minutes. Absorbance at 405 nm was detected on a BioTek plate reader.

[0188] All graphs were generated using GraphPad Prism 9; ELISA EC 50 values were determined using a sigmoidal, 4PL, X as log(concentration) fit, where the lower constraint was equivalent to the absorbance value from wells containing fully digested HA. Hyaluronidase activity was determined based on the known activity value of Acro PH20 at 108,422 units / mg (manufacturer information) and the EC 50 value determined at pH 5.35. Activity at pH 5.35: EC 50 ratio was used to determine all subsequent activities at different pHs for all samples, controls, and standards.

[0189] Example 1

[0190] Evolution of human HYAL1 mutants

[0191] Figure 1 An overview of the design / test portion of the workflow for evolving HLYAL1 into a HYAL mutant with a broader pH activity range, which can be used for the delivery of therapeutics for subcutaneous administration, is shown. After design, DNA encoding multiple HYAL1 mutants was ordered from Genscript. After delivery of this DNA, the HYAL1 mutants were expressed in ExpiCHO-S cells. After expression, samples were collected and activity was tested using a turbidity assay. As described below, the expression level was also evaluated using a HYAL1 His-tag HTRF expression assay.

[0192] Figure 2 Shows the results of the first round of screening. This is a library constructed mainly from literature reports on sequence homology and the effects of mutations on HYAL1 and other hyaluronidase homologs. The figure is sorted by FIOP from highest to lowest activity. FIOP stands for (fold improvement over parent) and refers to the activity of each mutant relative to the starting point of each round (e.g., in the first round, the starting point is wild-type HYAL1). FIOP is simply calculated as (activity of the variant) / (activity of the parent). The points on the graph show similar FIOP values with respect to expression levels. Activity was measured using turbidimetry, and expression data were collected using a commercial HTRF assay kit.

[0193] Figure 3 Shows data very similar to Figure 2 In this case, library variants were screened at both pH 4 (top) and pH 5 (bottom). During the initial rounds of evolution, we tested HYAL mutants at various pHs to identify mutants that were active at pH values closer to the target pH of 5.5.

[0194] Named "Evolution Tracker" Figure 4A - Figure 4I Shows the progress of HYAL1 evolution in a round-by-round format. Each box represents a separate library, and each column of boxes represents a round of evolution (matching the table at the top). The "fold improvement" row in the table shows how the HYAL1 backbone in that round compares to the backbone of the previous round. Rosetta refers to the RosettaCommons software available from http: / / RosettaCommons.org.

[0195] Figure 5 Shows in vivo data that confirm the activity of the engineered HYAL1 mutants in a mouse skin spreading model. In this experiment, wild-type HYAL1, the round 3 HYAL1 mutant, and a commercially used hyaluronidase (PH20) were compared. Each enzyme was mixed with a colored dye and injected intradermally into mice. Then the dye spreading was monitored over time.

[0196] Figure 6 and Figure 7Shows the workflow for expressing, purifying, and crystallizing the HYAL1 mutant and the resulting structure. The enzyme was expressed in ExpiCHO-S cells, treated with endoglycosidase to remove most of the glycans on the protein, and then purified. The purified material was screened for crystallization conditions. Suitable conditions were identified, and HYAL1 crystals were obtained. The crystal structure confirmed that during evolution, we had introduced several new N-linked glycosylation sites during HYAL1 evolution. The ASU is the smallest part of the crystal structure to which symmetry operations can be applied to generate the complete unit cell (crystal repeating unit). R 工作 is a measure of the agreement between the crystallographic model and the experimental X-ray diffraction data. R 自由 is a statistical quantity introduced by Axel T. Brünger in 1992 to evaluate the quality of a model from X-ray crystallography data.

[0197] Figure 8 Shows that in the 8th round of HYAL1 evolution, the focus became reducing the immunogenicity of the mutations we had introduced up to that point. ExpiVax software from ExpiVax, Providence, RI, USA was used to scan the regions in which mutant HYAL1 had been prepared. The software provides epitope scores that can be used to evaluate the immunogenicity of each possible 9-mer peptide in the scanned regions.

[0198] Figure 9 Using the EpiVax software (see Figure 8 ), we identified 7 epitope regions that had an increased risk of immunogenicity due to the mutations we had introduced. Depending on the score, these regions were each classified as low risk, medium risk, or high risk.

[0199] Figure 10 Outlines the method for designing additional HYAL1 mutations that reduce the immunogenicity risk at the sites identified by EpiVax analysis. This involves designing point mutations computationally (using RosettaCommons software) and then evaluating the effect of these mutations on the EpiVax immunogenicity scores. In this way, mutations are filtered by predicted stability (RosettaCommons software) and predicted reduction in immunogenicity (EpiVax). Mutations that pass these filters and meet the required criteria are used to design a new library of point mutants.

[0200] Figure 11 Shows the results of the 8th round of point mutant library screening. The top graph shows the number of epitopes removed by each mutation introduced. The graph is divided into individual regions (A, B, C, D, E, and FG) that match the regions mapped on the structure in Figure 9 . In Figure 11The figure on the bottom shows the activity and expressed FIOP of each mutant. In this case, we looked for mutants with an expressed and active FIOP close to 1.0 and selected them for combination in the final library.

[0201] Figure 12 The results of library screening are shown in which point mutations that reduce immunogenicity are combined. Library design efforts were made to maximize our chances of reducing the number of epitopes of interest. We designed triple-mutant mutants where the mutations were from each of three regions predicted to be at "high" immunogenic risk. We then designed mutants that also included a fourth mutation from one of the other regions, then mutants with a fifth mutation, and so on.

[0202] The results of the screening rounds and the HYAL1 mutants obtained are summarized in Table 2.

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215] References

[0216] 1De Cock, E. et al. Time Savings with Rituximab Subcutaneous Injection versus Rituximab Intravenous Infusion: A Time and Motion Study in Eight Countries. PLoS One 11, e0157957, doi:10.1371 / journal.pone.0157957 (2016).

[0217] 2Wasserman, R. L. Recombinant human hyaluronidase - facilitated subcutaneous immunoglobulin infusion in primary immunodeficiency diseases. Immunotherapy 9, 1035 - 1050, doi:10.2217 / imt - 2017 - 0092 (2017).

[0218] 3Wynne, C. et al. Comparison of subcutaneous and intravenous administration of trastuzumab: a phase I / Ib trial in healthy male volunteers and patients with HER2 - positive breast cancer. J Clin Pharmacol 53, 192 - 201, doi:10.1177 / 0091270012436560 (2013).

[0219] 4Usach, I., Martinez, R., Festini, T. & Peris, J. E. Subcutaneous Injection of Drugs: Literature Review of Factors Influencing Pain Sensation at the Injection Site. Adv Ther 36, 2986 - 2996, doi:10.1007 / s12325 - 019 - 01101 - 6 (2019).

[0220] 5Bittner, B., Richter, W. & Schmidt, J. Subcutaneous Administration of Biotherapeutics: An Overview of Current Challenges and Opportunities. BioDrugs 32, 425 - 440, doi:10.1007 / s40259-018-0295-0(2018).

[0221] 6Meyer, K. & Chaffee, E. The Mucopolysaccharides of Skin. Journal of Biological Chemistry 138, 491 - 499, doi:10.1016 / s0021-9258(18)51374-0(1941).

[0222] 7Meyer, K. The biological significance of hyaluronic acid and hyaluronidase. Physiological Reviews 27, 335 - 359, doi:10.1152 / physrev.1947.27.3.335(1947).

[0223] 8McClean, D. Studies on diffusing factors: The hyaluronidase activity of testicular extracts, bacterial culture filtrates and other agents that increase tissue permeability. The Biochemical Journal 35, 159 - 183, doi:10.1042 / bj0350159(1941).

[0224] 9Duran-Reynals, F. Tussue Permeability and the Spreading Factors in Infection: A Contribution to the Host: Parasite Problem. Bacteriological Reviews 6, 197 - 252, doi:10.1128 / br.6.4.197-252.1942(1942).

[0225] 10Papakonstantinou,E.,Roth,M.&Karakiulakis,G.Hyaluronic acid:A keymolecule in skin aging.Dermatoendocrinol 4,253-258,doi:10.4161 / derm.21923(2012).

[0226] 11Frost,G.I.Recombinant human hyaluronidase(rHuPH20):An enablingplatform for subcutaneous drug and fluid administration.Expert Opinion onDrug Delivery 4,427-440,doi:10.1517 / 17425247.4.4.427(2007).

[0227] 12Reitinger,S.,Müllegger,J.,Greiderer,B.,Nielsen,J.E.&Lepperdinger,G.Designed human serum hyaluronidase 1variant,HYAL1ΔL,exhibits activity upto pH 5.9.Journal of Biological Chemistry284,19173-19177,doi:10.1074 / jbc.C109.004358(2009).

[0228] Although the present invention has been described herein with reference to illustrative embodiments, it is to be understood that the invention is not limited thereto. Persons having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within its scope. Accordingly, the invention is limited only by the appended specification and claims herein.

Claims

1. A human hyaluronidase 1 (HYAL1) mutant, which comprises an amino acid sequence having at least 95% identity with the amino acid sequence of amino acids 20 to 433 of the HYAL1 amino acid sequence shown in SEQ ID NO: 2, wherein the HYAL1 mutant further comprises a combination of amino acid substitutions selected from the following: (a) Amino acid substitutions D68N, M246L, D323N, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, and W433E; (b) Amino acid substitutions D68N, M246L, D323N, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, and V412R; (c) Amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, V412R, R238C, I287P, F345T, and L377S; (d) Amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, V412R, R238C, F345T, and L377S; and (e) Amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, P23V, S304T, L379M, W433E, R194D, D299E, V412R, N235D, I287P, F345T, and L377S; and wherein the HYAL1 mutant (i) includes at least one N-glycosylation site comprising the sequence asparagine-X-serine / threonine, where X is any amino acid other than proline, and the site is glycosylated, and (ii) exhibits hyaluronidase activity between pH 3.5 and pH 5.

5.

2. The HYAL1 mutant according to claim 1, wherein the HYAL1 mutant comprises the amino acid substitutions D68N, M246L, D323N, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, and W433E.

3. The HYAL1 mutant according to claim 1, wherein the HYAL1 mutant comprises the amino acid substitutions D68N, M246L, D323N, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, and V412R.

4. The HYAL1 mutant according to claim 1, wherein the HYAL1 mutant comprises the amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, V412R, R238C, I287P, F345T, and L377S.

5. The HYAL1 mutant according to claim 1, wherein the HYAL1 mutant comprises the amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, V412R, R238C, F345T, and L377S.

6. The HYAL1 mutant according to claim 1, wherein the HYAL1 mutant comprises the amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, P23V, S304T, L379M, W433E, R194D, D299E, V412R, N235D, I287P, F345T, and L377S.

7. The HYAL1 mutant according to claim 1, wherein at least one N-glycosylation site generated by the substitution of D68N, D323N, G235N, or R326S is glycosylated.

8. The HYAL1 mutant according to claim 1, wherein the N-glycosylation sites generated by the substitutions of D68N, D323N, G235N, and R326S are each glycosylated.

9. The HYAL1 mutant according to claim 1, wherein the native N-glycosylation site and the N-glycosylation sites generated by the substitutions of D68N, D323N, G235N, and R326S are each glycosylated.

10. A human hyaluronidase 1 (HYAL1) mutant, which comprises an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, wherein the HYAL1 mutant (i) includes at least one N-glycosylation site comprising the sequence asparagine-X-serine / threonine, where X is any amino acid other than proline, and the site is glycosylated, and (ii) exhibits hyaluronidase activity between pH 3.5 and pH 5.

5.

11. A human hyaluronidase 1 (HYAL1) mutant, which comprises an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, wherein the HYAL1 mutant (i) includes at least one N-glycosylation site comprising the sequence asparagine-X-serine / threonine, where X is any amino acid other than proline, and the site is glycosylated, and (ii) exhibits hyaluronidase activity between pH 3.5 and pH 5.

5.

12. The HYAL1 mutant according to any one of claims 1 to 11, wherein the hyaluronidase is conjugated to a polymer.

13. The HYAL1 mutant according to claim 12, wherein the polymer is dextran or polyethylene glycol.

14. A composition, which comprises the HYAL1 mutant according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

15. The composition according to claim 14, wherein the composition further comprises a therapeutic agent.

16. The composition according to claim 15, wherein the therapeutic agent comprises a small molecule, a peptide, a macrocyclic peptide, a protein or a protein complex.

17. The composition according to claim 16, wherein the small molecule comprises an antibiotic or an anti-inflammatory agent.

18. The composition according to claim 16, wherein the peptide comprises an insulinotropic peptide, growth hormone, insulin or an insulin mutant.

19. The composition according to claim 15, wherein the therapeutic agent comprises an antibody, an antigen-binding protein, an scFv, a Fab or a fusion protein comprising the Fc domain of an antibody.

20. A method for increasing the diffusion of a therapeutic agent in a tissue of a subject, which comprises: administering to the tissue of the subject the composition comprising the HYAL1 mutant according to claim 14 in an amount sufficient to increase the diffusion of the therapeutic agent; and administering to the tissue a composition comprising the therapeutic agent.

21. A method for increasing the diffusion of a therapeutic agent in a tissue of a subject, which comprises: Administering to the tissue of a subject a composition comprising the therapeutic agent and an amount of the HYAL1 mutant according to claim 14 sufficient to increase the diffusion of the therapeutic agent in the tissue.

22. Use of the HYAL1 mutant according to any one of claims 1-13 for the manufacture of a medicament for use in combination with a therapeutic agent for treating a disease or disorder.

23. Use of the HYAL1 mutant according to any one of claims 1-13 for the manufacture of a medicament comprising hyaluronidase and a therapeutic agent for treating a disease or disorder.

24. Use of the HYAL1 mutant according to any one of claims 1-13 for increasing the diffusion of a therapeutic agent in the tissue of a subject for treating a disease or disorder.

25. Use of a composition comprising the HYAL1 mutant according to any one of claims 1-13 and a therapeutic agent for treating a disease or disorder.

26. A nucleic acid molecule encoding the HYAL1 mutant according to any one of claims 1-13.

27. The nucleic acid molecule according to claim 26, wherein the HYAL1 mutant according to claim 1 is fused to a signal sequence that targets the HYAL1 mutant to the endoplasmic reticulum.

28. An expression vector comprising the nucleic acid molecule according to claim 26 or 27.

29. A host cell comprising the nucleic acid molecule according to claim 26 or 27 or the expression vector according to claim 28.

30. The host cell according to claim 29, wherein the host cell is a mammalian host cell or a recombinant yeast host cell modified to produce complex N-glycans.

31. The host cell according to claim 30, wherein the mammalian host cell is a Chinese hamster ovary cell.

32. The host cell according to claim 30, wherein the recombinant yeast host cell is Pichia pastoris.

33. A method for producing a HYAL1 mutant, comprising: (a) introducing into a host cell the nucleic acid molecule encoding the HYAL1 mutant according to any one of claims 26 or 27, or the expression vector comprising the nucleic acid molecule encoding the HYAL1 mutant according to claim 28, to produce a recombinant host cell; (b) culturing the recombinant host cell in a culture medium under conditions sufficient for the recombinant host cell to express the HYAL1 mutant and secrete it into the culture medium; and (c) obtaining the HYAL1 mutant from the culture medium.

34. The method according to claim 33, wherein the host cell is a mammalian host cell or a recombinant yeast host cell modified to produce complex N-glycans.

35. The method according to claim 34, wherein the mammalian host cell is a Chinese hamster ovary cell.

36. The method according to claim 34, wherein the recombinant yeast host cell is Pichia pastoris.

37. The method according to claim 33, wherein the HYAL1 mutant comprises amino acids 1 to 433 of an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.

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