EP4608450A1

EP4608450A1 - Human hyaluronidase 1 mutants

Info

Publication number: EP4608450A1
Application number: EP23883336.2A
Authority: EP
Inventors: Benjamin N. BELL; Johnathon Tres BRAZELL; Keith A. Canada; Bernhard H. Geierstanger; James Alan GOGGINS; Laura J. KINGSLEY; Jeffrey C. Moore; Cameron L. NOLAND; Roy David ROW; Allan SOLIS; Deeptak Verma; Jianzhong Wen
Original assignee: Merck Sharp and Dohme LLC
Current assignee: Merck Sharp and Dohme LLC
Priority date: 2022-10-24
Filing date: 2023-10-19
Publication date: 2025-09-03
Also published as: KR20250093374A; CN120322249A; WO2024091417A1

Abstract

Human hyaluronidase 1 (HYAL1) mutants that are highly expressed and display hyaluronidase activity from pH 3.5 to pH 5.5 are disclosed.

Description

HUMAN HYALURONIDASE 1 MUTANTS

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file, created on February 21, 2023, is named 25596-WO-PCT_SL.XML and is 50,765 bytes in size.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to human hyaluronidase 1 (HYAL1) mutants that are highly expressed and display hyaluronidase activity from pH 3.5 to pH 5.5.

(2) Description of Related Art

Subcutaneous (SC) delivery of biologic drugs is a faster, less painful, and more convenient alternative to traditional intravenous injection or drip^1-4. Furthermore, many SC formulations are amenable to home administration via various devices, benefitting patients and healthcare workers alike¹-⁴. However, the accepted maximal tolerable volume for an SC injection is around 1.5 mL which is not compatible with the large dosages typical of most biologies⁴. Coformulation with hyaluronidase (HAase) is a well-established method to substantially increase the tolerable volume for SC delivery of biologies and other molecules, up to 500 mL in some cases²-⁵. Indeed, bovine and ovine HAases have been used clinically as a “spreading factor” to enhance tissue permeability since the 1950s⁶'⁹. In the past 20 years, recombinant human PH20 (rHuPH20) HAase, marketed under the tradename HYLENEX, has enabled even greater access to novel SC formulations of oncology and immunology therapies.

HAases act by degrading hyaluronic acid (HA) polymers, one of the main structural components of the extracellular matrix in the dermis layer of the skin¹⁰. Cleavage of these polymers by HAases release sequestered water which allows for spreading of injected formulations within the dermal layer and absorption by underlying capillaries and lymphatic vessels¹¹. In humans, there are five HAase isoforms: HYAL1-4 andHYAL5 (more commonly called PH20 or SPAM1). PH20 is the only isoform that has high enzymatic activity at neutral pH, severely limiting the clinical prospects of other human HAases. Efforts to increase the pH range of HYAL1 have had modest success, in which activity could be observed outto pH 5.9¹²

BRIEF SUMMARY OF THE INVENTION

The present invention provides human hyaluronidase 1 (HYAL1) mutants, which have been evolved through iterative rounds of protein engineering to have greatly improved hyaluronidase activity and importantly hyaluronidase activity within an expanded pH range compared to wild-type HYAL1 . In addition, the HYAL1 mutants have improved expression yields relative to wildtype HYAL1 . Theses HYAL1 mutants are useful for increasing the absorption of therapeutic agents into tissue and have the potential to reduce tissue damage in cases of extravasation of a therapeutic agent The HYAL1 mutants of the present invention provide a HYAL1 mutant with a pH-controlled activity profile, which reduces the effect of active hyaluronidase activity in the skin following the dispersion of a co-administered therapeutic agent through the skin, thus, reducing the unwanted side effects of persistent hyaluronidase activity following dispersion of the therapeutic agent.

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 set forth in SEQ ID NO: 2, wherein the hyaluronidase 1 mutant further comprises a combination of amino acid substitutions selected from the group consisting of : (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, V412RR238C, I287P, F345T, andL377S; (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 hyaluronidase 1 mutant (i) comprises at least one 7V-glycosylation site comprising the consensus sequence asparagine-X-serine/threonine, wherein X is any amino acid other than proline, that is glycosylated and (ii) displays hyaluronidase activity from pH 3.5 to pH 5.35 (or pH5.5).

In particular 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.

In particular 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.

In particular 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, V412RR238C, I287P, F345T, and L377S.

In particular 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.

In particular embodiments, the HYAL1 mutant comprises the amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, P23 V, S304T, L379M, W433E, R194D, D299E, V412R, N235D, I287P, F345T, and L377S.

In further embodiments, at least one A-glycosylation site created by the D68N, D323N, G235N, or R326S substitution is glycosylated. In further embodiments, the N- glycosylation sites created by the D68N, D323N, G235N, and R326S substitutions are each glycosylated. In further embodiments, the native A-glycosylation sites and the A-glycosylation sites created by the D68N, D323N, G235N, andR326S substitutions are each glycosylated.

The present invention further provides a HYAL1 mutant comprising an amino acid sequence with at least 95% (and in specific embodiments, 96%, 97%, 98%, 99% and 100%) identity to the amino acid sequence set forth 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) comprises at least one A-glycosylation site comprising the sequence asparagine-X- serine/threonine, wherein X is any amino acid other than proline, that is glycosylated and (ii) displays hyaluronidase activity from pH 3.5 to pH 5.35 (or pH5.5).

The present invention further provides a HYAL1 mutant comprising an amino acid sequence with at least 95% (and in specific embodiments, 96%, 97%, 98%, 99% and 100%) identity to the amino acid sequence set forth in set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, 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, that is glycosylated and (ii) displays hyaluronidase activity from pH 3.5 to pH 5.5.

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

The present invention further provides a HYAL1 mutant disclosed herein and a pharmaceutically acceptable carrier. In a further embodiment, the composition further comprises a therapeutic agent, thus in particular 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, a small molecule, a peptide, a macrocyclic peptide, a protein, or a protein complex. In particular embodiments, the small molecule comprises an antibiotic or an anti-inflammatory agent. In particular embodiments, the peptide comprises an insulinotropic peptide, a growth hormone, insulin, or an insulin mutant. In particular embodiments, 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.

In a further embodiment, the present invention provides compositions comprising a tumor-specific CAR-T cell or CAR-NK cell conjugated to a HYAL1 mutant disclosed herein. See for example, 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).

The present invention further provides a method for increasing the diffusion of a therapeutic agent in a tissue of a subject, comprising administering to the tissue of a subject a composition comprising a HYAL1 mutant disclosed herein in an amount sufficient to increase 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 may be administered consecutively or concurrently to the subject.

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

The present invention further provides for the use of a HYAL1 mutant disclosed herein for the manufacture of a medicament for use with a therapeutic agent for treatment of a disease or disorder.

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

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

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

The present invention further provides a nucleic acid molecule encoding a HYAL1 mutant disclosed herein. The present invention further provides a nucleic acid molecule encoding a HYAL1 mutant disclosed herein fused to a signal sequence that targets the HYAL1 mutant to the endoplasmic reticulum.

The present invention further provides an expression vector comprising a nucleic acid molecule encoding a HYAL1 mutant disclosed herein that, in specific embodiments, fused to a signal sequence that targets the HYAL1 mutant to the endoplasmic reticulum.

The present invention further provides a host cell comprising a nucleic acid molecule encoding a HYAL1 mutant disclosed herein that, in specific embodiments, is fused to a signal sequence that targets 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 a HYAL1 mutant disclosed herein that, in specific embodiments, is fused to a signal sequence that targets 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 A-glycans.

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

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

In a further embodiment, the host cell is a mammalian host cell, which in a further embodiment may be a Chinese hamster ovary cell. In a further embodiment, the host cell is a recombinant yeast host cell modified to produce complex A-gly cans, which in a further embodiment may be the recombinant yeast host cell Pichia pastoris.

In a further embodiment of the method, the HYAL1 mutant comprises amino acids 1 to 433 of the amino acid sequence setforth 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 comprise a signal sequence that targets the HYALl mutantto the endoplasmic reticulum.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig- 1 shows an overview of the design/test parts of the workflow for evolving human hyaluronidase 1 (HYAL1) into a HYAL1 mutant with a broader pH range of activity that would be useful for use in delivery of a therapeutic agent administered subcutaneously.

Fig. 2 shows the results of the first round of screening. This was a library built primarily from sequence homology and literature reports of mutation impacts on HYAL1 and other hyaluronidase homologs. The plot is ranked from highest to lowest activity FIOP. FIOP stands for (Fold Improvement Over Parent) and refers to each mutant’s activity relative to the starting point for each round (in round 1 for example the starting point was the wildtype HYAL1). FIOP is calculated simply as (Activity of Variant)/(Activity of Parent). The dots on the plot show a similar FIOP value for the expression level. The activities were measured using a turbidity assay and the expression data was collected using a commercial HTRF assay kit.

Fig- 3 shows data very similar to Fig. 2. In this case the library variants were screened at both pH 4 (top) and pH 5 (bottom).

Figs. 4 A - Fig. 41 show the progress of the HYAL1 evolution in a round-by- round format. Each of the boxes represents an individual library, and each column of boxes represents a round of evolution (matching the table that runs across the top). The “Fold Improvement” row in the table shows how the HYAL1 backbone for that round compared to the backbone of the previous round. Rosetta refers to RosettaCommons.org software for modeling mutations.

Fig- 5 shows in vivo data that demonstrates activity of an engineered HYAL1 mutant in a mouse skin-spreading model.

Fig- 6 showthe workflowthatwas used to express, purify, and crystallize HYAL1 mutants.

Fig. 7 shows the resulting structure from the workflow of Fig. 6. The term “ASU” means “asymmetric unit”.

Fig. 8 shows that in round 8 of the HYAL1 evolution the focus became reducing the immunogenicity of the mutations that had been introduced up to that point. ExpiVax software (EpiVax Inc.) was used to scan regions of HYAL1 where mutations had been made. The software provides an epitope score that can be used to assess 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.

Fig. 9 shows seven epitope regions that had increased immunogenicity risk as a result of the mutations that had been introduced during the evolution process. Each of those regions was classified as low, medium, or high risk, depending on the score. EpiVax software (see Fig. 8) was used for the analysis.

Fig. 10 summarizes the approach that was taken to design additional HYAL1 mutations that would reduce the immunogenicity risk of the sites identified by EpiVax analysis. The peptide sequences from top to bottom are represented by SEQ NO ID: 27 to SEQ ID NO: 33. Fig. 11 shows the results of the round 8 point-mutation library screen. The plot on the top shows the number of epitopes that were removed by each mutation that was introduced. The plot is divided into regions (A, B, C, D, E, and FG) that match the regions that were mapped on the structure shown in Fig. 9.

Fig. 12 shows the results of the library screen in which immunogenicity-reducing point mutations were combined. The plot shows the activity and expression FIOPs of each of the mutants shown in Fig. 12.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the terms “A-glycan” and “glycoform” are used interchangeably and refer to an A-linked oligosaccharide, for example, one that is attached by an asparagine-A- acetylglucosamine linkage to an asparagine residue of a polypeptide. N-linked glycoproteins contain an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in the protein. The predominant sugars found on glycoproteins are glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), A-acetylgalactosamine (GalNAc), N-acetyl glucosamine (GlcNAc) and sialic acid (Sia). Sialic acids are a class of a-keto acid sugars with a nine-carbon backbone. The most common member of this group is A-acetylneuraminic acid (Neu5Ac or NANA) found in animals and some prokaryotes. The processing of the sugar groups occurs co-translationally in the lumen of the ER and continues post-translationally in the Golgi apparatus for N-linked glycoproteins.

N-gl yeans have a common pentasaccharide core of MangGlcNAc^ comprising a mannose linked at its reducing end to the nonreducing end of a chitobiose core (GlcNAp 1- 4GlcNAc) in a P 1 ,4 linkage and one mannose residue linked to the P 1 ,4 linked mannose in an al, 3 linkage and the other mannose linked to the pi,4-linked mannose in an al, 6 linkage, represented by the structure showing the Mang GlcNAc2 linked to an asparagine residue comprising an A-glycosylation site in a glycoprotein The GlcNAc residue at the reducing end may also be linked to a fucose residue in an al, 6 linkage. Usually, N-glycan structures are presented with the nonreducing end to the left and the reducing end to the right or the nonreducing end at the top and the reducing end at the bottom. The reducing end of the N-glycan is the end that is attached to the Asn residue comprising the glycosylation site on the protein. -glycans differ with respect to the number of branches (antennae) comprising peripheral sugars (e.g. , GlcNAc, galactose, fucose and sialic acid) that are added to the Man3GlcNAc2 (“Mang”) core structure, which is also referred to as the “trimannose core”, the “pentasaccharide core”, the trimannosyl core”, or the “paucimannose core”.

A-glycans are classified according to their branched constituents (e.g., high mannose, complex, or hybrid). A “high mannose” type A-glycan comprises five or more mannose residues. A “complex” type A-gly can typically has at least one GlcNAc residue attached in a P 1 ,2 linkage to the nonreducing end of the mannose residue at the nonreducing end of the 1 ,3 mannose arm and at least one GlcNAc residue attached in a P 1 ,2 linkage to the nonreducing end of the mannose residue at the nonreducing end of the 1 ,6 mannose arm of the trimannose core. Complex -glycans may further include galactose (“Gal”) or N- acetylgalactosamine (“GalNAc”) residues that are optionally 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 A-gly cans may also have intrachain substitutions comprising “bisecting” GlcNAc residue and core fucose (“Fuc”) residue. Complex -glycans may also have multiple antennae on the “trimannose core,” often referred to as “multiantennary N-glycans.” A “hybrid” N-gly can comprises at least one GlcNAc attached in a P 1 ,2 linkage to the nonreducing end of the mannose residue at the nonreducing end of the 1,3 mannose arm of the trimannose core and zero or more mannoses attached to the nonreducing end of the mannose on the nonreducing end of the 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 residues. The various A-glycans are also referred to as “glycoforms.”

With respectto complex A-gly cans, the terms "G-2", "G-l", "GO", "Gl", "G2", "Al ", and "A2" mean the following. "G-2" refers to an N-glycan structure that can be characterized as Mang GlcNAc^; the term "G-l " refers to an A-glycan structure that can be characterized as GlcNAcMangGlcN ^; the term "GO" refers to an A-glycan structure that can be characterized as GlcNAc2MangGlcNAc2; the term "Gl " refers to an N-gly can structure that can be characterized as GalGlcNAc2Man3 GIcNAc^; the term "G2" refers to an A-glycan structure that can be characterized as Gal2GlcNAc2Man3GlcNAc2; the term "Al " refers to an N- glycan structure that can be characterized as SiaGal2GlcNAc2Man3GlcNAc2; and, the term " A2" refers to an A-glycan structure that can be characterized as Sia2Gal2GlcNAc2Man3GlcNAc2- Unless otherwise indicated, the terms “G-2", "G-l", "GO", "Gl", "G2", "Al", and "A2" refer to A-glycan species that lack fucose attached to the GlcNAc residue at the reducing end of the A-glycan. When the term includes an "F", the "F" indicates that the A -gley an species contains a fucose residue on the GlcNAc residue at the reducing end of the A-glycan. For example, GOF, GIF, G2F, A1F, and A2F all indicate that the A-glycan further includes a fucose residue attached to the GlcNAc residue at the reducing end of the A-glycan. Lower eukaryotes such as yeast and filamentous fungi do not normally produce A-glycans that produce fucose.

Complex A-glycans include biantennary A-gly cans, bisected A-gly cans, and multiantennary A-glycans. With respect to a biantennary A-glycan, the A-glycan comprises a 1,6 mannose arm and a 1 ,3 mannose arm in which the 1 ,6 mannose arm and the 1 ,3 mannose arm each comprises one GlcNAc residue linked in a P 1 ,2 linkage to the mannose residue at the nonreducing end of the arm. Each GlcNAc residue may be independently further attached to a galactose residue and each galactose residue may be independently further attached to a sialic acid residue. Thus, biantennary A-glycans may include A-glycans having the short-hand formula GlcNAc2Man3GlcNAc2, Galp| _2 GlcNAc2Man3GlcNAc2, or Sia _2)Gal _ 2)GlcNAc2Man3GlcNAc2- The term "1-2" refers to 1 or2 sugar residues.

With respect to multiantennary A-glycans, the term "multiantennary A-glycan" refers to a biantennary A-glycan that further comprises (i) a GlcNAc residue attachedin a pi,4 linkage to the nonreducing end of the mannose residue comprising the non-reducing end of the 1,6 arm or the 1,3 arm of the A-glycan or (ii) a GlcNAc residue is attached in a P 1,4 linkage to the nonreducing end of the mannose residue comprising the non-reducing end of the 1 ,6 arm and a GlcNAc residue is attached in a P 1 ,4 linkage to the nonreducing end of the mannose residue comprising the non-reducing end of the 1,3 arm of the A-glycan. Each GlcNAc residue may be independently further attached to a galactose residue and the galactose residue maybe independently further attached to a sialic acid residue. Thus, multiantennary A-glycans canbe characterized by the short-hand formulas GlcNAcp _4)Man3GlcNAc2, Gal -^GlcNAcp. 4)Man3GlcNAc2, or Sia _4)Gal _4)GlcNAcQ_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.

With respect to bisected A-glycans, the term "bisected A-gly can" refers to N- glycans in which the reducing end of a GlcNAc residue is linked in a P 1 ,4 linkage to the nonreducing end of the central mannose residue at the nonreducing end of the trimannose core. A bisected A-glycan may be characterized by the formula GlcNAc3Man3GlcNAc2 wherein each mannose residue is linked at its non-reducing end to a GlcNAc residue. In contrast, when a multiantennary A-glycan is characterized as GlcNAc3Man3GlcNAc2, the short-hand formula indicates that two GlcNAc residues are linked to the mannose residue at the non-reducing end of one of the two arms of the A-glycan and one GlcNAc residue is linked to the mannose residue at the non-reducing end of the other arm of the A-glycan. While the GlcNAc residues at the end of the 1,3 and 1,6 arms of the bisected A-gly can may each be further linked to a galactose residue, which may be further linked to a sialic acid residue, the bisecting GlcNAc residue is not further extended.

Table 1 provides a list of representative biantennary and bisected A-glycans, which includes the linkage to the Asn residue in the A-glycosylation site comprising the consensus sequence Asn-X-Ser/Thr, wherein X may be any amino acid other than proline.

Abbreviations used herein are of common usage in the art, see, e.g. , abbreviations of sugars, above. Other common abbreviations include “PNGase”, or “glycanase” or “glucosidase” which all referto peptide A-glycosidase F (EC 3.2.2.18). As used herein, the term "glycoprotein" refers to any protein having one or more

7V-glycans attached thereto and/or one or more O-glycans. Thus, the term refers both to proteins that are generally recognized in the art as a glycoprotein and to proteins which have been genetically engineered to contain one or more TV-linked glycosylation sites and/or ( -linked glycosylation sites.

As used herein, a “humanized glycoprotein” or a “human-like glycoprotein” refers alternatively to a protein having attached thereto A-glycans having fewer than four mannose residues, and synthetic glycoprotein intermediates (which are also useful and can be manipulated further in vitro or in vivo) having at least five mannose residues. In specific embodiment, glycoproteins produced according to the invention contain at least 30 mole %, preferably at least 40 mole % and more preferably 50, 60, 70, 80, 90, or even 100 mole % of the Man5GlcNAc2 intermediate, at least transiently. This may be achieved, e.g., by engineering a host cell of the invention to express a “better”, z.e., a more efficient glycosylation enzyme. For example, a mannosidase is selected such that it will have optimal activity under the conditions present at the site in the host cell where proteins are glycosylated and is introduced into the host cell preferably by targeting the enzyme to a host cell organelle where activity is desired.

The term “recombinant host cell”, as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a 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 which resides in a living tissue or organism. Preferred host cells are yeasts and fungi.

As used herein, the term “hyaluronidase” or “HYAL” refers to hyaluronoglucosaminidase, (EC 3.2.1.35) which catalyzes hydrolysis of glycosaminoglycans including hyaluronans. Hyaluronidase 1 (HYAL1) is a species of hyaluronidase found in lysozymes. Other species of hyaluronidases include HYAL2, HYAL3, HYAL4 andHYAL5 (also known as SPAM1 orPH20).

Four different purified hyaluronidases have been approved for use in the United States, three of animal origin and one recombinant. They are indicated as adjuvants in subcutaneous fluid administration for achieving hydration, for increasing the dispersion and absorption of other injected drugs, or for improving resorption of radiopaque agents, in subcutaneous urography. The three naturally-sourced hyaluronidases are orthologs of human HYAL5 (PH20) obtained from testicular preparations. They are sold under the trade names VITRASE (ovine, FDA-approved 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.

As used herein, the term "antibody" or “immunoglobulin” as used herein refers to a glycoprotein comprising at least two heavy chains (HCs) and two light chains (LCs) interconnected by disulfide bonds. Each HC is comprised of a heavy chain variable region or domain (V_H) and a heavy chain constant region or domain. Each light chain is comprised of an LC variable region or domain (VL) and a LC constant domain. In certain naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. In general, the basic antibody structural unit for antibodies is a Y-shaped tetramer comprising two HC/LC pairs (2H). Each tetramer includes two identical pairs of polypeptide chains, each pair having one LC (about 25 kDa) and HC chain (about 50-70 kD a) (H+L). Each HC:LC pair comprises one VJJ: one VL pair. The one Vjj:one

V pair may be referred to by the term “Fab”. Thus, each antibody tetramer comprises two Fabs, one per each arm of the Y-shaped antibody.

The LC constant domain is comprised of one domain, CL. The human VJJ includes seven family members: Vjjl, VJJ2, VJJ3, VJJ4, VJJ5, VJJ6, andVjj7; and the human V_L includes 16 family members: V_K1, V_K2, V_K3, V_K4, V_K5, V_K6, V^l, V>2, V^3, V>4, V^5, V^6, V 7, V^8, V^9, and V^10. Each of these family members can be further divided into particular subtypes. The VJJ and VL can be further subdivided into regions of hypervariability, termed complementarity determining region (CDR) areas, interspersed with regions that are more conserved, termed framework regions (FR). Each VJJ and VL is composed of three CDR regions and four FR regions, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Numbering of the amino acids in a VH may be determined using the Kabat numbering scheme. See Beranger, et al., Ed. Ginetoux, Correspondence between the IMGT unique numbering for C-DOMAIN, the IMGT exon numbering, the Eu and Kab at numberings: Human IGHG, Created: 17/05/2001, Version: 08/06/2016, which is accessible atwww.imgt.org/IMGTScientificChart/Numbering/ Hu IGHGnb er . html) .

The constant regions of the antibodies 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 (Cl q) of the classical complement system. Typically, the numbering of the amino acids in the heavy chain constant domain begins with number 118, which is in accordance with the Eu numbering scheme. The Eu numbering scheme is based upon the amino acid sequence of human IgGl (Eu), which has a constant domain that begins at amino acid position 118 of the amino acid sequence of the IgGl described in Edelman et al., Proc. Natl. Acad. Sci. USA. 63 : 78-85 (1969), and is shown for the IgGl, IgG2, IgG3, and IgG4 constant domains in Beranger et al., op. cit.

As used herein, production by recombinant means by using recombinant DNA methods means the use of the well-known methods of molecular biology for expressing proteins encoded by cloned DNA.

As used herein, the term "Fc domain”, or “Fc” as used herein is the crystallizable fragment domain or region obtained from an antibody that comprises the CH2 and CH3 domains of an antibody. In an antibody, the two Fc domains are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. The Fc domain may be obtained by digesting an antibody with the protease papain. Typically, amino acids in the Fc domain are numbered according to the Eu numbering convention (See Edelmann et al., Biochem. 63 : 78-85 (1969)).

As used herein, the term "antigen" as used herein refers to any substance, or portion thereof, which induces an immune response in the body.

As used herein, the term “antigen binding fragment” refers to a polypeptide or polypeptides comprising a fragment of a full-length antibody, which retains the ability to specifically bind to the antigen bound by the full length antibody, and/or to compete with the full length antibody for specifically binding to the antigen. Examples of antigen binding fragments include but are not limited to Fab fragment, Fab’ fragment, F(ab’)2 fragment, Fv region, and scFv. As used herein, the term "Fab fragment" refers to an antigen binder comprising one antibody light chain and the CHI and VJJ of one antibody heavy chain. 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.

As used herein, the term "Fab¹ fragment" refers to an antigen binder comprising one antibody light chain and a portion or fragment of one antibody heavy chain that contains the Vjjand the CHI domain up to a region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

As used herein, the term "F(ab')2 fragment" refers to an antigen binder comprising two antibody light chains and two heavy chains containing the VJJ and the CHI domain up to a region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. An F(ab')2 fragment thus is composed of two Fab' fragments that are 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.

As used herein, the term "Fv region" refers to an antigen binder comprising the variable regions from both the heavy and light chains of an antibody but lacks the constant regions.

As used herein, the term “ScFv” or “single-chain variable fragment” refers to a fusion protein comprising a VJJ and VL fused or linked together by a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VJJ with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.

As used herein, the term "diabody" refers to an antigen binder comprising a small antibody fragment with two antigen-binding regions, which fragments comprise a heavy chain variable domain (VJJ) connected to a light chain variable domain (V ) in the same polypeptide chain (VJJ-VL or VL-VJJ) 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 complementarity domains of another chain and create two antigen-binding regions. Diabodies are described more fully in, e.g., 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 generally see Holliger and Hudson (2005) Nat. Biotechnol. 23 : 1126- 1136.

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

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

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

As used herein, the term "extracellular antigen binding domain," "extracellular domain," or "extracellular ligand binding domain" when used in reference to a CAR refers to the part of a CAR that is located outside of the cell membrane and is capable of binding to an antigen, target or ligand.

As used herein, the term "hinge region" when used in reference to a CAR refers to the part of a CAR that connects two adjacent domains of the CAR protein, e.g., the extracellular domain and the transmembrane domain.

As used herein, the term "transmembrane domain" refers to the portion of a CAR that extends across the cell membrane and anchors the CAR to cell membrane. As used herein, the term "intracellular T cell receptor-activating signaling domain", "cytoplasmic signaling domain," or "intracellular signaling domain" refers to the part of a CAR that is located inside of the cell membrane and is capable of transducing an effector signal.

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 the addition of extra genetic material in the form of DNA or RNA to the total genetic material of the cell. According to 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.

As used herein, the term "stimulatory molecule" refers to a molecule expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the T cell receptor (TCR) complex in a stimulatory way for at least some aspect of the T cell signaling pathway. Stimulatory molecules comprise two distinct classes of cytoplasmic signaling sequence, those that initiate antigen-dependent primary activation (referred to as "primary signaling domains"), and those that act in an antigen-independent manner to provide a secondary co-stimulatory signal (referred to as "co-stimulatory signaling domains").

As used herein, the term "immune cell" or "immune effector cell" refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid-derived phagocytes. According to particular embodiments, the engineered immune cells are T cells, and are referred to as CAR-T cells because they are engineered to express CARs comprising an ScFv that targets a human tumor; or the engineered immune cells are NK cells, and are referred to as CAR-NK cells because they are engineered to express CARs comprising an ScFv disclosed herein that targets a human tumor. In the present invention, said CAR-T and CAR-NK cells are conjugated to a HYAL1 mutant disclosed herein.

As used herein, the term "isolated” antibodies or antigen-binding fragments thereof are at least partially free of other biological molecules from the cells or cell cultures in which they are produced. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antibody or antigen-binding fragment may further be at least partially free of recombinant host cell components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term "isolated" is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or fragments.

As used herein, the term "monoclonal antibody" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprisingthe 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 multitude of different antibodies having different amino acid sequences in their variable domains that are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be madeby the hybridoma method first described by Kohler et al., Nature 256: 495 (1975) or may be madeby recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques describedin Clackson et al., Nature 352: 624-628 (1991), and Marks et al., J. Mol. Biol. 222: 581-597 (1991), for example. See also Presta, J. Allergy Clin. Immunol. 116: 731 (2005).

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

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

As used herein, the term “library” as used herein is, typically, a collection of related but diverse polynucleotides that are, in general, 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 and/or heavy chain immunoglobulins, which are diverse but related in their nucleotide sequence; for example, which immunoglobulins are functionally diverse in their abilities to form complexes with other immunoglobulins, and bind a particular antigen.

As used herein, the term “polynucleotides” discussed herein form part of the present invention. A "polynucleotide", "nucleic acid " or "nucleic acid molecule" include DNA and RNA, single- or double-stranded. Polynucleotides e.g., encoding a HYAL1 mutant of the present invention, may, in an embodiment of the invention, be flanked by natural regulatory (expression control) sequences, or may be associated with 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.

Polynucleotides e.g., encoding a HYAL1 mutant of the present invention, may be operably associated with a promoter. A “promoter” or “promoter sequence” is, in an embodiment of the invention, a DNA regulatory region capable of binding an RNA polymerase in a cell (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. A promoter sequence is, in general, bounded at its 3 ' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at any level. Within the promoter sequence maybe found a transcription initiation site (conveniently defined, for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. The promoter may be operably associated with other expression control sequences, including enhancer and repressor sequences or with a nucleic acid of the invention. Promoters which may be used to control gene expression include, butare not limited to, cytomegalovirus (CMV) promoter (U.S. PatentNos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist, etal., 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 etal., 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 P- lactamase promoter (Villa-Komaroff etal., Proc. Natl. Acad. Sci. USA 75: 3727-3731 (1978)), or the tac promoter (DeBoer etal., 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, PGK (phosphoglycerol kinase) promoter or the alkaline phosphatase promoter.

As used herein, the terms "vector", "cloning vector" and "expression vector" include a vehicle (e.g., a plasmid) by which a DNA orRNA sequence can be introduced into a host cell so as to transform the host and, optionally, promote expression and/or replication of the introduced sequence. Polynucleotides encoding a HYAL1 mutant of the present invention may, in an embodiment of the invention, be in a vector.

As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that not all progeny will have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

As used herein, the term "control sequences" or “regulatory sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for expression in eukaryotes, for example, include a promoter, operator or enhancer sequences, transcription termination sequences, and polyadenylation sequences for expression of a messenger RNA encoding a protein and a ribosome binding site for facilitating translation of the messenger RNA. As used herein, a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence, e.g., a regulatory sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates 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 so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

As used herein, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

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

As used herein, the term "treat" or "treating" means to administer a therapeutic agent, such as a composition containing any of the human HYAL1 mutants of the present invention, topically, subcutaneously, intramuscularly, intradermally, or systemically to an individual in need. The amount of a therapeutic agent that is effective to treat a disease or disorder, including in specific embodiments, a cancer or proliferative disease, in the individual may vary according to factors such as the disease or disorder state, age, and/or weight of the individual, and the ability of the therapeutic agent to elicit a desired response in the individual. Whether the therapeutic objective has been achieved can be assessed by the individual and/or any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of the treatment. Thus, the terms denote that a beneficial result has been or will be conferred on a human or animal individual in need.

As used herein, the term "treatment," as it applies to a human or veterinary individual, refers to therapeutic treatment, as well as diagnostic applications. "Treatment" as it applies to a human or veterinary individual, encompasses contact of the HYAL1 mutant or a composition of the present invention to a human or animal subject.

As used herein, the term “therapeutically effective amount” refers to a quantity of a specific substance sufficient to achieve a desired effect in an individual being treated. For instance, this may be the amount necessary to inhibit or reduce the severity of a disease or disorder in an individual.

As used herein, the term “disease” or “disorder” refers to a pathological condition in an organism resulting from, e.g., infection, dysfunction, cancer, or genetic defect, and characterized by identifiable symptoms.

Human hyaluronidase 1 (HYAL1) mutants

The present invention provides human hyaluronidase 1 (HYAL1) mutants, which have been evolved through iterative rounds of protein engineering to have greatly improved hyaluronidase activity and activity within an expanded pH range compared to wild-type HYAL1 . Wild-type HYAL1 has maximal activity at pH 3.5 and little to no detectable activity at pH 5.5, the HYAL1 mutants of the present invention have high activity in the pH range 5-6 with little to no activity above pH 7, which distinguishes the HYAL1 mutants from other commercial hyaluronidases. In addition, the HYAL1 mutants have improved expression yields relative to wildtype HYAL1.

The HYAL1 mutants of the present invention are useful for increasing the absorption of therapeutic agents into tissue and to reduce tissue damage in cases of extravasation of a therapeutic agent. The HYAL1 mutants of the present invention are highly active at pH 5.5 (formulation pH) and inactive at pH 7.4 (skin pH), thus, providing a switchably active, hyaluronidase. The HYAL1 mutants have a narrower spreading range compared to the FDA approved hyaluronidase product HYLENEX. Following introducing a HYAL1 mutant in a composition comprising a pH 5.3 or 5.5 buffer into the skin, the enzyme dissipates in skin and the local environment increases to the normal pH of skin at pH 7.0 or 7.2 at which the activity falls. The HYAL1 mutants of the present invention provide a hyaluronidase with a pH controlled activity profile in skin, which reduces the duration of active hyaluronidase in the skin following the dispersion of a co-administered therapeutic agent through the skin, thus, reducing the unwanted side effects of persistent hyaluronidase activity following dispersion of the therapeutic agent.

Thus, 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 set forth in SEQ ID NO: 2, wherein the hyaluronidase 1 mutant further comprises a combination of amino acid substitutions selected from the group consisting of : (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, andV412R; (c) amino acid substitutions D68N, M246L, S330K, L347V, H262N, D290T, R326S, D276K, F20D, A182E, G235N, P23V, S304T, L379M, W433E, R194D, D299E, V412RR238C, I287P, F345T, andL377S; (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, P23 V, S304T, L379M, W433E, R194D, D299E, V412R, N235D, I287P, F345T, and L377S; and wherein the hyaluronidase 1 mutant (i) comprises at least one A-gly cosylation site comprising the sequence asparagine-X-serine/threonine, wherein X is any amino acid other than proline, that is glycosylated and (ii) displays hyaluronidase activity from pH 3.5 to pH 5.5.

In particular 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. In particular 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.

In further embodiments, at least one A-glycosylation site created by the D68N, D323N, G235N, orR326S substitution is glycosylated. In further embodiments, the N- glycosylation sites created by the D68N, D323N, G235N, andR326S substitutions are each glycosylated. In further embodiments, the native A-glycosylation sites and the A-glycosylation sites created by the D68N, D323N, G235N, andR326S substitutions are each glycosylated.

The present invention further provides a HYAL1 mutant comprising an amino acid sequence with at least 95% (and in specific embodiments, 96%, 97%, 98%, 99% and 100%) identity to the amino acid sequence setforth 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) comprises at least one A-glycosylation site comprising the sequence asparagine-X- serine/threonine, wherein X is any amino acid other than proline, that is glycosylated and (ii) displays hyaluronidase activity from pH 3.5 to pH 5.5.

The present invention further provides a HYAL1 mutant comprising an amino acid sequence with at least 95% (and in specific embodiments, 96%, 97%, 98%, 99% and 100%) identity to the amino acid sequence setforthin set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, wherein the hyaluronidase 1 mutant (i) comprises at least one A-glycosylation site comprising the sequence asparagine-X-serine/threonine, wherein X is any amino acid other than proline, that is glycosylated and (ii) displays hyaluronidase activity from pH 3.5 to pH 5.5.

The present invention further provides a HYAL1 mutant disclosed herein and a pharmaceutically acceptable carrier. In a further embodiment, the composition further comprises a therapeutic agent, thus in particular 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, a small molecule, a peptide, a macrocyclic peptide, a protein, or a protein complex. In particular embodiments, the small molecule comprises an antibiotic or an anti-inflammatory agent. In particular embodiments, the peptide comprises an insulinotropic peptide, a growth hormone, insulin, or an insulin mutant. In particular embodiments, the therapeutic agent comprises an antibody, an antigen-binding protein, an scFv, Fab, or a fusion protein comprising the Fc domain of an antibody.

The present invention further provides a method for increasing the diffusion of a therapeutic agent in a tissue of a subject, comprising administering to the tissue of a subject a composition comprising a HYAL1 mutant disclosed herein in an amount sufficient to increase 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 consecutively or concurrently to the subject.

The present invention further provides for the use of a HYAL1 mutant disclosed herein for the manufacture of a medicament comprising the hyaluronidase and a therapeutic agent for treatment of a disease or disorder. The present invention further provides for the use of a HYAL1 mutant disclosed herein for increasing the diffusion of a therapeutic agent in a tissue of a subject for the treatment of a disease or disorder.

The present invention further provides a nucleic acid molecule encoding a HYAL1 mutant disclosed herein. In a particular embodiment, the HYAL1 mutant encoded by the nucleic acid molecule comprises an amino acid sequence with at least 95% (and in specific embodiments, 96%, 97%, 98%, 99% and 100%) identity to the amino acid sequence set forth 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.

The present invention further provides a nucleic acid molecule encoding a HYAL1 mutant disclosed herein fused to a signal sequence that targets the HYAL1 mutant to the endoplasmic reticulum. In particular embodiments, the nucleic acid molecule comprises amino acids 1 to 433 of the amino acid sequence setforth 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.

The present invention further provides an expression vector comprising a nucleic acid molecule encoding a HYAL1 mutant disclosed herein that, in specific embodiments, is fused to a signal sequence that targets the HYAL1 mutant to the endoplasmic reticulum.

The present invention further provides a host cell comprising a nucleic acid molecule encoding a HYAL1 mutant disclosed herein that, in specific embodiments, is fused to a signal sequence that targets 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 a HYAL1 mutant disclosed herein that, in specific embodiments, is fused to a signal sequence that targets 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 A-gly cans.

In a further embodiment, the mammalian host cell is a Chinese hamster ovary cell and the recombinant yeast host cell is Pichia pastoris. The present invention further provides a method for producing a HYAL1 mutant disclosed herein, comprising: (a) introducing a nucleic acid molecule encoding the HYAL1 mutant, or an expression vector disclosed herein comprising a nucleic acid molecule encoding the HYAL1 mutant, into a host cell to produce a recombinant host cell; (b) cultivating the recombinant host cell in a culture medium under conditions sufficient for the recombinant host cell to express and secrete the HYAL1 mutant into the culture medium; and (c) obtaining the HYAL1 mutant from the culture medium.

In a further embodiment, the host cell is a mammalian host cell, which in a further embodiment may be a Chinese hamster ovary cell. In a further embodiment, the host cell is a recombinant yeast host cell modified to produce complex A-gly cans, which in a further embodiment may be the recombinant yeast host cell is Pichia pastoris.

In a further embodiment of the method, the HYAL1 mutant comprises amino acids 1 to 433 of the amino acid sequence setforth 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 comprise a signal sequence (amino acids 1-19) that targets the HYAL1 mutant to the endoplasmic reticulum.

TV-linked glycosylation of hyaluronidases (such as the HYAL1 mutants disclosed herein) can be very important for their catalytic activity and stability. While altering the type of glycan modifying a mutant can have dramatic effects on a protein's antigenicity, structural folding, solubility, and stability, most enzymes are not thought to require glycosylation for optimal enzyme activity. Hyaluronidases are thus unique in this regard, in that removal of N- linked glycosylation can result in near complete inactivation of the hyaluronidase activity. For such hyaluronidases, the presence of TV-linked glycans is critical for generating an active enzyme. In one aspect of the invention, complex glycosylation capped with sialic acid residues is described whereas others capped with galactose, GlcNAc, or mannose residues are contemplated as well. In particular embodiments, the HYAL1 mutants disclosed herein comprise an TV-glycan selected from A-glycans set forth in Table 1. In certain embodiments, the HYAL1 mutant comprises an TV-glycan comprising terminal sialic acid residues. The present invention further provides compositions in which the predominant TV-gly can is selected from the TV-glycans set forth in Table 1. In certain embodiments, the predominant TV-gly can is an TV-glycan comprising terminal sialic acid residues. In particular embodiments, the HYAL1 mutant comprises three wild-type N-glycan sites and mutations that generate at least one non-native A-glycosylation site. In particular embodiments, the HYAL1 mutant comprises three wild-type A-glycan sites and mutations that generate two or three non-native TV-gly cosylation sites. In particular embodiments, the HYAL1 mutant comprises at least five A-gly cans. In particular embodiments, the HYAL1 mutant comprises at least six A-glycans.

Formulations of the hyaluronidase 1 mutants are also provided. The HYAL1 mutants may be formulated in lyophilized forms and stabilized solutions for example. Formulations containing specific metal ions, such as calcium, magnesium, or sodium, are useful for optimal activity at neutral pH. In addition to stabilized solution formulations, slow-release formulations are contemplated herein for extended removal of glycosaminoglycans or extended promotion of spreading or diffusion of agents such as pharmacologies. Also provided herein are kits providing for pre-packaged syringes of HYAL1 mutants for the administration of small volumes of the HYAL1 mutants for intraocular surgical procedures and other small volume procedures. Balanced salt formulations for ex vivo use in artificial reproductive technology procedures are also provided.

Methods for the use of the HYAL1 mutants in the removal of glycosaminoglycans are also provided. Hyaluronidases including the HYAL1 mutants open channels in the interstitial space through degradation of glycosaminoglycans that generally permit the diffusion of molecules less than about 500 nm in size. These channels can remain relatively open for a period of 24-48 hours depending on the dose and formulation. 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 and other molecules less than about 500 nm in size. In addition, without being restricted to a particular theory or mechanism of action, it is believed that the formation of such channels can facilitate bulk fluid flow within an interstitial space, which can in turn promote the dispersion or movement of a solute (such as a detectable molecule or other diagnostic agent, an anesthetic or other tissue-modifying agent, a pharmacologic or pharmaceutically effective agent, or a cosmetic or other esthetic agent) that is effectively carried by the fluid in a process sometimes referred to herein as “convective transport” or simply convection. Such convective transport can substantially exceed the rate and cumulative effects of molecular diffusion and can thus cause the therapeutic or other administered molecule to more rapidly and effectively perfuse a tissue. Furthermore, when a molecule such as a therapeutic or other agent (such as a small molecule drug or a larger molecule or complex) is co-formulated or co-administered with a HYAL1 mutant 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 into or around other internal tissues, organs or other relatively confined spaces within the body), then the fluid associated with the administered dose can both provide a local driving force (i.e. hydrostatic pressure) as well as lower impedance to flow (by opening channels within the interstitial matrix) — both of which would tend to increase fluid flow, and with it convective transport of the therapeutic agent or other molecule contained within the fluid. As described and illustrated in more detail herein, and as will be appreciated by those of skill in the art, these aspects of the use of the HYALl mutants can have substantial utility for improving the bioavailability as well as manipulating other pharmacokinetic and/or pharmacodynamic characteristics of co-formulated or co-administered agents.

In specific embodiments, a single short acting dose is preferable. Temporary removal of glycosaminoglycans can be used to enhance the delivery of solutions and drugs into and/or through interstitial spaces. This can be useful for the diffusion of anesthesia and for the administration of therapeutic fluids, molecules and proteins. Subcutaneous, intradermal and intramuscular administration of molecules in the presence of HYAL1 mutants (and/or other glycosaminogly canases) also facilitate their systemic distribution more rapidly. Such methods are very useful when intravenous access is not available or where more rapid systemic delivery of molecules is needed. By way of illustration, delivery of other large molecules such as Factor VIII, that are poorly bioavailable upon subcutaneous administration, may be injected with HYAL1 mutants to increase their availability.

In the context of non-intravenous parenteral injections (such as intradermal, subcutaneous, intramuscular and other injections into spaces other than the vasculature), a HYAL1 mutant and another agent (e.g. a co-formulation or a mixture comprising a HYAL1 mutant and another agent such as a diagnostic agent, an anesthetic agent, a pharmacologic agent, an esthetic agent, or combinations thereof) in a volume of liquid (e.g. a pharmaceutical excipient or other solution) can be introduced into a site or sites within the body by injection or infusion. Without wishing to be bound by theory, it is believed that several forces can be brought into motion to enhance delivery of the pharmacologic or other agent (the extent of which depends in part on the particular composition, volume and site of administration for example). These driving forces can include an increase in hydrostatic pressure as a volume of fluid is effectively forced into a contained space (such as the sub-Tenon's space, or a site of intradermal, subcutaneous, intramuscular or other non-IV parenteral injection), a subsequent increase in convective transport of solutes (or convection) as fluid flow is increased down its pressure gradient (and dissolved molecules or macromolecular complexes are carried with it), as well as an increase in diffusion and/or permeation mediated by degradation of glycosaminoglycans and concomitant channel formation within the downstream intercellular matrix or interstitial space.

As will be appreciated by those of skill in the art, HYAL1 mutants can thus be used to effectively promote delivery of a number of anesthetics, diagnostics, pharmacologies and/or other agents to the posterior segments of the eye for treating conditions such as retinal detachments, retinal vein occlusions, proliferative retinopathies, diabetic retinopathies, inflammatory conditions (such as uveitis, choroiditis, retinitis and the like), as well as degenerative diseases, vascular diseases and various tumors. Again as will be appreciated by those of skill in the art, a variety of pharmacologic or pharmaceutically effective agents can be usefully applied to treating such posterior segment conditions and diseases, including, by way of illustration, anesthetic and pharmacologic agents such as those described and illustrated below.

Co-formulations or co-administrations of a HYAL1 mutant with other substances may also be envisioned for injectable pens for small volume or rapid subcutaneous administration. Examples such as Epipen™, insulin, and other fluids can be formulated. The methods of the invention include administration of the HYAL1 mutant or pharmaceutical compositions containing the HYAL1 mutant prior to, simultaneously with or following 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 a site the same as the site of administration of the therapeutic molecule.

Without wishing to be bound by theory, it is believed that the ability of HYAL1 mutants to cause the degradation of a portion of the glycosaminoglycans in the interstitial spaces between cells results in a temporary opening up of channels within the interstitium, which in turn tends to increase interstitial fluid flow and to concomitantly facilitate the diffusion and/or convective solute transport (convection) of dissolved components within the interstitial fluid (such as anesthetics, drugs and other pharmacologic agents, labels and diagnostic agents, and the like). As will be appreciated by those of skill in the art, HYAL1 mutants of the present invention can be applied to enhance the bioavailability (and potentially improve other pharmacokinetic and/or pharmacodynamic properties) of a number of pharmacologic and other agents that are useful for treating or diagnosing various disease conditions or otherwise modifying one or more tissues in vivo. Illustrative categories of such therapeutic agents include: anti-cancer agents, anti- infectives, anesthetics, anti-inflammatories, cytokines, antibodies and other proteins, nucleic acids, macromolecular complexes, and numerous other molecules and pharmacologic agents which modify cellular or other physiologic activities, including various categories of agents (and exemplary members thereof) described herein and in the art.

While the diffusion and convective transport of even small molecules can be enhanced by opening interstitial channels and increasing fluid flow, in the case of larger pharmacologic or other agents, such as many biotherapeutics (including antibodies and other proteins, large nucleic acids, macromolecular complexes (such as liposomes and other macromolecular carriers), as well as gene therapy vectors and the like)), the size of the molecules and the presence of interstitial components such as glycosaminoglycans substantially impairs the diffusion and/or convection of the agents. Several consequences potentially limiting the agent's usefulness can result. For example, the pharmacokinetics of the agent can be effectively impaired by a slowing of absorption and thus distribution of the agent. In addition, trapping of a portion of the agent at or near the site of administration both limits its bioavailability and can also cause toxicity as a result of a potentially sustained and high local dose. In the latter regard, local toxicity that may be associated with painful or other side effects is a problem with many large biomolecules that are administered by non-intravenous injection, such as by subcutaneous, intradermal or intramuscualar injection. As a result, a number of pharmacologic agents have pharacokinetic (PK) and/or pharmacodynamic (PD) profiles that can be enhanced by coformulating the agent with a HYALl mutant and/or co-administering the agent with a HYALl mutant, which may be provided before, coincident with or after the agent, and administered at the same or a different site, which parameters would be the subject of optimization in standard models (such as animal models typically used to assess the pharmacokinetics and pharmacodynamics of the agent). Any of a variety of therapeutics and pharmacologies as well as other agents (such as cosmetic or esthetic formulations) that are typically administered by parenteral administration can thus be enhanced using HYAL1 mutants. Among non-IV parenterals, HYAL1 mutants can also be used to allow the agents to be administered by more convenient routes, and/or with greater efficiency. By way of illustration (and without limitation), by reformulating and/or co-administering agents with a HYAL1 mutant (either local or systemic and either before, coincident with or after administration of the agent) agents that are typically administered by subcutaneous injection can instead be administered by intradermal injection, and agents that are typically administered by intramuscular injection can instead be administered by subcutaneous or intradermal injection. Alternatively, agents can be administered by the same route, but with improved pharmacokinetics and/or pharmacodynamics using HYAL1 mutants.

The use of HYAL1 mutants to reformulate IV drugs and other agents as non-IV parenterals also enables delivery of the agents using any of a variety of new injection devices designed to ease and/or speed delivery, and to facilitate self-administration. Such devices include, for example, ultra-sharp and microneedle devices (such as those being developed by Becton Dickinson and others) as well as needle-free injection devices (such as Biojector™ and other devices available from Bioject; IntraJect™ and other devices available from Aradigm; Medijector™ devices and the like). A number of devices (such as the Biojector) are particularly useful for facilitating intradermal injections which tend to require more experience when using standard needles (due to the potential for penetrating the dermis during needle placement, and delivering the agent to an underlying tissue site such as the subcutaneous layer). For some pharmacologic agents, such as vaccines for example (e.g. a DNA-based or other vaccine), it can be particularly advantageous to deliver the agent into the dermal as opposed to sub-dermal layers since there tends to be a relatively high concentration of antigen-presenting cells (APCs) localized within the dermis.

In the context of those agents that are typically delivered by intradermal injection (whether by standard needles or other newer devices), or those many other agents which are not currently but could be delivered by intradermal injection, the local co-introduction of a HYAL1 mutant can be used to enhance delivery of the pharmacologic or other agent delivered and to promote its dispersal or spreading within the dermis. In the case of a vaccine, where the dermis may be the primary target tissue, given the local abundance of APCs, the HYAL1 mutant can thus facilitate dispersal of the vaccine within the target tissue, thereby increasing the probability and extent of interactions between a vaccine and APCs (which can significantly potentiate the generation of an immunomodulatory response). In the case of other agents, the dermis may not be the primary target tissue but is rather a tissue into which a dose of the agent is introduced from which it is desired that the agent be absorbed into another tissue, typically the bloodstream. In the latter case, the bloodstream may itselfbe the target tissue of interest (e.g. for blood- modulatory factors as described herein and in the art), or the bloodstream may itself be a delivery tissue by which the agent is transported to a distal target tissue (e.g. a tissue in the body supplied by the bloodstream). In either of these latter cases (in which delivery is intradermal but it is desired that the agent be delivered to the bloodstream), the HYAL1 mutant (and potentially a volume of liquid in which it is injected) can facilitate dispersal of the agent first within the dermis and thence into the vasculature draining the dermis and eventually into the larger blood supply), as described and illustrated herein.

The use of HYAL1 mutants to enhance the pharmacokinetics and/or pharmacodynamics of other therapeutic or pharmacologic agents can also be applied to agents delivered by routes other than non-IV parenteral administration. For example, without wishing to be bound by theory, it is believed that the presence of HYAL1 mutants in interstitial spaces within the body (which can be achieved by local and/or systemic administration of HYAL1 mutants) tends to promote interstitial channels and an increase in fluid flow within the interstitial space that in turn facilitates both diffusion and convective transport of agents dissolved with the interstitial fluid (such as pharmacologic and other agents). By way of illustration, agents that are administered directly into the bloodstream (e.g. by intravenous injection), or orally (and thus enter the bloodstream after uptake from the gastrointestinal tract for example), can still be subject to constraints in the post-absorption i.e. distribution phase of their pharmacokinetics. Without wishing to be bound by theory, it is believed thatHYALl 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 space (which effectively forms the intermedium between almost all pharmacologic delivery routes and the target cells).

In addition, it is believed that changes in oncotic pressure brought about by the administration of HYAL1 mutants can contribute to enhancing the delivery of pharmacologic agents. Again, without wishing to be bound by theory, the presence of a HYAL1 mutant and concomitant degradation along the interstitial side of a vascularized tissue, would tend to decrease the oncotic pressure within the interstitial space which in turn would enhance fluid filtration from the vasculature into the interstitial space.

The potential to decrease interstitial pressure is considered to be particularly important in the context of many cancers in which interstitial pressure within the tumor (tumor interstitial pressure of tumor interstitial fluid (TIF)) is elevated. High TIF can result in relative impedance to fluid flow from the vasculature toward the center of a tumor, thereby limiting the amount of an anti-cancer agent that effectively reaches a tumor, particularly sites that are deeper within a tumor mass. The introduction of HYAL1 mutants to tumor interstitia would thus tend to enhance the delivery of locally delivered as well as systemically available anti-cancer agents which can more readily penetrate the tumor when interstitial oncotic pressure is reduced and diffusion and/or convective transport increased. Measurements of TIF and hydraulic conductivity (K) within tumors (e.g. using tagged molecules such as albumin labeled with Evan's Blue dye) can be used to quantitatively assess the effect of various concentrations of HYAL1 mutants on the fluid dynamics of tumors in vivo, as illustrated herein and/or in the art.

Further exacerbating the reduced fluid dynamics within many tumors, and highlighting an additional potential benefit of applying HYAL1 mutants to tumors, is the fact that many tumors exhibit accumulation of glycosaminoglycans, particularly hyaluronan (which may be due to the fact that lymphatics, which are the predominant route for hyaluronan catabolism, are impaired or lacking in many tumors). Such excess hyaluronan can contribute to impeding hydraulic conductivity. The introduction of HYAL1 mutants can thus be used to counteract the accumulation of glycosaminoglycans in many tumors, improving hydraulic conductivity within the tumor and effectively rendering them more susceptible to anti-tumor agents (whether locally or systemically delivered).

As will be appreciated by those of skill in the art, the principles described herein can also be applied to numerous other pharmacologies and other agents that are desired to be delivered to sites within the body, such as for the prevention, diagnosis and/or treatment of disease or to otherwise modulate physiological functions.

In addition to their use in potential improvements and/or reformulations of a variety of parenterally-administered pharmacologies and/or agents, HYAL1 mutants can also be usefully employedin connection with non -parenteral agents (such as agents formulated as pills, liquids or other forms for ingestion and typical 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 diffusion and/or convective transport within the interstitium (either systemically or locally (e.g., by local or targeted administration of a HYALl mutant)) can be applied to improve the extent and/or rate at which non-parenterals (as well as parenterals) reach desired target cells.

In addition, a number of agents that are typically administered non-paren terally (e.g. orally) may be reformulated, or their active ingredients reformulated, for use in parenteral administrations that are rendered more effective and/or safe in combination with HYAL1 mutants. To illustrate this broadly applicable approach, the ability of HYAL1 mutants to facilitate targeted delivery to particular tissues (such as the ability of HYAL1 mutants to provide for transdermal delivery to tissues within the posterior of the eye) can be used to deliver any of a variety of agents (including agents previously administered systemically) directly and preferentially to a localized site of interest within the body. This can not only provide for more desirable and/or more rapidly achieved concentrations at the site of interest, but can substantially reduce potential problems and limitations associated with systemic administration in which concentrations at undesired sites may equal or even exceed concentrations at the desired target site (potentially triggering undesired side effects as well as wasting agent). In some cases, agents that are not widely used or are not used for certain indications or in certain patients, for example, may be effectively applied to benefit additional patients by being co-formulated or coadministered with HYAL1 mutants as described and illustrated herein.

As will be appreciated by those of skill in the art, the ability to employ HYAL1 mutants to enhance, speed and/or target biodistribution of co-formulated and/or co-administered agents, and to manipulate other aspects of their pharmacokinetics or pharmacodynamics (e.g. in order to improve their risk: benefit profile or facilitate their use by patients, family or health care professionals), provides a major opportunity for improving drugs and other agents that are used treat, diagnose or prevent diseases.

Without being limited to a particular set of applications, HYAL1 mutants as described herein can be used to effectively achieve bolus or bolus-like delivery of any number of pharmacologic and other agents by non-intravenous parenteral routes, as well as by other routes of administration (e.g. by enhancing the delivery of agents into and/or through a target tissue after they leave the bloodstream (whether they were introduced into the bloodstream directly (such as by IV administration) or indirectly (such as by oral or non-IV parenteral administration)).

Without being restricted to a specific mechanism of action or aspect thereof, it is believed that the ability of these enzymes to temporarily degrade components of the interstitial matrix between cells (which accounts for a substantial portion of the fluid space in the body and a correspondingly large space that must be traversedin order for pharmacologies and other agents to reach most target cells) can significantly promote the delivery of agents to target cells by one or more of several potentially synergistic means.

First, while the bulk of the interstitial fluid exhibits some degree of flow, that flow is often restrained or impeded by glycosaminoglycans such as hyaluronan and other interstitial components. The ability of HYAL1 mutants to open channels within such interstitial spaces, as described and illustrated herein, can be used to decrease impedance and increase the extent and rate of “downstream” flow resulting from any given “upstream” pressure.

Second, in the case of non-IV parenteral injections of HYAL1 mutants and another pharmacologic or other agent, the volume of the non-IV parenteral injection can be used to increase the upstream driving pressure or pressure head (e.g. by increasing the hydrostatic pressure within a confined space), which can further promote flow.

Third, since the volume of fluid comprising the introduced HY AL 1 mutant and other therapeutic agent is effectively driven down the increased pressure gradient (i.e. the increased gradient created by elevating the hydrostatic pressure associated within the injected “bolus” and simultaneously decreasing the interstitial pressure within the surrounding tissue), solutes within the bolus can be effectively carried (e.g. by convective transport) into the adjacent tissue. This injected fluid or bolus can thusbe efficiently and relatively quickly moved into the adjacent tissue and deliver whatever pharmacologic or other agent was introduced at or near the same site of introduction (e.g. by co-formulating or co-administering the agent in combination with the HY AL 1 mutant).

Methods of Evolving HYAL1

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

The HYAL1 mutants disclosed herein may be obtained by subjecting the polynucleotide encoding the parent sequence to mutagenesis and/or directed evolution methods. An exemplary directed evolution technique is 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; WO 98/27230; WO 00/42651; WO 01/75767 andU.S. Pat. No. 6,537,746. Other directed evolution procedures that can be used include, among others, staggered extension process (StEP), in vitro recombination (Zhao etal., 1998, Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell et al, 1994, PCR Methods Appl. 3 :S136-S140), and cassette mutagenesis (Black etal, 1996, Proc. Natl. Acad. Sci. USA 93 :3525-3529).

The clones obtained following mutagenesis treatment are screened for HY AL 1 mutants having the desired improved enzyme property. Wild-type HYAL1 are only active at very acidic pHs with no activity at a pH greater than pH 4.5 and the expression level of wild-type human HYAL1 is not high enough for large scale manufacturing. Furthermore, evolution of the HYAL1 may introduce an immunogenicity risk requiring the HYAL1 mutants to be measured for immunogenicity risk. The desired improved property of the HYAL1 mutants is (i) active at pH > 5.5, (ii) have a specific activity greater than 30,000 units/mg, (iii) stable activity (greaterthan 80%) for 12-30 months at 2-8 °C, and (iv) low immunogenicity risk.

Measuring enzyme activity from the expression libraries may be performed at different pHs over time using a turbidity assay or an ELISA activity assay. Relative expression of the HYAL1 mutants may be determined by expressing the HYAL1 mutants as fusion proteins having a C-terminal His-6 tail (SEQ ID NO: 46) and measuring expression levels using a His-tag Homogeneous Time Resolved Fluorescence (HTRF) Expression Assay. Immunogenicity risk may be determined by using a computer program such as the ExpiVax software to scan the regions of the HYAL1 mutants comprising the mutations. The computer program provides an epitope score for the scanned regions that can be used to assess the immunogenicity of each possible 9-mer peptide in the scanned regions. Clones containing a polynucleotide encoding a HYAL1 mutant are then isolated, sequenced to identify the nucleotide sequence changes (if any), and used to express the enzyme in a host cell.

Where the sequence of the polypeptide is known, the polynucleotides encoding the enzyme can be prepared by standard solid-phase methods, according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be individually synthesized, thenjoined (e.g., by enzymatic or chemical litigation methods, or polymerase mediated methods) to form any desired continuous sequence. For example, polynucleotides and oligonucleotides of the invention can be prepared by chemical synthesis using, e.g., the classical phosphoramidite method described by Beaucage etal, 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 synthetic methods. According to the phosphoramidite method, oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors. In addition, essentially 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., ExpressGenlnc. Chicago, Ill., Operon Technologies Inc., Alameda, Calif., and many others.

HYAL1 mutants expressed in a host cell may be recovered from the cells and or the culture medium using any one or more of the well-known techniques for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting-out, ultracentrifugation, and chromatography.

Chromatographic techniques for isolation of the HYAL1 mutants include, among others, reverse phase chromatography high performance liquid chromatography (RP-HPLC), ion exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art.

In some embodiments, affinity techniques may be used to isolate the improved HYAL1 mutants. For affinity chromatography purification, the protein sequence can be tagged with a recognition sequence to enable 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 others that will be apparent to those having skill in the art. Antibodies may also be used as affinity purification reagents. Any antibody that specifically binds the HYAL1 mutant may be used.

All the experiments in this work have been reproduced at least 2 times (N=3). Consistent results were obtained. Statistical analyses were performed using GraphPad Prism 8 with all the collected data points included.

GENERAL METHODS

HYAL1 Transient Transfection andExpression

Expi-CHO-S cells (Gibco, Cat# A29127) were cultured according to manufacturer guidelines. Two days prior to transfection, cells were seeded to 1.5 x 10^ cells/mL in Expi-CHO Expression Medium (Gibco, Cat# A2910003) in non-baffled 250 mL shake flasks (Thermo Scientific, Cat# 4115-0125). On the day of transfection, cells were counted using a Countess II Cell Counter and then diluted to 6 x 10^ cells/mL in fresh ExpiCHO Expression Media. Cells were then transferred to the inner 60 wells of 96-well culture plates (Corning, Cat# PDW20CS) at a volume of 800 pL per well. The outermost wells of each plate were filled with 800 pL of sterile phosphate-buffered saline (PBS).

Transfections were carried out using ExpiCHO-S Transfection Kits (Gibco, Cat# A29133) according to the manufacturer protocol. DNA vectors comprising an open reading frame encoding amino acids 23-433 ofHYALl or mutant thereof linked at the N-terminus to a non-native signal peptide comprising amino acid sequence of SEQ ID NO: 25 and linked at the C-terminus to TG-His6 peptide (SEQ ID NO: 26), OptiMEM, and Expif ectamine were combined in separate 96-well plates and then transferred to the cell culture plates. Cultures were sealed with gas-permeable membranes (E&K Scientific, Cat# T896100-S) and incubated at 37 °C, 8% CO₂ with shaking at 900 revolutions per minute (rpm). On the day following the transfection, ExpiCHO-S Enhancer- 1 and Enhancer-2 were added according to the manufacturer protocol ata total volume of 200 pL per well. On the 6^th day after transfection, cells were harvested via centrifugation at 2000 x g for 10 minutes. Supernatants were then transferred to fresh plates and stored at 4 °C until assayed for Hyal-1 activity and expression level.

HYAL1 Activity Screen HYAL1 and mutants thereof relative activities were measured using a turbidity assay. Cell culture supernatants containing H Y AL 1 variants were diluted between2X- 500X into reaction buffer (20 mM Sodium Phosphate, 77 mMNaCl, 0.01%BSA, pH 4.0, 4.5, 5.0, 5.4, 5.5, 6.0, 6.5, or 7.5). Reactions were then carried out in a 96-well plate (Biorad, Cat# HSP9601) by mixing 25 pL of each Hyal-1 sample with 25 pL of 0.3 mg/mL hyaluronic acid (Sigma Aldrich, Cat# H7630) (pH adjusted to match reaction buffer) and incubating the sealed plates at 37 °C for the duration of the reaction (between 15 minutes and 18 hours depending on pH and evolution round). Reactions were then quenched by heating the plates on a PCR thermocycler at 95 °C for 5 minutes. After quenching, 30 pL of each sample was mixed with 150 pL of an acidic albumin solution (24 mMNaOAc, 79 mMHOAc, 0.1%BSA, pH 3.75) in clear 96-well plates. Plates were incubated on the benchtop for 5 minutes and then turbidity was measured at 600 nm on a Spectramax i3x.

HYAL1 His-tag HTRF Expression Assay

HYAL1 relative expression levels were measured using a His-tag based detection kit (Cisbio, Cat# 64HISPEH). Cell culture supernatants containing Hyal-1 variants were diluted between IX - 200X in the kit assay buffer, and 10 pL of each sample was transferred to a white ’A-area 96-well plate (Corning, Cat# 3693). Followingthe kit manufacturer protocol, 5 pL of XL665 was added followed by 5 pL of the Gold Eu conjugate (samples were mixed via pipetting with each reagent addition. Plates were then sealed with foil plate seals and incubated at room temperature for 2 hours on an orbital shaker (250 rpm). HTRF measurements were then collected according to the vendor protocol using a PerkinElmer EnVision HRTF plate reader.

HYAL1 recombinant expression in ExpiCHO-S cells

HYAL1 mutants were expressed recombinantly in ExpiCHO-S™ cells (Thermo Fisher Scientific) based on manufacturer recommendations. Briefly, 0.2-pm filter sterilized plasmid DNA encoding each HYAL1 mutant was acquired at a maxi- or giga-prep scale and sequence verified by Sanger sequencing from Elim Biopharm (Hayward, CA). DNA complexes were formed first by mixing 160 pg plasmid DNA with 8 mL cold OptiPRO™ SFM (Thermo Fisher Scientific) and mixed gently inverting. Cold ExpiFectamine™ CHO Reagent (Thermo Fisher Scientific; 640 pL) was added to 7.4 mL cold OptiPRO™ SFM and gently inverted. Next, the diluted ExpiFectamine™ CHO Reagent was added to the diluted plasmid DNA and after 1 minute this mixture was added to 200 mL ExpiCHO-S cell cultures diluted to 6 x 10⁶ cells/mL. Cultures were placed in a shaking incubator (Infors HT Multitron) at 37 °C, 8 % CO2, 80% humidity, shaking at 120 rpm. The next day, 48 mL ExpiCHO™ Feed and 1.2 mL ExpiFectamine™ CHO Enhancer were added to each culture and incubated for an additional 6 days.

On day 7, cultures were pelleted at 500 x g and culture supernatants were filtered using vacuum 0.2-pm bottle-top filters (Thermo Fisher Scientific). Filtered ExpiCHO™ supernatants were buffered to a final concentration of 10 mM HEPES using 1 M HEPES [pH 7.0] (Teknova) and 20 mM imidazole using 4 M imidazole [pH 8.0] (Advanced BioReagents). These supernatants were applied by gravity to 3 -4 mL Ni Sepharose excel resin (Cytiva) preequilibrated with bindingbuffer (20 mM HEPES, 100 mMNaCl, 20 mM imidazole [pH 7.0]). Resins were subsequently washed with 3 column volumes (CVs) of binding buffer, 3 CVs of binding buffer with 50 mM imidazole, and3 CVs of binding buffer with 75 mM imidazole. Finally, HYAL1 mutant samples were eluted with binding buffer with 250 mM imidazole. HYAL1 mutants were found predominantly in the 75 mM imidazole wash or elution fractions and pure protein fractions were pooled following analysis by SDS-PAGE. HYAL1 mutant samples were concentrated to ~1 mL using Amicon® Ultra 15 mL centrifugal filters (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 mMNaCl [pH 7.0] using an AKTA avant 25 FPLC system (Cytiva). Pure HYAL1 mutant fractions were pooled following analysis by SDS-PAGE and used for activity and biophysical analyses.

Biotinylated Hyaluronic Acid (BHA) ELISA Plate Coating

96-well Nunc microplates (Thermo Fisher) were coated with 100 pL/well of 1.2 pg/mL biotinylated Hyaluronic Acid (BHA) (Creative PEGWorks) along with 2.2 pg/mL Sulfo- TV-hydroxysulfosuccinimide(Sulfo-NHS) (Thermo Fisher) and 1.5 pg/mLl-Ethyl-3-(3- Dimethylaminopropyl)carbodiimide, Hydrochloride (EDCA) (Thermo Fisher) in sodium phosphate buffer(100 mMPhosphate buffer, 2 MNaCl, 50 mMMgSO₄ [pH 5.8]). Plates were incubated at 4 °C for 24 hours. Plates were then washed 3x on a BioTek plate washer with PBST (PBS + 0.05% v/v Tween 20). Plates were stored at -20 °C in storage solution (2 MNaCl, 50 mM MgSO₄ in PBS [pH 7.4]) and were used within one week of coating.

Hyaluronidase ELISA Activity Assay

HYAL1 constructs were analyzed alongside control hyaluronidases: rHuPH20 Hylenex (Halozyme), rHuPH20 (Aero BioSystems), rHu-HYALl (R&D Systems), and bee venom hyaluronidase. Serial dilutions were prepared in different buffers depending on the pH analyzed: histidine buffer (20 mM histidine, 130 mMNaCl, 1 mMCaC12 and 0.04% Tween- 20®) for pH 5.0-7.5, sodium acetate buffer (20 mM sodium acetate, 130mMNaCl, 1 mMCaC12 and 0.04% BSA) for pH 4.0 and4.5, and sodium formate buffer (20 mM, 130 mMNaCl, 1 mM CaCl₂ and 0.04% BSA) for pH 3.0 and 3.5.

Serial dilutions and transfers to ELISA plates were performed using an automated liquid handler (Agilent Bravo). Samples were further diluted by lOx by addition of 20 pL of diluted protein into 180 pL of buffer. Serial dilutions (100 pL each) were transferred to BHA- coated ELISA plates and incubated at 37 °C for 1 hour. Plates were then washed 3x with PBST buffer via BioTek plate washer. Subsequently, 200 pL of 6 M guanidine HC1 was added as a stop solution to quench hyaluronidase activity, incubated at room temperature (RT) for 5 minutes and washed 3x with PBST. Afterwards, 100 pL/well of 0.1 pg/mL horseradish peroxidase (HRP)-conjugated streptavidin (Thermo Fisher) in PBS was added and placed on a shaker at room temperature for 30 minutes. Plates were then washed 3xwith PBST and 100 pL of KLP ABTS® peroxidase substrate (contains 2, 2’-azino-di(3-ethylbenzthiazoline- 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.

All graphs were generated using GraphPad Prism 9; ELISA EC50 values were determined using a sigmoidal, 4PL, Xis log(concentration) fitwith a lower bound constraint equivalentto the absorbance valuefrom wells containing fully digested HA. Hyaluronidase activity was determined based on the known activity value for Aero PH20 of 108,422 Units/mg (manufacturer communication) and the determined EC50 value at pH 5.35. The activity £650 ratio at pH 5.35 was used to determine all subsequent activities at different pH for all samples, controls, and standards. EXAMPLE 1

Evolution of human HYAL1 mutants

Fig- 1 shows an overview of the design/test parts of the workflow for evolving HLYAL1 into a HYAL mutant with a broader pH range of activity that would be useful for use in delivery of a therapeutic agent administered subcutaneously. Once designs were made, DNA encoding a plurality of HYAL1 mutants was ordered from Genscript. After delivery of that DNA, the HYAL1 mutants were expressed in ExpiCHO-S cells. After expression, samples were collected and tested for activity using a turbidity assay. Expression levels were also assessed using HYAL1 His-tag HTRF Expression Assay as described below.

Fig. 2 shows the results of the first round of screening. This was a library built primarily from sequence homology and literature reports of mutation impacts on HYAL1 and other hyaluronidase homologs. The plot is ranked from highest to lowest activity FIOP. FIOP stands for (Fold Improvement Over Parent) and refers to each mutant’s activity relative to the starting point for each round (in round 1 for example the starting point was the wild-type HYAL1). FIOP is calculated simply as (Activity of Variant)/(Activity of Parent). The dots on the plot show a similar FIOP value for the expression level. The activities were measured using a turbidity assay and the expression data was collected using a commercial HTRF assay kit.

Fig- 3 shows data very similar to Fig. 2. In this case the library variants were screened at both pH 4 (top) and pH 5 (bottom). During the first several rounds of evolution we tested HYAL mutants at various pHs in an effort to identify mutants that were active at pH values closer to a target pH of pH5.5.

Fig. 4 A - Fig. 41 entitled “Evolution Tracker” show the progress of the HYAL1 evolution in a round-by-round format. Each of the boxes represents an individual library, and each column of boxes represents a round of evolution (matching the table that runs across the top). The “Fold Improvement” row in the table shows how the HYAL1 backbone for that round compared to the backbone of the previous round. Rosetta refers to RosettaCommons software, available from http://RosettaCommons.org.

Fig- 5 shows in vivo data that demonstrates activity of an engineered HYAL 1 mutant in a mouse skin-spreading model. In this experiment, wildtype HYAL1 , a round 3 HYAL1 mutant, and the commercially used hyaluronidase (PH20) were compared. Each of the enzymes was mixed with a colored dye and intradermally injected into mice. The dye spreading was then monitored overtime.

Fig. 6 and Fig. 7 show the workflow that was used to express, purify, and crystallize HYAL1 mutants and the resulting structure. The enzyme was expressed in ExpiCHO- S cells, treated with endoglycosidases to remove the majority 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 the course of the evolution we had introduced several new TV-linked glycosylation sites during the evolution of HYAL1. ASU is the smallest part of a crystal structure to which symmetry operations can be applied in order to generate the complete unit cell (the crystal repeating unit). R_WOrk is ^a measure of the agreement between the crystallographic model and the experimental X-ray diffraction data. Rf_reeis a statistical quantity introduced in 1992 by Axel T. Briinger to assess the quality of a model from X-ray crystallographic data.

Fig- 8 shows that in round 8 of the HYAL1 evolution the focus became reducing the immunogenicity of the mutations that we introduced up to that point. ExpiVax software from ExpiVax, Providence, RI, USA was used to scan regions of HYAL1 where mutations hadbeen made. The software provides an epitope score that can be used to assess the immunogenicity of each possible 9-mer peptide in the scanned regions.

Fig. 9 Using the EpiVax software (see Fig. 8) we identified? epitope regions that had increased immunogenicity risk as a result of the mutations we had introduced. Each of those regions was classified as low, medium, or high risk, depending on the score.

Fig. 10 summarizes the approach that was taken to design additional HYAL1 mutations that would reduce the immunogenicity risk of the sites identified by EpiVax analysis. This involved designing point mutations in silico (using RosettaCommons software) and then evaluating the impact of those mutations on the EpiVax immunogenicity scores. In this way, mutations were filtered by predicted stability (RosettaCommons software) and predicted reduction of immunogenicity (EpiVax). Mutations that passed these filters and met the desired criteria were used to design a new point mutant library.

Fig. 11 shows the results of the round 8 point-mutation library screen. The plot on the top shows the number of epitopes that were removed by each mutation that was introduced. The plot is divided into regions (A, B, C, D, E, and FG) that match the regions that were mapped on the structure in Fig. 9. The plot on the bottom Fig. 11 shows the activity and expression FIOPs of each of the mutants. In this case, we looked for mutations that had expression and activity FIOPs near 1.0 and selected them for combination in the final library.

Fig. 12 shows the results of the library screen in which immunogenicity-reducing point mutations were combined. The library was designed as an effort to maximize our chances of reducing the number of epitopes of concern. We designed three-mutation mutants with mutations from each of the three regions with predicted “high” immunogenicity 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. The results of the selection rounds and the HYAL1 mutants obtained are summarized in Table 2.

REFERENCES

1 De 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/joumal.pone.0157957 (2016).

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

3 Wynne, 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).

4 Usach, 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/sl2325-019-01101-6 (2019).

5 Bittner, 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).

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

7 Meyer, K. The biological significance of hyaluronic acid and hyaluronidase. Physiological Reviews 27, 335-359, doi:10.1152/physrev.1947.27.3.335 (1947). 8 McClean, 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/bj 0350159 (1941).

9 Duran-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).

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

11 Frost, G. I. Recombinant human hyaluronidase (rHuPH20): An enabling platform for subcutaneous drug and fluid administration. Expert Opinion on Drug Delivery 4, 427-440, doi: 10.1517/17425247.4.4.427 (2007).

12 Reitinger, S., Miillegger, J., Greiderer, B., Nielsen, J. E. & Lepperdinger, G. Designed human serum hyaluronidase 1 variant, HYAL1AL, exhibits activity up to pH 5.9. Journal of Biological Chemistry 284, 19173-19177, doi:10.1074/jbc.C109.004358 (2009).

While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the specification and the claims attached herein.

Claims

WHAT IS CLAIMED:

1 . A human hyaluronidase 1 (HYAL1) mutant comprising an amino acid sequence having at least 95% identity to the amino acid sequence of amino acids 20 to 433 of the amino acid sequence for HY AL 1 set forth in SEQ ID NO: 2, wherein the HYAL1 mutant further comprises a combination of amino acid substitutions selected from the group consisting of:

(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, P23 V, S304T, L379M, W433E, R194D, D299E, V412R, N235D, I287P, F345T, and L377S; and wherein the HYAL1 mutant (i) comprises at least one A-glycosylation site comprising the sequence asparagine-X-serine/threonine, wherein X is any amino acid other than proline, that is glycosylated and (ii) displays hyaluronidase activity from pH 3.5 to pH 5.5.

2. The HYAL1 mutant of 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, andW433E.

3. The HYAL1 mutant of claim 1 , wherein the HYAL1 mutant comprises the amino acid substitutions D68N, M246L,D323N, S330K,L347V,H262N, D290T, R326S, D276K, F20D, Al 82E, G235N, P23 V, S304T, L379M, W433E, R194D, D299E, and V412R.

4. The HYAL1 mutant of 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, V412RR238C, I287P, F345T, and L377S.

5. The HYAL1 mutant of 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 of 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 of claim 1 , wherein at least one A-glycosylation site created by the D68N, D323N, G235N, orR326S substitution is glycosylated.

8. The HYALl mutant of claim 1, wherein the A-glycosylation sites created by the D68N, D323N, G235N, and R326S substitutions are each glycosylated.

9. The HYAL1 mutant of claim 1 , wherein the native A-glycosylation sites and the A-glycosylation sites created by the D68N, D323N, G235N, andR326S substitutions are each glycosylated.

10. A human hyaluronidase 1 (HYAL1) mutant comprising an amino acid sequence with at least 95% identity to the amino acid sequence set forth in SEQ IDNO: 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 HY AL 1 mutant (i) comprises at least one 7V-glycosylation site comprising the sequence asparagine-X- serine/threonine, wherein X is any amino acid other than proline, that is glycosylated and (ii) displays hyaluronidase activity from pH 3.5 to pH 5.5.

11. A human hyaluronidase 1 (HYAL1) mutant comprising an amino acid sequence with at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, wherein the HYALl 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, that is glycosylated and(ii) displays hyaluronidase activity from pH 3.5 to pH 5.5.

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

13. The HYALl mutant of claim 12, wherein the polymer is dextran or polyethylene glycol.

14. A composition comprising a HYAL1 mutant of any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

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

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

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

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

19. The composition of claim 15, wherein the therapeutic agent comprises an antibody, an antigen-binding protein, an scFv, 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, comprising: administering to the tissue of a subject a composition comprising the HYAL1 mutant of claim 14 in an amount sufficient to increase 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, comprising: administering to the tissue of a subject a composition comprising the therapeutic agent and an amount of the HYAL1 mutant of claim 14 in an amount sufficient to increase diffusion of the therapeutic agent in the tissue.

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

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

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

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

26. A nucleic acid molecule encoding the HYAL1 mutant of any one of claims

1-13.

27. The nucleic acid molecule of claim 26, wherein the HYAL1 mutant of 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 of claim 26 or 27.

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

30. The host cell of claim 29, wherein the host cell is a mammalian host cell or a recombinant yeast host cell modified to produce complex A-gly cans.

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

32. The host cell of claim 30, wherein the recombinant yeast host cell sPichia pastor is.

33. A method for producing a HYAL1 mutant, comprising:

(a) introducing a nucleic acid molecule of any one of claims 26 or 27 encoding the HYAL1 mutant, or the expression vector of claim 28 comprising a nucleic acid molecule encoding the HYAL1 mutant, into a host cell to produce a recombinant host cell;

(b) cultivating the recombinant host cell in a culture medium under conditions sufficient for the recombinant host cell to express and secrete the HYAL1 mutant into the culture medium; and

(c) obtaining the HYAL1 mutant from the culture medium.

34. The method of claim 33, wherein the host cell is a mammalian host cell, or a recombinant yeast host cell modified to produce complex 7V-glycans.

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

36. The method of claim 34, wherein the recombinant yeast host cell sPichia pastor is.

37. The method of claim 33, wherein the HYAL1 mutant comprises amino acids 1 to 433 of an amino acid sequence with at least 95% identity to the amino acid sequence set forth 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.