Combination therapy with dupilumab and hyaluronidases

Abstract

Provided are combination dosing regimens comprising administering a monoclonal antibody, such as dupilumab, and hyaluronidase. Combinations and compositions containing the monoclonal antibody and hyaluronidase are provided. The dosing regimens and combinations with dupilumab are for treating or preventing allergic diseases such as atopic dermatitis (eczema), asthma and nasal polyps which result in chronic sinusitis, eosinophilic esophagitis, and prurigo nodularis.

Description

[0001] COMBINATION THERAPY WITH DUPILUMAB AND HYALURONIDASES

[0002] FIELD OF THE INVENTION

[0003] The inventions herein relate to compositions and combination dosing regimen comprising administering Dupilumab and hyaluronidase. Provided are dosing regimens and combinations for treating or preventing allergic diseases such as atopic dermatitis (eczema), asthma and nasal polyps which result in chronic sinusitis, eosinophilic esophagitis, and prurigo nodularis.

[0004] INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0005] An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The electronic file was created on March 4, 2024 is 3.75 kilobytes in size, and is titled Halozyme-PH20.xml.

[0006] BACKGROUND

[0007] Patient surveys have shown that the majority of patients would prefer to receive a long- acting injectable regimen at fewer intervals rather than taking intravenous injections. Currently, all available monoclonal antibodies are administered via intravenous administration.

[0008] A safety concern when treating patients with injectable suspensions is injection site reactions. Combining a monoclonal antibody with other drug products can alter the injection site reaction profile of a monoclonal antibody. Combining a monoclonal antibody with other drug products also may alter the pharmacokinetic (pK) profile of a monoclonal antibody.

[0009] Long-acting injectable treatments are limited by patient experience and side effects. Achieving an injectable suspension with a high concentration of drug in order to dose less frequently and overcome the non-compliance problem with treatment regimens, whilst maintaining product stability, avoiding drug-drug interactions, and avoiding patient side effects is desirable.

[0010] There is a need in the art for a treatment and prevention with monoclonal antibodies that can be dosed at fewer intervals without increasing injection site reactions.

[0011] SUMMARY

[0012] Provided are combination dosing regimens, comprising administering hyaluronidase; and administering a monoclonal antibody, in specific embodiments dupilumab. Also provided are methods of treating or preventing allergic diseases such as atopic dermatitis (eczema), asthma and nasal polyps which result in chronic sinusitis, eosinophilic esophagitis, and prurigo nodularis, comprising administering to a patient in need of treatment or prevention the combination dosing regimen as described herein. Provided are combinations and kits. comprising a composition comprising a soluble hyaluronidase; and a monoclonal antibody, in specific embodiments dupilumab.

[0013] Soluble hyaluronidase can be administered with a monoclonal antibody to allow a larger amount of the monoclonal antibody to be administered to a patient at in a single dose that the amounts of monoclonal antibody administered alone. In specific embodiments, the monoclonal antibody is dupilumab. Thus, methods, regimens, and combinations provided herein can be administered less frequently.

[0014] BRIEF DESCRIPTION OF DRAWING

[0015] The following detailed description of embodiments of the hyaluronidase formulations, in some embodiments high volume administration, will be better understood when read in conjunction with the appended drawings of exemplary embodiments.

[0016] FIG. 1 provides the duration of subcutaneous injection times with dupilumab and dupilumab + rHuPH20.

[0017] FIG. 2 provides post-injection measurements of back leakage after subcutaneous injections with dupilumab and dupilumab + rHuPH20.

[0018] FIG. 3 provides post-injection measurements of bleb volume after subcutaneous injections with dupilumab and dupilumab + rHuPH20.

[0019] FIG. 4 provides post-injection measurements of bleb area after subcutaneous injections with dupilumab and dupilumab + rHuPH20.

[0020] FIG. 5 provides post-injection measurements of bleb height after subcutaneous injections with dupilumab and dupilumab + rHuPH20.

[0021] FIG. 6 provides post-injection swelling measurements after subcutaneous injections with dupilumab and dupilumab + rHuPH20.

[0022] FIG. 7 provides post-injection induration measurements after subcutaneous injections with dupilumab and dupilumab + rHuPH20.

[0023] FIG. 8 provides the concentration versus time after subcutaneous injections with 2 mL of dupilumab 5 mL dupilumab + rHuPH20.

[0024] FIG. 9 provides the dose-normalized concentration versus time after subcutaneous injections of 2 mL and 5 mL dupilumab.

[0025] FIGS. 10-14 are photographic images of the injection site before and after subcutaneous administration of dupilumab + rHuPH20. FIGS. 15-18 are photographic images of the injection site before and after subcutaneous administration of dupilumab.

[0026] FIG. 19 is a photographic image of the site before subcutaneous administration of dupilumab.

[0027] FIG. 20 is a schematic of the model structure, differential equations, and description of the parameters used for simulating SC dupilumab administration with rHuPH20.

[0028] FIGS. 21A and 21B show simulated change in plasma concentration of dupilumab after IV administration compared to SC administration with rHuPH20.

[0029] FIG. 22 shows plasma concentration and Cmax for SC administration of bococizumab with and without rHuPH20.

[0030] FIG. 23 shows plasma concentration and Cmax for SC administration of tocilizumab with and without rHuPH20.

[0031] FIG. 24 shows the difference in Cmin for amivantamab administered SC with rHuPH20 relative to IV administration without rHuPH20.

[0032] DETAILED DESCRIPTION

[0033] A. DEFINITIONS

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to w hich the invention(s) belong. All patents, patent applications, published applications and publications, GenBank® sequences, databases, websites, and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. If there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0035] As used herein the term ‘combination dosing regimen’ refers to at least two components administered together to a patient.

[0036] As used herein, the term ‘treatment’ or ‘treating’ refers to alleviating the specified condition, eliminating or reducing the symptoms of the condition, slowing or eliminating the progression, invasion, or spread of the condition and reducing or delaying the reoccurrence of the condition in a previously afflicted subject. As used herein, the term ‘prevention’ or 'preventing’ refers to precluding developing a disease, disorder, or condition or reducing the risk of developing the disease, disorder, or condition or reducing the symptoms thereof.

[0037] As used herein the term ‘injection site reaction’ means side effects at or near the spot where the infusion / inj ection was received. This includes pain or discomfort, redness, swelling, itching, bruising, lumps, infection complications (cellulitis or abscess), and irritation.

[0038] As used herein, a soluble hyaluronidase is a hyaluronidase of form thereof that is not GPI anchored, and that is soluble under physiological conditions and is secreted upon expression. Hyaluronidases, such as ovine and bovine hyaluronidases occur as soluble hyaluronidases. Human PH20 hyaluronidase does not occur as a soluble hyaluronidase. It is known in the art that removal of all or a part of the GPI anchor results in soluble forms.

[0039] As used herein the term “rHuPH20” refers to the soluble hyaluronidase composition produced upon expression in a mammalian cell, such as a CHO cell, or other cell that effects glycosylation, of nucleic acid encoding residues 36-482 of SEQ ID NO: 1. For expression in cells the encoding nucleic acid is linked to the native (residues 1-35 of SEQ ID NO: 1) or a heterologous signal sequence for trafficking and secretion of the encoded polypeptides. The resulting secreted soluble glycoprotein is a heterogeneous mixture of polypeptides, including polypeptides that terminate at residues 479, 480, 481, and 482, and are composed of residues 36-479, 36-480, 36-481, and 36-482 with reference to SEQ ID NO:1. Shorter C-terminally truncated forms also may be included.

[0040] As used herein, “combination therapy” refers to a treatment in which a subject if given two or more therapeutic agents, such as at least two or at least three therapeutic agents, for treating a single disease.

[0041] As used herein, “hyaluronidase activity" refers to the ability to enzymatically catalyze the cleavage of hyaluronic acid. The United States Pharmacopeia (USP) XXII assay for hyaluronidase determines hyaluronidase activity indirectly by measuring the amount of higher molecular weight hyaluronic acid, or hyaluronan, (HA) substrate remaining after the enzyme is allowed to react with the HA for 30 min at 37 °C (USP XXII-NF XVII (1990) 644-645 United States Pharmacopeia Convention. Inc, Rockville. MD). A Reference Standard solution can be used in an assay to ascertain the relative activity, in units, of any hyaluronidase. In vitro assays to determine the hyaluronidase activity of hyaluronidases, such as PH20, including soluble PH20 and esPH20, are known in the art and described herein. Exemplary assays include the micro turbidity assay that measures cleavage of hyaluronic acid by hyaluronidase indirectly by detecting the insoluble precipitate formed when the uncleaved hyaluronic acid binds with serum albumin and the biotinylated- hyaluronic acid assay that measures the cleavage of hyaluronic acid indirectly by detecting the remaining biotinylated-hyaluronic acid non-covalently bound to microtiter plate wells with a streptavidin-horseradish peroxidase conjugate and a chromogenic substrate. Reference Standards can be used, for example, to generate a standard curve to determine the activity in Units of the hyaluronidase being tested.

[0042] As used herein, specific activity refers to Units of activity7per mg protein. The milligrams of hyaluronidase is defined by the absorption of a solution of at 280 nm assuming a molar extinction coefficient of approximately 1.7, in units of M-l cm-1.

[0043] As used herein, “neutral active” refers to the ability of a PH20 polypeptide to enzymatically catalyze the cleavage of hyaluronic acid at neutral pH (e.g. at or about pH 7.0).

[0044] As used herein, a “GPI-anchor attachment signal sequence” is a C-terminal sequence of amino acids that directs addition of a preformed GPI-anchor to the polypeptide within the lumen of the ER. GPI-anchor attachment signal sequences are present in the precursor polypeptides of GPI-anchored polypeptides, such as GPI-anchored PH20 polypeptides. The C-terminal GPI-anchor attachment signal sequence typically contains a predominantly hydrophobic region of 8-20 amino acids, preceded by a hydrophilic spacer region of 8-12 amino acids, immediately downstream of the ©-site, or site of GPI-anchor attachment. GPI- anchor attachment signal sequences can be identified using methods well known in the art, such as but not limited to, in silico methods and algorithms (see, e.g. Udenfriend et al. (1995) Methods Enzymol. 250:571-582, Eisenhaber et al., (1999) J. Biol. Chem. 292: 741-758, Fankhauser et al., (2005) Bioinformatics 21 : 1846-1852. Omaetxebarria et al., (2007) Proteomics 7: 1951-1960, Pierleoni et al., (2008) BMC Bioinformatics 9:392), including those that are readily available on bioinformatic websites, such as the ExPASy Proteomics tools site (e.g. the World Wide Web site expasy.ch / tools / ).

[0045] As used herein, sequence identity refers to the relatedness between or among polypeptides among nucleic acid molecules. Sequence identity7can be assessed by aligning two sequences and counting the number of differences between the aligned portion and the sequence to which it is compared. Whether any two molecules have nucleotide sequences or amino acid sequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” or “homologous” can be determined using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux (1984) Nucleic Acids Research 12:387), BLASTP, BLASTN, FASTA (Altschul (1990) J. Mol. Biol. 215:403); Guide to Huge Computers, Bishop, ed., Academic Press, 1994, and Carrillo (1988) SIAM J. Applied Math 48: 1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity . Other commercially or publicly available programs include, DNAStar "McgAlign" program and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program. Percent homology or identity of proteins and / or nucleic acid molecules can be determined, for example, by comparing sequence information using a GAP computer program (e.g. Needleman (1970) J. Mol. Biol. 48:443, as revised by Smith and Waterman (1981) Adv. Appl. Math. 2:482. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e. nucleotides or amino acids), which are similar, divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov (1986) Nucl. Acids Res. 14:6745, as described by Schwartz and Dayhoff, eds.. Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

[0046] Therefore, as used herein, the term “identity” or “homology” represents a comparison between a test and a reference polypeptide or polynucleotide.

[0047] As used herein, the term at least “90% identical to” refers to percent identities from 90 to 99.99 relative to the reference nucleic acid or amino acid sequence of the polypeptide. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polypeptide length of 100 amino acids are compared. No more than 10% (i.e. 10 out of 100) of the amino acids in the test polypeptide differs from that of the reference polypeptide. Similar comparisons can be made between test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of a polypeptide or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g. 10 / 100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. At the level of homologies or identities above about 85- 90%. the result should be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often by manual alignment without relying on software.

[0048] As used herein, an aligned sequence refers to the use of homology (similarity and / or identity ) to align corresponding positions in a sequence of nucleotides or amino acids. Typically, two or more sequences that are related by 50% or more identity are aligned. An aligned set of sequences refers to 2 or more sequences that are aligned at corresponding positions and can include aligning sequences derived from RNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence.

[0049] As used herein, “denaturing condition” or “denaturation condition” refers to any condition or agent that, when exposed to a protein, affects or influences the degradation or denaturation of the protein, generally as a result of a loss or partial loss of the tertiary or secondary structure of the protein. Denaturing conditions can result in effects such as loss or reduction in activity, loss or reduction of solubility, aggregation and / or crystallization.

[0050] As used herein, “resistance to a denaturation condition” refers to any amount of decreased reduction or elimination of a property or activity of the protein associated with or caused by denaturation. For example, denaturation is associated with or causes increased crystallization or aggregation, reduced solubility or decreased activity. Hence, resistance to denaturation means that the protein exhibits decreased aggregation or crystallization, increased solubility or increased or greater activity (e.g. hyaluronidase activity) when exposed to a denaturing condition compared to a reference protein (e.g. unmodified enzyme).

[0051] As used herein, stability of a modified PH20 hyaluronidase means that it exhibits resistance to denaturation caused by a denaturation condition or denaturing agent.

[0052] For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections that follow.

[0053] B. Overview

[0054] Provided are combination dosing regimens, comprising administering a hyaluronidase; and administering an a monoclonal antibody, in specific embodiments dupilumab. The agents in the dosing regimens can be administered sequentially, intermittently, serially, in the same composition, and / or in other combinations of the agents. The combination dosing regimens described herein are dosing regimens suitable to be provided to a patient in order to treat or prevent cancer. In some embodiments, the combination dosing regiments described herein are dosing regimens suitable to be provided to a patient in order to treat or prevent allergic diseases such as atopic dermatitis (eczema), asthma and nasal polyps which result in chronic sinusitis, eosinophilic esophagitis, and prurigo nodularis.

[0055] In an embodiment of the combination dosing regimens provided are dosing regimens suitable for treating a cancer. In an alternative embodiment, the combination dosing regimen provided herein is for preventing cancer. The combination dosing regimen is administered to a patient in need of treatment for cancer. In an embodiment of the combination dosing regimens provided are dosing regimens suitable for treating allergic diseases such as atopic dermatitis (eczema), asthma and nasal polyps which result in chronic sinusitis, eosinophilic esophagitis, and prurigo nodularis. In an alternative embodiment, the combination dosing regimen provided herein is for preventing allergic diseases such as atopic dermatitis (eczema), asthma and nasal polyps which result in chronic sinusitis, eosinophilic esophagitis, and prurigo nodularis. The combination dosing regimen is administered to a patient in need of treatment of an allergic disease such as atopic dermatitis (eczema), asthma and nasal polyps which result in chronic sinusitis, eosinophilic esophagitis, and prurigo nodularis.

[0056] In embodiments herein, provided are combination dosing regimens in which a monoclonal antibody and a soluble hyaluronidase are administered. In some embodiments, the monoclonal antibody is dupilumab.

[0057] C. Therapeutic Monoclonal Antibodies

[0058] In an embodiment, the combination dosing regimens contemplate the administration of a hyaluronidase and therapeutic monoclonal antibody to a human subject, in some embodiments the monoclonal antibody is dupilumab. In an embodiment, the combination dosing regimen contemplates the administration of a hyaluronidase and biosimilar monoclonal antibody approved by one or more drug regulatory authority.

[0059] The term “antibody” is used in the broadest sense and specifically includes monoclonal antibodies (including full length monoclonal antibodies), multi-specific antibodies (e g. bispecific antibodies), and antibody fragments that exhibit a desired biological activity or function.

[0060] An antibody generally comprises two heavy chains and two light chains, each comprising a variable domain and a constant domain. Each variable domain contains a hypervariable region containing three complementarity determining regions (CDR) flanked by four segments of the framework region. As used herein, the “framework region” contains all four segments FR1, FR2, FR3, and FR4 that flank a set of three hypervariable regions.

[0061] Antibodies can be chimeric, humanized, or human, for example, and can be antigenbinding fragments of these. Antibodies are generally produced by immunizing an animal with an antigen, and can be produced by recombinant technology, or by synthesis of the amino acid sequence, for example. “Antibody fragments” comprise a portion of a full-length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; singlechain antibody molecules; and multispecific antibodies such as bispecific antibodies, for example formed from antibody fragments. “Functional fragments” substantially retain binding to an antigen of the full-length antibody and retain a biological activity.

[0062] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies of the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. 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, monoclonal antibodies may be made by the hybridoma method first described by Kohler (1975), Nature 256:495, or may be made by recombinant DNA methods (see, e g., U.S. Patent No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries.

[0063] “Chimeric” antibodies (immunoglobulins) contain a portion of a heavy and / or light chain identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567). A “humanized antibody” as the term is used herein, is a subset of chimeric antibodies.

[0064] “Humanized” forms of non-human (e g. murine) antibodies are chimeric antibodies that contain minimal sequence derived from nonhuman immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient or acceptor antibody) in which variable domain hypervariable region residues of the recipient antibody are replaced by hypervariable region residues from a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. The hypervariable regions can be complementarity-determining regions (CDR) defined by sequence, or hypervariable loops (HVL) defined by structure, or both. In some embodiments, the variable domain framework regions are derived from a consensus sequence variable domain, for example, containing at each residue an amino acid compiled as most abundant at that position in a class or subclass of human immunoglobulin variable domains, for example, in a Kabat compilation. In some instances, one or more amino acids of the variable domain framework region (FR) of the human immunoglobulin or consensus sequence is replaced with one or more corresponding residues of the non -human donor antibody and / or one or more amino acids of the donor antibody hypervariable regions is replaced w ith one or more corresponding human residues of the human recipient variable domain. In some instances, one or more residues of the variable domain framework regions and / or hypervariable regions is a residue not found at the corresponding position in the recipient antibody or in the donor antibody. Modifications to the amino acid sequence of the variable domain framework regions and hypervariable regions are generally made to further refine antibody performance, for example, improve binding affinity. In general, the humanized antibodies used to produce the pan-specific antibodies described herein will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable region residues (CDR or HVL) correspond to those of a nonhuman immunoglobulin and all or substantially all of the framework region (FR) residues correspond to those of a human variable domain consensus sequence, and may include one or more amino acid substitutions. In some embodiments, the number of amino acid substitutions in the human consensus framework region is typically no more than 10, and may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions in the heavy chain variable domain framework regions, and 0, 1, 2, 3, 4, 5, 6, 7, 8. 9, or 10 substitutions in the light chain variable domain framework region. The humanized antibody optionally comprises at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin.

[0065] An “Fv” fragment is an antibody fragment that contains a complete antigen recognition and binding site, and generally comprises a dimer of one heavy and one light chain variable domain in tight association that can be covalent in nature, for example in a single chain variable domain fragment (scFv). It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain comprising only three hypervariable regions specific for an antigen has the ability to recognize and bind antigen.

[0066] "‘Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, where these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0067] A “Fab” fragment includes a variable domain and a constant domain of the light chain and a variable domain and the first constant domain (CHI) of the heavy chain. A Fab' fragment includes one or more cysteine carboxy terminal linkages to the heavy or light chains. F(ab')2 antibody fragments comprise a pair of Fab fragments that are generally covalently linked near their carboxy termini by hinge cysteines. Other chemical couplings of antibody fragments are also known.

[0068] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between the two variable domains on the same chain, the variable domains are forced to pair with complementary domains of another chain, creating two antigen-binding sites.

[0069] The term “hypervariable region’" when used herein refers to the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a “complementarity -determining region” or “CDR” (defined by sequence as residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain (Kabat (1991) Sequences of Proteins of Immunological Interest, National Institutes of Health) and / or those residues from a hypervariable loop. In one example. HVL residues can include. 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53- 55 (H2) and 96-101 (H3) in the heavy7chain variable domain.

[0070] “Framework region” or “FR” residues are those variable domain residues flanking the hypervariable region residues as herein defined. In general, a variable domain contains three hypervariable regions flanked by four sequences of the framework region (FR1, FR2, FR3, and FR4).

[0071] The term “consensus sequence” as used herein, refers to an artificial variable domain sequence comprising at each position the residue that is most abundant at that position in the variable domains of a group of antibodies of a particular class. The consensus variable domain sequences do not have any known antibody binding specificity or affinity.

[0072] In embodiments herein, a monoclonal antibody is provided in a suspension. The suspension includes any suitable suspension of a monoclonal antibody, such as those exemplified. Combinations and treatment regimens are provided herein in which a monoclonal antibody is administered in combination with a soluble hyaluronidase. In some embodiments the monoclonal antibody is dupilumab.

[0073] D. Soluble hyaluronidases

[0074] Soluble hyaluronidases include any that, upon expression, are secreted from a cell and exist in soluble form. Such soluble hyaluronidases include, for example, but are not limited to, bacterial soluble hyaluronidases, non-human soluble hyaluronidases, such as bovine PH20 and ovine PH20, human soluble PH20, and variants thereof. Generally soluble forms of PH20 are produced using protein expression systems that facilitate correct N-glycosylation to ensure the polypeptide retains activity, since glycosylation is important for the catalytic activity and stability of hyaluronidases. Such cells include, for example Chinese Hamster Ovary (CHO) cells (e.g. DG44 CHO cells).

[0075] Soluble PH20 hyaluronidase is available and sold, for example, under the trademark ENHANZE®. ENHANZE® technology' provides to a drug delivery' technology, employing the soluble hyaluronidases to facilitate the delivery of injected drugs and fluids. When coformulated with other drugs or administered with other drugs, the ENHANZE® technology reduces treatment burden for patients. It can allow' for large volume subcutaneous injection with increased dispersion and absorption of co-administered therapies. ENHANZE® technology has been marketed with biologies; it has not, prior to the instant description, been used for delivery of specific small molecules, nor for delivery of therapeutics for treatment and / or prevention of cancer. rHuPH20 refers to the composition produced upon expression in a cell, such as CHO cell, of nucleic acid encoding residues 36-482 of SEQ ID NO: 26, generally linked to the native or a heterologous signal sequence (residues 1-35 of SEQ ID NO: 26). rHuPH20 is produced by expression of a nucleic acid molecule, such as encoding amino acids 1-482 (set forth in SEQ ID NO: 26) in a mammalian cell. Translational processing removes the 35 amino acid signal sequence. As produced in the culture medium there is heterogeneity at the C -terminus such that the product, designated rHuPH20, includes a mixture of species that can include any one or more of the polypeptides 36-480, 36-481, and 36-482 of SEQ ID NO: 26, and some shorter polypeptides, in various abundance. rHuPH20 and forms of soluble hyaluronidase are produced in cells, such as CHO cells, for example DG44 CHO cells, that facilitate N-glycosylation. PH20 is a glycoprotein, and as known in the art, requires glycosylation retain activity'. See, e.g. U.S. Patent Nos. 8,927,249 and 9,284,543 (and PCT Publication No. WO 2010 / 077297), which describe the effects of glycosylation and partial glycosylation and elimination of glycosylation on the activity of soluble forms of PH20. These patents and publications also describe and exemplify I soluble C-terminally truncated forms of PH20.

[0076] 1. Forms of Soluble Human PH20

[0077] Soluble hyaluronidases include bovine and ovine PH20, and recombinant and humanized forms thereof. Human PH20 in nature includes a GPI anchor and exists linked to sperm cells; it is not soluble. C -terminally -truncated forms thereof are soluble. Soluble forms of recombinant human PH20 have been produced and can be used in the compositions, combinations and methods described herein. Descriptions of and production of such soluble forms of PH20 are described, for example, in U.S. Patent Nos. 7,767.429; 8,202,517; 8,431,380; 8.431,124; 8.450,470; 8.765.685; 8,772.246; 7,871,607; 7,846,431; 7.829,081; 8,105,586; 8,187,855; 8,257,699; 8,580,252; 9,677,061 ; and 9,677,062, each incorporated by reference herein. The soluble hyaluronidases, thus include forms of human PH20, which are neutral active hyaluronidases, and which require glycosylation for activity.

[0078] SEQ ID NO: 1 sets forth the sequence of the precursor polypeptides; the mature PH20 polypeptide (residues 36-509); soluble forms also include those with amino acid truncations at the N-terminal, such as deletions of the first one, two, three, or fours residues, such that the resulting polypeptides have an N-terminus, for example, at residue 36, 37, 38, 39, or 40, and a C-terminus at a residue from 465 to 500, and variants thereof, including, but not limited to, variants discussed below, variants known in the art, and allelic variants.

[0079] Hyaluronidases for use in the compositions, combinations and methods herein are soluble neutral active hyaluronidases. Exemplary thereof are the soluble C-terminally truncated forms of mature human PH20. Soluble forms that have hyaluronidase activity, include but are not limited to, those that are truncated at residues from 465 to 500 of sequence ID No. l, and that are, upon expression, secreted. Exemplary' thereof are polypeptides that have sequence 36-465, 36-466, 36-467, 36-468, 36-469, 35-470, 36-471, 36-472, 36-474, 36- 475, 36-476, 35-477. 36-478, 36-479, 36-480. 36-481, 36-482, 36-483 35-484. 36-485, 36- 486, 36-487, 36-488, 36-489, 36-490, 35-491, 36-492, 36-493, 36-494, 36-495, 36-496, 36- 497, 35-498, 36-499, and 36-500 of SEQ ID NO: 1, as well as N-terminally truncated forms of each of the preceding that lack two to five residues at the N-terminus, such as for example 37-368, 38-468, and any others that exhibit hyaluronidase activity’ at neutral pH, such as pH in the range of 7.0-7.4.

[0080] Thus, such soluble forms include truncated forms of the mature form of human PH20 lacking all or a portion of the C-terminal GPI anchor, so long as the hyaluronidase is soluble and retains hyaluronidase activity. Soluble forms are secreted upon expression in mammalian cells, and are encoded with a signal sequence, such are residues 1-35 of SEQ ID NO. 1 or a heterologous signal sequence that is cleaved by the cell to effect secretion. Soluble forms are forms that, when expressed in a cell, lack the signal peptide. Also included among soluble hyaluronidases are variants of the soluble PH20 polypeptides that exhibit hyaluronidase activity7. Variants include polypeptides having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity7to any of the PH20 polypeptides 36-465, 36-466, 36-467, 36-468, 36-469, 35-470, 36-471, 36-472, 36-474, 36- 475, 36-476, 35-477, 36-478, 36-479, 36-480, 36-481, 36-482, 36-483 35-484, 36-485, 36- 486, 36-487, 36-488, 36-489, 36-490, 35-491, 36-492, 36-493, 36-494, 36-495, 36-496, 36- 497, 35-498, 36-499. and 36-500 of SEQ ID NO: 1. Amino acid variants include conservative and non-conservative insertions, or deletions, or replacements, and include the modifications, singly or combinations of the modifications detailed, for example, in U.S. Patent No. 11,041,149 and International PCT publication No. WO 2013 / 102144. U.S. Patent No.

[0081] I I,041.149 and International PCT publication No. WO 2013 / 102144 describe a systematic analysis and results identifying the effects of amino acid modifications at each residue in PH20 to thereby provide a structure / function map of PH20; a skilled person can identify replacement residues and consequent alterations in properties and activities, such as for effecting increases in enzymatic activity, stability in denaturing conditions, and also residues whose replacement or deletion decreases or eliminates enzymatic activity.

[0082] It is understood that residues that are important or otherwise required for the activity of a hyaluronidase, such as any described above or known to those of skill in the art, are generally invariant and, except for possible conservative amino acid substitutions, cannot be changed. These include, for example, active site residues. For example, amino acid residues

[0083] I I I, 113 and 176 (corresponding to residues in the mature PH20 polypeptide) of a human PH20 polypeptide, or soluble form thereof, are generally invariant and are not altered. Other residues that confer glycosylation and formation of disulfide bonds required for proper folding also can be invariant.

[0084] The soluble human PH20 hyaluronidase is GPI-anchored and is rendered soluble by truncation at the C-terminus by removal of all or a part of the GPI anchor. Such truncation can remove all of the GPI anchor attachment sequence or can remove only some of the GPI anchor attachment sequence. The resulting polypeptide, however, is soluble. In instances where the soluble hyaluronidase retains a portion of the GPI anchor attachment signal sequence, 1, 2, 3, 4, 5, 6, 7 or more amino acid residues in the GPI anchor attachment signal sequence can be retained, provided the polypeptide is soluble. Polypeptides containing one or more amino acids of the GPI anchor are termed extended soluble hyaluronidases. One of skill in the art can determine whether a polypeptide is GPI-anchored using methods well known in the art. Such methods include, but are not limited to, using known algorithms to predict the presence and location of the GPI anchor attachment signal sequence and co-site, and performing solubility analyses before and after digestion with phosphatidylinositol-specific phospholipase C (PI-PLC) or D (PI-PLD).

[0085] Extended soluble hyaluronidases, which terminate for example, at residues 495, 496, 497, 498, 499, and 500, with reference to SEQ ID NO: 1, such as those set forth in SEQ ID NO: 61-66, can be produced by making C -terminal truncations to any naturally GPI-anchored hyaluronidase such that the resulting polypeptide is soluble and contains one or more amino acid residues from the GPI anchor attachment signal sequence (see. e.g. U.S. Patent No. 8,927,249). These include hyaluronidases that are neutral active, soluble, contain amino acid substitutions, and have at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% or more sequence identity to any of SEQ ID NO: 61-66.

[0086] Typically, for use in the compositions, combinations, and methods herein, a soluble human hyaluronidase, such as a soluble human PH20, is used, such as a PH20 and variants having, for example, at least 91% or 95% or 98% sequence identity thereto, including those with 1 to 5 N-terminal residues deleted. Hyaluronidases used in the regimens, combinations, compositions, and methods herein can be recombinantly produced or can be purified or partially purified from natural sources, such as, for example, from testes extracts. Methods for production of recombinant proteins, including recombinant hyaluronidases, are well known in the art.

[0087] Recombinant soluble forms of human PH20 have been generated and can be used in the compositions, combinations and methods provided herein. For example, with reference to SEQ ID NO: 1, which sets forth the sequence of full length precursor PH20, which includes a signal sequence (residues 1-35), soluble forms include, but are not limited to, C-terminal truncated polypeptides of human PH20 set forth in SEQ ID NO: 1 having a C-terminal amino acid residue 467. 468, 469, 470, 471, 472. 473, 474. 475, 476, 477, 478, 479. 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 of the sequence of amino acids set forth in SEQ ID NO: 1, or polypeptides that exhibit at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity thereto, when aligned with the unmodified sequence of the soluble PH20, have activity at neutral pH, and are soluble (secreted into the medium when expressed in a mammalian cell). Soluble forms of human PH20 generally include those that contain amino acids 36-464 set forth in SEQ ID NO: 1 and terminate at any of residues, 465-500, and optionally include a 1- 3 amino acid deletion at the N-terminus ( / .e. lack residues 36. 36-37, or 36-38 of SEQ ID NO: 1). For example, when expressed in mammalian cells, the 35 amino acid N-terminal signal sequence (residues 1-35 of SEQ ID NO: 1) is cleaved during processing, and a soluble form of the protein is secreted. Thus, the mature soluble polypeptides include those that contain ammo acids 36 to 467. 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481. 482, 483, and up to and including 500 of SEQ ID NO: 1. Exemplary of soluble hyaluronidases are soluble human PH20 polypeptides that are 442, 443, 444, 445, 446 or 447 amino acids in length, such as set forth those set forth above, and variants thereof that have, for example, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and retains hyaluronidase activity7. The generation of such soluble forms of recombinant human PH20 are described, for example, in U.S. Patent No. 7,767.429; 8,202,517; 8,431,380; 8.431,124; 8.450,470; 8.765.685; 8,772,246; 7,871,607; 7,846,431; 7,829,081; 8,105,586; 8,187,855; 8,257,699; 8,580,252; 9,677,061; and 9,677,062.

[0088] Generally soluble forms of PH20 are produced using protein expression systems that facilitate correct N-glycosylation to ensure the polypeptide retains activity7, since glycosylation is important for the catalytic activity7and stability of hyaluronidases. Such cells include, for example Chinese Hamster Ovary (CHO) cells (e.g. DG44 CHO cells).

[0089] The composition that recombinantly produced from mammalian cells, such as CHO cells, has been referred to rHuPH20. It refers to the composition produced upon expression in a cell, such as CHO cell, of nucleic acid encoding residues 36-482 of SEQ ID NO: 1, generally linked to the native (residues 1-35 of SEQ ID NO: 1) or a heterologous signal sequence. rHuPH20 is produced by expression of a nucleic acid molecule, such as encoding amino acids 1-482 (set forth in SEQ ID NO: 1) or 36 to 482 with a heterologous signal sequence. Post translational processing removes the 35 amino acid signal sequence, resulting in polypeptide or a mixture of polypeptides, including those set forth in SEQ ID NO: 3 and 44-49. As produced in the culture medium there is heterogeneity at the C-terminus such that the product, designated rHuPH20, includes a mixture of species that can include any one or more of SEQ ID NO: 3 and 44-49 in various abundance. Generally, the soluble hyaluronidases, rHuPH20 is produced in cells that facilitate correct N-glycosylation to retain activity, such as CHO cells (e.g. DG44 CHO cells). Human soluble PH20 hyaluronidase requires glycosylation for activity. When produced recombinantly from a vector encoding residues 36-582, the most abundant species is the 446 amino acid polypeptides corresponding to residues 36-481 of SEQ ID NO: 1. The particular distribution of resulting polypeptides can depend upon the particular method of production. An exemplary method for production of high levels of PH20 is detailed, for example in U.S. Patent Nos. 8,187,855 and 8,343,487.

[0090] 2. Glycosylation of hyaluronidases

[0091] Glycosylation, including N- and O-linked glycosylation, of some hyaluronidases, including the soluble PH20 hyaluronidases, can be important for their catalytic activity and stability. For some hyaluronidases, removal of N-linked glycosylation can result in near complete inactivation of the hyaluronidase activity. For such hyaluronidases, the presence of N-linked glycans can be important for generating an active enzyme. N-linked oligosaccharides fall into several primary types (oligomannose, complex, hybrid, sulfated), all of which have (Man) 3-GlcNAc-GlcNAc- cores attached via the amide nitrogen of Asn residues that fall within -Asn-Xaa-Thr / Ser-sequences (where Xaa is not Pro). Glycosylation at an -Asn-Xaa-Cys-site has been reported for coagulation protein C. In some instances, a hyaluronidase, such as a PH20 hyaluronidase, can contain N-glycosidic and O- glycosidic linkages. For example, PH20 has O-linked oligosaccharides as well as N-linked oligosaccharides. There are six potential N-linked glycosylation sites at N82, N166, N235, N254, N368, N393 of human PH20 exemplified in SEQ ID NO: 1.

[0092] 3. Variants

[0093] As discussed above, variants of PH20 are known to those of skill in the art, or readily can be prepared in view of the skill and knowledge in the art. Variants include those with amino acid replacements, insertions, and deletions. Variants of the soluble PH20 polypeptides that have altered properties, such as increased stability and / or activity, have been produced. U.S. Patent No. 9,447,401 and family members U.S. Patent Nos. 10,865,400, 11,041,149 and 11,066,656 describe and provide a structure / function map of human PH20 detailing the effects of amino acid replacements at every residue in the catalytic domain of PH20. These patents provide about 7000 examples in which the effects of replacing each amino acid with 15 other amino acids on activity and stability were identified and described. By virtue of those patents, and earlier publications / patents, describing virtually all variants of soluble PH20 polypeptides are known in the art. A skilled person readily can prepare soluble hyaluronidases and variants thereof and know the properties of the resulting hyaluronidase.

[0094] Other variants also are known to those of skill in the art, and can be used in the combinations, regimens, and methods described herein. For example, see, International PCT Publication No. W02020 / 022791 and W02020197230A which are incorporated by reference, and which describe modified PH20 polypeptides. These polypeptides, which include variants of the PH20 polypeptides that generally span residues 38-468, and include replacements, insertions, and deletions. The variants include for example one or more amino acid residues changes S343E, I344N, M345T, M348K, K349E, L353A, L354I, N356E, and I361T (with reference to SEQ ID NO: 1), and others, including about 15 amino acid variations, and truncations at the N-terminus and C-terminus. Variants that contain such modifications and others are set forth in SEQ ID NO: 60-115 of International PCT publication No. W02020 / 022791. Exemplary of these polypeptides is the polypeptide of SEQ ID NO:99, therein, and reproduced below as SEQ ID NO:2. International PCT Publication No. W02021 / 150079 provides variant PH20 polypeptides described as having increased stability relative to unmodified PH20, such as those in rHuPH20. These variant polypeptides have been shown to have PH20 activity and are described as having use for subcutaneous co-administration with other agents.

[0095] E. Methods of Administration, Regimens, and Combinations

[0096] 1. Methods of Administration

[0097] In an embodiment, each of the hyaluronidase and monoclonal antibody, in a specific embodiment dupilumab, can be administered to a patient via injection. In an embodiment the hyaluronidase and monoclonal antibody is administered subcutaneously. For example, the hyaluronidase and monoclonal antibody can be administered to a patient subcutaneously in the abdominal tissue, leg or arm. The hyaluronidase and monoclonal antibody can be administered separately or in the same composition.

[0098] The compositions for administration to a patient via an injection (e.g. subcutaneously) also may comprise suitable inert additives, stabilizers, carriers, or excipients. In an embodiment, the injectable composition comprises histidine. In an embodiment, the injectable composition comprises sodium chloride. In an embodiment, the injectable composition comprises polysorbate. In an embodiment, the polysorbate comprises polysorbate 80. In an embodiment, the injectable composition comprises an antioxidant. In an embodiment, the antioxidant comprises methionine.

[0099] It is shown and described herein that when a monoclonal antibody is administered in combination with the hyaluronidase, dispersion of the co-injected drugs or co-delivered is enhanced. By depolymerizing hyaluronan, hyaluronidase temporarily facilitates dispersion byreducing the viscosity of interstices. The permeability barrier in these tissues is restored to pre-inj ection levels within 24 to 48 hours after injection of hyaluronidase. This allows for higher volumes in a single injection of the monoclonal antibody to be administered to the patient.

[0100] When administered in separate compositions, the hyaluronidase and monoclonal antibody are injected as close to the same site as possible. For example, in an embodiment, hyaluronidase is first injected to a patient at a first injection site and subsequently the monoclonal antibody is injected at the same injection site or at an injection site as close to the first injection site as possible.

[0101] In an embodiment, the monoclonal antibody is administered at a concentration of about 10 mg / kg or about 50 mg / kg. In an embodiment, the monoclonal antibody is administered a concentration of about 100 mg / kg.

[0102] 2. Regimens Provided are regimens for administration of a monoclonal antibody in combination with a soluble hyaluronidase. The monoclonal antibody generally is formulated as a solution for subcutaneous injection at effective concentrations, and the hyaluronidase is provided as a composition containing an effective concentration of soluble hyaluronidase for delivery of an effective amount of hyaluronidase in about 0.5 mL to 10 mL. such as 1 mL to 5 mL, or 1 mL to 3 mL. The monoclonal antibody and hyaluronidase can be administered separately or coformulated for administration in a single composition. When administered separately, they can be administered in any order, but generally the hyaluronidase is administered first. The monoclonal antibody and / or hyaluronidase can be provided as separate compositions, such as suspension and solutions, or can be provided as a co-formulation.

[0103] As described herein, the hyaluronidase and monoclonal antibody can be administered together or sequentially or any other defined regimen. For example, in some embodiments, the hyaluronidase is administered to a patient before the monoclonal antibody is administered i.e. in a first step hyaluronidase is administered to a patient; and in a second step the monoclonal antibody is administered to a patient. In an embodiment a monoclonal antibody is administered to the patient as soon as possible after hyaluronidase has been administered to the patient i.e. immediately after hyaluronidase has been administered to the patient.

[0104] In accord with regimens and compositions provided herein, the hyaluronidase is administered in an amount suitable to allow7a therapeutic dose of a monoclonal antibody to be administered to the patient in a single dose. In some embodiments, the therapeutic dose of the monoclonal antibody is from about lOmg to about 1500 mg. Exemplary7ranges include, but are not limited to, an amount suitable to allow' a dose from 10 mg to 2000 mg be administered to the patient, or dose of at least 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg. 90 mg, 100 mg, 150 mg. 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg. 800 mg, 850 mg. 900 mg, 950 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg or 2000 mg or more to be administered to a patient. In other embodiments, the hyaluronidase is administered in an amount suitable to allow7a dose of at least or at about 100 mg to be administered. It is understood that a skilled practitioner can determine a particular dose, which can depend upon various parameters including the mass of the patient, the age of the patient, and other conditions of the patient.

[0105] In an exemplary embodiment, a monoclonal antibody is administered at a dose of from 100 mg to 2000 mg, such as, but not limited to, a dose of from 900 mg to 2000 mg, such as for example, a dose of at least or at 10 mg to 1000 mg, such as for example, a dose of at least or at 10 mg, 20 mg. 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg or 2000 mg. Exemplary7thereof the monoclonal antibody is administered at a dose of 1000 mg to 2000 mg or in amounts in between such doses. For example, a monoclonal antibody is administered at a dose of about 1500 mg, or at a dose of about 1750 mg, or at a dose of about 1900 mg, or at a dose of about 1950 mg.

[0106] In an embodiment, the amount of the disclosed formulation administered to the subject is dependent on the subject being treated, the severity of the disorder or condition, the rate of administration, the disposition of the compounds, and / or the discretion of the prescnbing physician. In an embodiment, an effective dosage of the monoclonal antibody in the disclosed formulation is in the range of about 0.001 to about 100 mg per kg body weight per day, such as about 1 to about 35 mg / kg / day, in single or divided doses. For a 70 kg human, this would amount to about 0.05 to 7 g / day, such as about 0.05 to about 2.5 g / day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, for example by dividing such larger doses into several small doses for administration throughout the day. Low dose administration of a monoclonal antibody in combination with hyaluronidase is possible due to decreased residence time at the site of administration.

[0107] In an embodiment, the disclosed formulation is administered to the subject in multiple doses. Dosing may be about once, twice, three times, four times, five times, six times, or more than six times per day. Dosing may be about once a month, once every7two weeks, once a week, or once every7other day. In one embodiment, the disclosed formulation is administered about once per day to about 6 times per day. In one embodiment, the administration of the disclosed formulation continues for less than about 7 days. In yet another embodiment the administration continues for more than about 6, 10, 14, 28 days, two months, six months, or one year. In some cases, continuous dosing is achieved and maintained as long as necessary.

[0108] Hyaluronidases have been used clinically since the 1950s. For example, rHuPH20, approved by the FDA in 2004. has been shown to be well tolerated in clinical evaluation of doses of up to 96,000 U, wherein U is USP units. For example, for purposes herein, hyaluronidase is administered at a dose of from 2000 to 15,000 U, such as, but not limited to from 5,000 to 15,000 U, such as 6,000 to 12,000 U, 8,000 to 12,000 U, such as at or about a dose of about 10,000 U, for example 10.000 U. The hyaluronidase is administered in volumes that range from at or about 0.5 mL to 10 mL, such as 1 mL to 5 mL, or at or aboutl mL to 3 mL, such as a 1 mL; the volume is a function of the specific activity of a particular formulation of the hyaluronidase. The particular amount depends upon parameters understood by those of skill in the art. Co-administration of a monoclonal antibody with hyaluronidase, as noted, allows for administration of a higher doses and larger volumes of the monoclonal antibody potentially affording a longer interval between injections. This can increase the convenience of long-acting regimens and can result in better adherence to therapy and positively impact treatment outcomes and acceptability.

[0109] As discussed above, the combination of a monoclonal antibody and hyaluronidase can allow less frequent dosing compared to administration of the monoclonal antibody alone, in dosing regimens that do not include a hyaluronidase. For example, in accord with the instant disclosure, hyaluronidase and monoclonal antibody are administered once every 3 months to once every year, such as once every 3 months, once every' 4 months, once every 5 months or once every 6 months or other intervals that are longer than 3 months and less than 6 months, 9 months, or one year. In an exemplary regimen, hyaluronidase and monoclonal antibody are administered once every 3 months.

[0110] In an embodiment, combination dosing regimen is provided that comprises administering hyaluronidase; and a monoclonal antibody, wherein hyaluronidase is administered at a dose of from 4000 to 15,000 U; and the monoclonal antibody is administered at a dose of from 10 to 100 mg / kg wherein the combination dosing regimen is administered once every 3 months to once every 6 months.

[0111] In another embodiment, provided is a combination dosing regimen comprising administering hyaluronidase; and administering a monoclonal antibody, wherein hyaluronidase is administered at a dose of from 6000 to 12,000 U; and the monoclonal antibody is administered at a dose of from 10 to 100 mg / kg. Wherein the combination dosing regimen is administered once every 3 months to once every 6 months.

[0112] In another embodiment, provided a combination dosing regimen comprising administering hyaluronidase; and administering a monoclonal antibody, wherein hyaluronidase is administered at a dose of 10.000 U; and the monoclonal antibody is administered at a dose of from 10 mg / kg to 100 mg / kg. In this regime the monoclonal antibody has a concentration of 10 to 100 mg / kg, and the combination dosing regimen is administered once every 3 months.

[0113] The regimens provided herein are for treating or preventing cancer, comprising administering to a patient in need of treatment or prevention a combination dosing regimen described herein. Prevention, as described herein, includes reducing the risk of infection. Hence in embodiments herein, provide are methods of treating a cancer, the method comprising administering to a patient a combination dosing regimen described herein. In an alternative embodiment, provided is a method of preventing cancer, the method comprising administering to a human the combination dosing regimen described herein. In the first method, the patient has been diagnosed with cancer; in the latter, the subject has not been diagnosed with a cancer, but generally is a subject at risk of exposure to a monoclonal antibody.

[0114] In a further aspect, the combination dosing regimens as described herein are for use in the treatment or prevention of cancer. In an embodiment, the combination dosing regimens described herein are for use in the treatment of cancer. In an alternative embodiment, the present invention provides the combination dosing regimen as described herein for use in the prevention of cancer.

[0115] In an embodiment, cancers for treatment or prevention as described herein include but are not limited to, anaplastic large cell lymphoma (ALCL), peripheral T-cell lymphoma (PTCL), adult T-cell leukemia / lymphoma, cutaneous T-cell lymphoma (CTCL), extra-nodal NK-T-cell lymphoma, non-Hodgkin’s lymphoma, diffuse large B-cell lymphoma, particularly EBV-positive diffuse large B-cell lymphoma. B cell acute lymphoblastic leukemia, breast cancer, lung cancer, gastric cancer, ovarian cancer, colon cancer, gastric cancer, endometrial cancer, cervical cancer, colorectal cancer, esophageal cancer, squamous cell carcinoma, pancreatic cancer, prostate cancer, stomach cancer, thyroid cancer, glioma, melanoma, urinary bladder cancer, urogenital cancer and uterine cancer.

[0116] 3. Combinations, Compositions and Kits

[0117] Provided herein are compositions, combinations, and kits. The combinations comprise a composition containing the hyaluronidase: and a composition that is suspension comprising a monoclonal antibody. The compositions comprising the monoclonal antibody are formulated as a suspension in amounts for administering a dose of the monoclonal antibody, such as in an amount that is 10 mg to 2000 mg, such as such as, but not limited to, a dose of from 10 mg to 1000 mg, such as for example, a dose of at least or at 10 mg, 20 mg, 30 mg. 40 mg, 50 mg. 60 mg. 70 mg. 80 mg. 90 mg. 100 mg, 150 mg. 200 mg, 250 mg. 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg or 2000 mg as detailed above. Each of the compositions can be formulated for single dosage or multiple dosage administration or for dilution as appropriate.

[0118] The hyaluronidase is formulated for administration of a dose from 2000 to 15,000 U. such as, but not limited to from 5,000 to 15,000 U, such as 6,000 to 12,000 U, 8,000 to 12,000 U, such as at or about a dose of about 10,000 U, for example 10,000 U. Compositions containing hyaluronidase for administration are well known, and generally are formulated at a pH of about 7 to about 7.4, in appropriate buffers, salts, stabilizers and surfactant as needed. See e.g. U.S. Patent No. 7,767,429. Variants, as described herein, that are more stable in denaturing conditions, such as those described in U.S. Patent No. 9,447,401 and family members and variants designed for increased activity and / or stability can be formulated. The hyaluronidase and monoclonal antibody can be co-formulated as suspensions or mixed prior to use for administration in a single composition. The compositions containing both are formulated to deliver an appropriate dose of each.

[0119] The combinations can contain the two compositions or the single co-formulations, and optionally instructions for use. The combinations can be packaged as kits. Exemplary combinations and kits can include a syringe or other container containing the hyaluronidase, and a syringe or other container containing the monoclonal antibody. Alternatively, the monoclonal antibody and hyaluronidase can be provided in a dual compartment container, such as a dual compartment where the compositions are separated, such as by a membrane that can be punctured prior to administration. In these aspects, the hyaluronidase and monoclonal antibody are as described herein.

[0120] The syringe or other container may be sized and shaped to hold a volume of the hyaluronidase corresponding to a volume selected from:

[0121] (a) 3 mL to 5 rnE, 3 mL to 10 mL, 3 mL to 15 mL, 3 mL to 20 mL, 3 mL to 25 mU, 3 ml to 30 mE, 3 mL to 35 mL, 3 mL to 40 mL, 3 mL to 45 mL, 3 mL to 50 mL, 5 mL to 10 mL, 5 mL to 15 mL, 5 mL to 20 mL, 5 mL to 25 mL, 5 mL to 30 mL, 5 mL to 35 mL. 5 mL to 40 mL; 5 mL to 45 mL. 5 mL to 50 mL, 10 mL to 15 mL; 10 mL to 20 mL; 10 mL to 25 mL; 10 mL to 30 mL; 10 mL to 35 mL; 10 mL to 40 mL, 10 mL to 50 mL;

[0122] (b) about 3 mL to about 5 mL, about 3 mL to about 10 mL, about 3 mL to about

[0123] 15 mL, about 3 mL to about 20 mL, about 3 mL to about 25 mL, about 3 ml to about 30 mL, about 3 mL to about 35 mL. about 3 mL to about 40 mL, about 3 mL to about 45 mL, about 3 mL to about 50 mL. about 5 mL to about 10 mL. about 5 mL to about 15 mL, about 5 mL to about 20 mL, about 5 mL to about 25 mL, about 5 mL to about 30 mL, about 5 mL to about 35 mL, about 5 mL to about 40 mL; about 5 mL to about 45 mL, about 5 mL to about 50 mL. about 10 mL to about 15 mL; about 10 mL to about 20 mL; about 10 mL to about 25 mL; about 10 mL to about 30 mL; about 10 mL to about 35 mL; about 10 mL to about 40 mL, about 10 mL to about 50 mL; (c) at least about 3 mL, at least about 3.5 mL, at least about 4 mL, at least about

[0124] 4.5 mL, at least about 5.5 mL, at least about 6 mL, at least about 6.5 mL, at least about 7 mL, at least about 7.5 mL, at least about 8 mL, at least about 8.5 mL, at least about 9 mL, at least about 9.5 mL, at least about 10 mL, at least about 10.5 mL, at least about 11 mL, at least about 11.5 mL. at least about 12 mL, at least about 12.5 mL, at least about 13 mL, at least about 13.5 mL, at least about 14 mL, at least about 14.5 mL, at least about 15 mL, at least about 15.5 mL, at least about 16 mL, at least about 16.5 mL, at least about 17 mL, at least about 17.5 mL, at least about 18 mL, at least about 18.5 mL, at least about 19 mL, at least about 19.5 mL. at least about 20 mL, at least about 25 mL, at least about 30 mL, at least about 35 mL, at least about 40 mL, at least about 45 mL, at least about 50 mL; and

[0125] (d) at least 3 mL, at least 3.5 mL, at least 4 mL, at least 4.5 mL, at least 5.5 mL, at least 6 mL, at least 6.5 mL, at least 7 mL, at least 7.5 mL, at least 8 mL, at least 8.5 mL, at least 9 mL, at least 9.5 mL, at least 10 mL, at least 10.5 mL, at least 11 mL, at least

[0126] 11.5 mL, at least 12 mL, at least 12.5 mL, at least 13 mL, at least 13.5 mL, at least 14 mL, at least 14.5 mL, at least 15 mL, at least 15.5 mL, at least 16 mL, at least 16.5 mL, at least 17 mL, at least 17.5 mL, at least 18 mL, at least 18.5 mL, at least 19 mL, at least 19.5 mL, at least 20 mL, at least 25 mL, at least 30 mL, at least 35 mL, at least 40 mL, at least 45 mL, at least 50 mL.

[0127] The hyaluronidase may be delivered at a rate of approximately 0.08-0.75 mL / sec. For example, this would provide target delivery time ranges of 13-120 seconds for a 10 mL dose volume. 10 mL of the hyaluronidase may be delivered at a rate of 0.33 mL / sec. In one embodiment, the hyaluronidase is delivered at a rate of:

[0128] (a) 0.5 mL / 10 sec., 0.75 mL / 10 sec., 1 mL / 10 sec., 1.25 mL / 10 sec., 1.5 mL / 10 sec., 1.75 mL / 10 sec, 2 mL / 10 sec., 2.25 mL / 10 sec, 2.5 mL / 10 sec., 2.75 mL / 10 sec, 3 mL / 10 sec., 3.25 mL / 10 sec, 3.5 mL / 10 sec., 3.75 mL / 10 sec, 4 mL / 10 sec., 4.25 mL / 10 sec, 4.5 mL / 10 sec., 4.75 mL / 10 sec. 5 mL / 10 sec;

[0129] (b) 2 mL / 30 sec., 2.5 rnL / 30 sec., 3 mL / 30 sec., 3.5 mL / 30 sec., 4 rnL / 30 sec., 4.5 mL / 30 sec., 5 mL / 30 sec., 5.5 mL / 30 sec.. 6 mL / 30 sec., 6.5 mL / 30 sec., 7 mL / 30 sec..

[0130] 7.5 rnL / 30 sec., 8 mL / 30 sec., 8.5 mL / 30 sec., 9 rnL / 30 sec., 9.5 mL / 30 sec., 10 mL / 30 sec., 10.5 mL / 30 sec.; and

[0131] (c) 4 mL / min, 5 mL / min, 6 mL / min, 7 rnL / min, 8 mL / min, 9 mL / min, 10 mL / min, 11 mL / min, 12 mL / min, 13 mL / min, 14 mL / min, 15 mL / min, 16 mL / min, 17 mL / min, 18 mL / min, 19 mL / min. 20 mL / min, 21 mL / min. The hyaluronidase may be delivered at a delivery time of approximately of 13-120 seconds. In one embodiment, the hyaluronidase is delivered at a delivery' time of:

[0132] (a) about 10 seconds, about 12 seconds, about 16 seconds, about 18 seconds, about 20 seconds, about 22 seconds, about 24 seconds, about 26 seconds, about 28 seconds, about 30 seconds, about 32 seconds, about 34 seconds, about 36 seconds, about 38 seconds, about 40 seconds, about 42 seconds, about 44 seconds, about 46 seconds, about 48 seconds, about 50 seconds, about 52 seconds, about 54 seconds, about 56 seconds, about 58 seconds, about 60 seconds, about 65 seconds, about 70 seconds, about 75 seconds, about 80 seconds, about 85 seconds, about 90 seconds, about 95 seconds, about 100 seconds, about 105 seconds, about 110 seconds, about 115 seconds, or about 120 seconds.

[0133] (b) about 10 seconds to about 120 seconds, about 12 seconds to about 115 seconds, about 16 seconds to about 110 seconds, about 18 seconds to about 105 seconds, about 20 seconds to about 100 seconds, about 22 seconds to about 95 seconds, about 24 seconds to about 90 seconds, about 26 seconds to about 85 seconds, about 28 seconds to about 80 seconds, about 30 seconds to about 75 seconds, about 32 seconds to about 70 seconds, about 34 seconds to about 65 seconds, about 36 seconds to about 60 seconds, about 38 seconds to about 58 seconds, about 40 seconds to about 56 seconds, about 42 seconds to about 54 seconds, about 44 seconds to about 52 seconds or about 46 seconds to about 50 seconds.

[0134] In an embodiment, the compositions and combinations described herein can comprise one or more of inactive ingredients, including but not limited to, a divalent cation, a buffer, a pH adjusting agent, an anti-oxidation agent, a tonicity modifier, a surfactant, and other inactive ingredients / agents described below. Provided below is a description of the inactive ingredients that can be included in the hyaluronidase compositions and combinations described herein. The inactive ingredients are exemplary only and provide a platform from which minor adjustments can be made. It is understood that very small changes in the concentrations of the various excipients and other components (e.g. ±15% of the stated concentrations), or small changes in pH, can be made while retaining some if not all of the hyaluronan degrading enzy me stability'. Further changes also can be made by adding or removing excipients. For example, the type of stabilizing surfactant can be changed.

[0135] Divalent Cation

[0136] In some embodiments, the hyaluronidase compositions and combinations provided herein comprise an amount of a divalent cation to achieve at least 50%. and generally at least 70%, of the initial enzymatic activity of the hyaluronidase at temperatures of between or approximately between 37° C. to 42° C., such as at least or about or approximately 37° C or 40° C, for at least three (3) days and generally at least one month (e.g. 4 weeks) as described herein. For example, the amount of divalent cation is an amount to achieve at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99% or more of the initial enzymatic activity of the hyaluronidase for at least three (3) days, and generally for at least 4 weeks at temperatures between or approximately between 37° C. to 42° C., such as at least or about or approximately 40° C.

[0137] For example, hyaluronidase compositions and combinations provided herein can contain an amount of Lys-Lys, salt, derivative, analogue or mimetic thereof, to achieve at least 50%, and generally at least 70%, of the initial enzymatic activity of the hyaluronandegrading enzyme at temperatures between or approximately between 37° C to 42° C, such as at least or about or approximately 40° C, for at least three (3) days and generally for at least 4 weeks. Such a hyaluronidase composition or combination provided herein may contain between or about between 5 mM to 300 mM Lys-Lys, such as 10 mM to 200 mM, 50 mM to 150 mM or 10 mM to 50 mM. For example, a hyaluronidase composition or combination provided herein may contain at least or about at least or 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 125 mM, 150 mM, 200 mM, 300 mM or more Lys-Lys.

[0138] In another example, the hyaluronidase compositions or combinations provided herein can contain an amount of MgCh, a derivative, an analogue or a mimetic thereof, to achieve at least 50%, and generally at least 70%, of the initial enzymatic activity of the hyaluronidase at temperatures between or approximately between 37° C to 42° C, such as at least or about or approximately 40° C, for at least three (3) days and generally for at least 4 weeks. The hyaluronidase compositions and combinations provided herein may contain between or about between 5 mM to 300 mM MgCh, such as 10 mM to 200 mM, 50 mM to 150 mM or 10 mM to 50 mM. For example, the hyaluronidase compositions and combinations provided herein may contain at least or about at least or 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 125 mM, 150 mM, 200 mM, 300 mM or more MgCh.

[0139] As discussed below, formulations containing a divalent cation (e.g. Lys-Lys), if necessary’, also can contain a tonicity modifier (e.g. NaCl).

[0140] If necessary', the pH of the hyaluronidase compositions and combinations described herein can be adjusted using acidifying agents to lower the pH or alkalizing agents to increase the pH. Exemplary acidifying agents include, but are not limited to, acetic acid, citric acid, sulfuric acid, hydrochloric acid, monobasic sodium phosphate solution, and phosphoric acid. Exemplary alkalizing agents include, but are not limited to, dibasic sodium phosphate solution, sodium carbonate, or sodium hydroxide.

[0141] Any buffer can be used in the compositions and combinations provided herein so long as it does not adversely affect the stability of the composition / combination and supports the requisite pH range required. Examples of particularly suitable buffers include Tris, succinate, acetate, phosphate buffers, histidine, citrate, aconitate, malate and carbonate. Those of skill in the art, however, will recognize that the compositions and combinations provided herein are not limited to a particular buffer, so long as the buffer provides an acceptable degree of pH stability, or “buffer capacity'’ in the range indicated. Generally, a buffer has an adequate buffer capacity within about 1 pH unit of its pK. Buffer suitability can be estimated based on published pK tabulations or can be determined empirically by methods well known in the art. The pH of the solution can be adjusted to the desired endpoint within the range as described above, for example, using any acceptable acid or base.

[0142] Buffers that can be included in the compositions and combinations provided herein include, but are not limited to, Tris (Tromethamine), histidine, phosphate buffers, such as dibasic sodium phosphate, and citrate buffers. For example, the buffer can be a histidine hydrochloride (histidine / HCl) buffer. Generally, the buffering agent is present in an amount herein to maintain the pH range of the composition or combination between or about between 6.5 to 7.8, for example between or about between 6.8 to 7.8 such as between or about between 7.0 to 7.6. Such buffering agents can be present in the compositions and combinations at concentrations between or about between 1 mM to 100 mM. such as 10 mM to 50 mM or 20 mM to 40 mM, such as at or about 30 mM. For example, such buffering agents can be present in the compositions and combinations in a concentration of or about or at least 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM. 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, or more.

[0143] In some examples, a buffering agent is not required. In an embodiment, the hyaluronidase compositions and combinations described herein comprise a surfactant. The surfactants generally are non-ionic surfactants. Surfactants that can be included in the compositions and combinations herein include, but are not limited to, partial and fatty' acid esters and ethers of polyhydric alcohols such as of glycerol, or sorbitol, poloxamers and polysorbates. For example, exemplary surfactants in the compositions and combinations herein include any one or more of poloxamer 188 (PLURONICS® such as PLURONIC® F68), TETRONICS®, polysorbate 20, polysorbate 80, PEG 400, PEG 3000, Tween® (e.g. Tween® 20 or Tween® 80), Triton® X-100, SPAN®. MYRJ®, BRIJ®, CREMOPHOR®. polypropylene glycols or polyethylene glycols. In some examples, the compositions and combinations herein contain poloxamer 188, polysorbate 20, polysorbate 80, generally poloxamer 188 (pluronic F68).

[0144] In the compositions and combinations provided herein, the total amount of the one or more surfactants as a percentage (%) of mass concentration (w / v) in the compositions and combinations herein can be, for example, between from or between about from 0.0% to 1.0%, such as between or about between 0.0% to 0.0005%, 0.0005% to 0.005%, 0.001% to 0.01%, 0.01% to 0.5%, 0.01% to 0.1% or 0.01% to 0.02%. For example, the compositions and combinations provided herein can contain at or about 0.001%, 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%. 0.04%. 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.08%. or 0.09% surfactant.

[0145] Anti-Oxidation Agent

[0146] The compositions and combinations provided herein also can contain antioxidants to reduce or prevent oxidation, in particular oxidation of the hyaluronidase. Exemplary antioxidants include, but are not limited to, cysteine, tryptophan and methionine. In particular examples, the anti-oxidant is methionine. The compositions and combinations provided herein can include an antioxidant at a concentration from between or from about between 5 mM to or to about 50 mM, such as 5 mM to 40 mM, 5 mM to 20 mM or 10 mM to 20 mM. For example, methionine can be provided in the compositions and combinations herein at a concentration from between or from about between 5 mM to or to about 50 mM, such as 5 mM to 40 mM, 5 mM to 20 mM or 10 mM to 20 mM. For example, an antioxidant, for example methionine, can be included at a concentration that is or is about or is at least 5 mM, 10 mM, 11 mM, 12 mM. 13 mM, 14 mM. 15 mM, 16 mM. 17 mM, 18 mM, 19 mM. 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 35 mM, 40 mM, 45 mM or 50 mM. In some examples, compositions and combinations described herein contain 10 mM to 20 mM methionine, such as or about or at least 10 mM or 20 mM methionine.

[0147] Tonicity Modifier

[0148] Optionally, the stable hyaluronidase compositions and combinations provided herein can contain a tonicity modifier.

[0149] For example, in some embodiments, a tonicity modifier is included in the compositions and combinations herein to produce a solution with the desired osmolality. The compositions and combinations provided herein have an osmolality of between or about between 245 mOsm / kg to 500 mOsm / kg. For example, the osmolality is or is about or at least 245 mOsm / kg, 250 mOsm / kg, 255 mOsm / kg, 260 mOsm / kg, 265 mOsm / kg, 270 mOsm / kg, 275 mOsm / kg, 280 mOsm / kg, 285 mOsm / kg, 290 mOsm / kg, 295 mOsm / kg, 300 mOsm / kg, 350 mOsm / kg, 400 mOsm / kg, 450 mOsm / kg or 500 mOsm / kg. Typically, a tonicity modified is included in the compositions and combinations herein that contain a divalent cation, such as Lys-Lys, in a concentration that is less than 100 mM, such as less than 80 mM, 70 mM. 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM or less. For example, a tonicity modified is included in the compositions and combinations herein that contain a divalent cation, such as Lys-Lys, at a concentration of between or about between 10 mM to 50 mM, such as about or approximately 10 mM. 15 mM, 20 mM. 25 mM, 30 mM, 35 mM, 40 mM, 45 mM or 50 mM.

[0150] Tonicity modifiers include, but are not limited to, glycerin, NaCl, amino acids, polyalcohols, trehalose, and other salts and / or sugars. For example, the compositions and combinations provided herein can optionally include NaCl as atonicity modifier. The NaCl can be included at a concentration of between or about between 0 mM to 200 mM, such as generally 30 mM to 100 mM, 50 mM to 160 mM, for example 50 mM to 120 mM or 80 mM to 140 mM. Generally, the NaCl is less than 150 mM. and generally less than 140 mM, 130 mM, 120 mM, 110 mM, 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM or less. The particular amount can be empirically determined in order to retain enzyme activity and / or tonicity7.

[0151] In another example, glycerin (glycerol) is optionally included in the compositions and combinations described herein. For example, the compositions and combinations provided herein typically contain less than 60 mM glycerin, such as less than 55 mM, less than 50 mM, less than 45 mM, less than 40 mM, less than 35 mM, less than 30 mM, less than 25 mM, less than 20 mM, less than 15 mM, 10 mM or less.

[0152] Other Agents or Excipients

[0153] The stable compositions and combinations provided herein can optionally contain one or more other agents, carriers, excipients or preservatives. For example, exemplar}7stabilizers that optionally can be included in the hyaluronidase compositions and combinations provided herein include, but are not limited to, amino acids, amino acid derivatives, amines, sugars, polyols, salts and buffers, surfactants, and other agents. For example, included among the ty pes of stabilizers that optionally can be contained in the formulations herein is an amino acid stabilizer or a hyaluronidase inhibitor (e.g. a hyaluronidase substrate, such as hyaluronan). Exemplary amino acid stabilizers, amino acid derivatives or amines include, but are not limited to, L- Arginine. Glutamine, glycine, Lysine, Methionine, Proline, Lys-Lys, Gly-Gly, Trimethylamine oxide (TMAO) or betaine. Exemplary of sugars and polyols include, but are not limited to, glycerol, sorbitol, mannitol, inositol, sucrose or trehalose. Exemplary of salts and buffers include, but are not limited to, magnesium chloride, sodium sulfate, Tris such as Tris (100 mM), or sodium Benzoate. Exemplary surfactants include, but are not limited to, poloxamer 188 (e.g. Pluronic® F68), polysorbate 80 (PS80), polysorbate 20 (PS20). Other stabilizers include, but are not limited to, hyaluronic acid (EIA), human serum albumin (HSA). phenyl butyric acid, taurocholic acid, polyvinylpyrolidone (PVP) or zinc.

[0154] In an embodiment, the hyaluronidase compositions and combinations also can optionally contain an amount of preservative(s) that, when combined with the components set forth above, result in a stable composition or combination. When included, the preservatives are present in a sufficient concentration to provide the anti-microbial requirements of, for example, the United States Pharmacopoeia (USP) and the European Pharmacopoeia (EP). Typically, formulations that meet EP (EPA or EPB) anti-microbial requirements contain more preservative than those formulated only to meet USP anti-microbial requirements. Generally, when included, the compositions and combinations provided herein contain preservative(s) in an amount that exhibits anti-microbial activity by killing or inhibiting the propagation of microbial organisms in a sample of the composition as assessed in an antimicrobial preservative effectiveness test (APET). Non-limiting examples of preservatives that can be included in the compositions and combinations provided herein include, but are not limited to, phenol, meta-cresol (m-cresol). methylparaben, benzyl alcohol, thimerosal, benzalkonium chloride, 4-chloro-l -butanol, chlorhexidine dihydrochloride, chlorhexidine digluconate, L-phenylalanine, EDTA, bronopol (2-bromo-2-nitropropane-l,3-diol), phenylmercuric acetate, glycerol (glycerin), imidurea, chlorhexidine, sodium dehydroacetate, ortho-cresol (o-cresol), para-cresol (p-cresol), chlorocresol, cetrimide, benzethonium chloride, ethylparaben, propylparaben or butylparaben and any combination thereof. In one example, the compositions and combinations contain at least one phenolic preservative. For example, the composition or combination contains phenol, m-cresol or phenol and m-cresol. When included in the compositions and combinations provided herein, the total amount of the one or more preservative agents as a percentage (%) of mass concentration (w / v) in the composition and combination can be, for example, between from or between about from 0.1% to 0.4%, such as 0.1% to 0.3%, 0.15% to 0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2% to 0.3%, or 0.3% to 0.4%, and generally less than 0.4% (w / v) preservative, for example, at least or about at least 0.1%, 0. 12%, 0. 125%. 0. 13%. 0. 14%. 0. 15%. 0. 16%, 0. 17%, 0. 175%, 0. 18%. 0. 19%. 0.2%, 0.25%, 0.3%, 0.325%, 0.35% but less than 0.4% total preservative.

[0155] Optionally, the compositions and combinations can include carriers such as a diluent, adjuvant, excipient, or vehicle with which the formulation is administered. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences’’ by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, generally in purified form or partially purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions also can be employed as liquid carriers, particularly for injectable solutions.

[0156] For example, pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances. Examples of aqueous vehicles include Sodium Chloride Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, com oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to parenteral preparations packaged in multiple-dose containers, which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN 80). A sequestering or chelating agent of metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0157] Compositions can contain along with an active ingredient: a diluent such as lactose, sucrose, di calcium phosphate, or carboxymethylcellulose: a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acacia, gelatin, glucose, molasses, polyvinylpyrrolidone, celluloses and derivatives thereof, povidone, crospovidones and other such binders know n to those of skill in the art.

[0158] For example, an excipient protein can be added to the composition or combination that can be any of a number of pharmaceutically acceptable proteins or peptides. Generally, the excipient protein is selected for its ability to be administered to a mammalian subject without provoking an immune response. For example, human serum albumin is generally well-suited for use in pharmaceutical formulations. Other known pharmaceutical protein excipients include, but are not limited to, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. The excipient is included in the composition or combination at a sufficient concentration to prevent adsorption of the protein to the holding vessel or vial. The concentration of the excipient will vary according to the nature of the excipient and the concentration of the protein in the composition or combination.

[0159] A composition or combination, if desired, also can contain minor amounts of wetting or emulsifying agents, or pH buffering agents, for example, acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.

[0160] 3. Methods of Administration

[0161] In an embodiment, each of the hyaluronidase and monoclonal antibody can be administered to a patient via injection. In an embodiment the hyaluronidase and monoclonal antibody are administered subcutaneously. For example, the hyaluronidase and monoclonal antibody can be administered to a patient subcutaneously in the abdominal tissue. The hyaluronidase and monoclonal antibody can be administered separately or in the same composition.

[0162] In an embodiment, the hyaluronidase and monoclonal antibody are administered topically by mucosal delivery. In an embodiment, the mucosal delivery is selected from the group consisting of buccal delivery', pulmonary delivery', ocular delivery, nasal delivery', intranasal delivery, vaginal delivery, and oral delivery. In an embodiment, the hyaluronidase is administered directly to a mucosal tissue of the human subject, including at the affected site. In an embodiment, the mucosal tissue is selected from the group consisting of anterior nostril, nasal sinus, vaginal, esophagus, urethral, sublingual and buccal.

[0163] In an embodiment, various delivery systems are known and can be used to administer the hyaluronidase in combination with a monoclonal antibody. For example, the hyaluronidase can be encapsulated in liposomes, microparticles, microcapsules for topical delivery. In addition, pulmonary administration can also be used, such as inhalers or nebulizers, and aerosol formulations.

[0164] If the hyaluronidase and monoclonal antibody comprise pulmonary or intranasal administration, the composition can be formulated in the form of an aerosol, spray, mist or drip. In particular, the hyaluronidase and monoclonal antibody can be provided by the use of suitable propellants (such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases). Aerosol sprays are delivered from pressurized packaging or sprayers. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve that can deliver a metered amount. Capsules and cartridges (made of, for example, gelatin) containing powder mixtures of compounds and suitable powder bases such as lactose or starch can be formulated and used in inhalers.

[0165] If the hyaluronidase and monoclonal antibody are administered topically, including directly to the affected site, the compositions can be in the form of ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion. With regard to non-sprayable topical dosage forms, it is generally employed to include a carrier or one or more excipients compatible with topical application, and the dynamic viscosity is preferably greater than the viscosity of water to a semi-solid or solid form. Suitable formulations include (but are not limited to) solutions, suspensions, emulsions, creams, ointments, powders, wipes, ointments, which are sterilized or used to affect various properties such as osmotic pressure, if necessary, adjuvants (such as preservatives, stabilizers, wetting agents, buffers or salts) are present in the composition.

[0166] Other suitable topical dosage forms include sprayable aerosol formulations, where the active ingredient, optionally combined with a solid or liquid inert carrier, is mixed and encapsulated with a pressurized volatile substance (such as a gaseous propellant such as freon) or encapsulated in a squeeze bottle. If necessary, a moisturizing agent or humectant may also be added to the composition.

[0167] In an embodiment, it may be necessary to locally administer the hyaluronidase to the affected site in need of treatment; this may be achieved by, for example, but not limited to, topical administration, local infusion, injection, or by means of an implant. The implant may be a porous or non-porous material, including membranes and matrices, such as silicone membranes, polymers, fibrous matrices or collagen matrices.

[0168] In an embodiment, the invention provides a composition for transdermal delivery containing the hyaluronidase, monoclonal antibody and a pharmaceutical excipient suitable for transdermal deliver . Compositions of the present invention can be formulated into preparations in solid, semi-solid, or liquid forms suitable for local or topical administration, such as gels, water soluble jellies, creams, lotions, suspensions, foams, powders, slurries, ointments, solutions, oils, pastes, suppositories, sprays, emulsions, saline solutions, dimethylsulfoxide (DMSO)-based solutions. In general, carriers with higher densities are capable of providing an area with a prolonged exposure to the active ingredients. In contrast, a solution formulation may provide more immediate exposure of the active ingredient to the chosen area.

[0169] The transdermal compositions also may comprise suitable solid or gel phase carriers or excipients, which are compounds that allow increased penetration of, or assist in the delivery of, the hyaluronidase and monoclonal antibody across the stratum comeum permeability barrier of the skin. There are many of these penetration-enhancing molecules know n to those skilled in the field of topical formulation. Examples of such carriers and excipients include, but are not limited to, humectants (e.g. urea), glycols (e.g. ethylene glycol, propylene glycol), alcohols (e.g. methanol, ethanol, propanol, including isopropanol and n- propanol; butanol, including n-butanol, isobutanol, tert-butanol, and sec-butanol; pentanol, including 1-pentanol, 2-pentanol, 3-pentanol, 2-methyl-l-butanol, 3 -methyl- 1 -butanol, 2- methyl-2-butanol, 2-methy 1-3 -butanol, and 2,2-dimethylpropanol; and hexanol, including hexan-l-ol, hexan-2-ol, hexan-3-ol, 2-methylpentan-l-ol, 3-methylpentan-l-ol, 4- methylpentan-l-ol. 2-methylpentan-2-ol, 3-methylpentan-2-ol, 4-methylpentan-2-ol. 2- methylpentan-3-ol , 3-methylpentan-3-ol, 2,2-dimethylbutan-l-ol , 2,3-dimethylbutan-l-ol , 3,3-dimethylbutan-l-ol, 2,3-dimethylbutan-2-ol , 3,3-dimethylbutan-2-ol, and 2-ethylbutan-l- ol), fatty acids (e.g. oleic acid, a-linolenic acid, linoleic acid, y-linolenic acid, palmitoleic acid), surfactants (e.g. isopropyl myristate and sodium lauryl sulfate), pyrrolidones (e.g. N- methyl-2-pyrrolidone, 2-pyrrolidone), glycerol monolaurate, sulfoxides (e.g. dimethyl sulfoxide, decylmethylsulfoxide), terpenes (e.g. menthol, 1,8-cineole, limonene, menthone, nerolidol), amines, amides, alkanes, alkanols, water, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols and polypropylene glycols.

[0170] Another exemplary formulation for delivery hyaluronidase and monoclonal antibody employs transdermal delivery devices (“patches’’). Such transdermal patches may be used to provide continuous or discontinuous infusion of the hyaluronidase in controlled amounts, either with or without another active pharmaceutical ingredient. The construction and use of transdermal patches for the delivery of pharmaceutical agents is known. See, e.g. U.S. Patent No. 5,023,252; 4,992,445 and 5.001,139 incorporated by reference herein. Such patches may be constructed for continuous, pulsatile, or on demand delivery of hyaluronidase and monoclonal antibody.

[0171] A diverse range of delivery methods may be utilized to cater to a wide range of medical needs. These methods offer patients and healthcare providers options based on the nature of the medication, dosage requirements, and the specific condition being treated. In some embodiments, an injector, or other handheld devices for injections that allow patients to self-administer preset doses, is used to administer the hyaluronidase and monoclonal antibody to the affected site. In some embodiments, the autoinjector is a prefilled syringe, a high volume autoinjector or a large volume autoinjector. The injector may be button actuated or needle guard actuated and may be configured to deliver a single dose or a plurality of doses.

[0172] A wearable administration apparatus may be utilized to administer the hyaluronidase and monoclonal antibody to the affected site in cases that require prolonged delivery. In some embodiments, an on-body injector, or other wearable device that delivers a larger volume of medication subcutaneously over an extended period, is used to administer the hyaluronidase and monoclonal antibody to the affected site. In some embodiments, a patch pump, or other wearable device adhering to the skin that delivers medication subcutaneously through an injection site, is used to administer the hyaluronidase and monoclonal antibody to the affected site. In some embodiments, a wearable infusion pump, or other device worn on the body for continuous subcutaneous infusion of medication, is used to administer the hyaluronidase and monoclonal antibody to the affected site.

[0173] An implantable administration apparatus may be utilized to administer the hyaluronidase and monoclonal antibody. The implantable apparatus may be made of porous or non-porous materials (such as silicone membranes, polymers, fibrous matrices, or collagen matrices) that release medication gradually over time. In some embodiments, a subcutaneous impact, or other implant specifically designed to be placed beneath the skin for controlled and sustained release of medication, is used to administer the hyaluronidase and monoclonal antibody to the affected site. In some embodiments, an intramuscular implant, or other implant designed for insertion into muscle tissue, providing a localized and sustained release of medication, is used to administer the hyaluronidase to the affected site. In some embodiments, an intradermal implant, or other implant placed within the dermal layer of the skin for targeted and controlled delivery of medication, is used to administer the hyaluronidase and monoclonal antibody to the affected site. In some embodiments, topical administration, or other application directly onto the skin, including creams, gels, ointments, and transdermal patches, is used to administer the hyaluronidase and monoclonal antibody to the affected site.

[0174] The compositions for administration to a patient via an injection (e.g. subcutaneously) also may comprise suitable inert additives, carriers, or excipients. In an embodiment, the injectable composition or combination comprises histidine. In an embodiment, the injectable composition comprises sodium chloride. In an embodiment, the injectable composition comprises polysorbate. In an embodiment, the polysorbate comprises polysorbate 80. In an embodiment, the injectable composition comprises an antioxidant. In an embodiment, the antioxidant comprises methionine.

[0175] In an embodiment, the injectable combination comprises a hyaluronidase and an antibody, including any antibody from Table A. In an embodiment, the antibody and hyaluronidase are mixed with an inert additive, carrier, or excipient selected from dextran (e.g. dextran 40). sodium chloride, sodium phosphate dibasic anhydrous, sodium phosphate monobasic monohydrate, and sucrose. In an embodiment, the antibody and hyaluronidase is mixed with each dextran (e.g. dextran 40), sodium chloride, sodium phosphate dibasic anhydrous, sodium phosphate monobasic monohydrate, and sucrose. In an embodiment, the antibody and hyaluronidase are mixed with an inert additive, carrier, or excipient selected from trehalose dihydrate, sodium citrate dihydrate, citric acid monohydrate, and polysorbate (e;g., polysorbate 80). In an embodiment, the antibody and hyaluronidase are mixed with each of trehalose dihydrate, sodium citrate dihydrate, citric acid monohydrate, and polysorbate (e;g., polysorbate 80). In an embodiment, the antibody and hyaluronidase are mixed with an inert additive, carrier, or excipient selected from polysorbate (e.g. polysorbate 20), sodium succinate, and sucrose. In an embodiment, the antibody and hyaluronidase are mixed with each of polysorbate (e.g. polysorbate 20), sodium succinate, and sucrose. In an embodiment, the antibody and hyaluronidase are mixed with an inert additive, carrier, or excipient selected from polysorbate (e.g. polysorbate 80), sodium chloride, sucrose, and tromethamine. In an embodiment, the antibody and hyaluronidase are mixed with each of polysorbate (e.g. polysorbate 80), sodium chloride, sucrose, and tromethamine. In an embodiment, the antibody and hyaluronidase are is mixed with an inert additive, carrier, or excipient selected from polysorbate (e.g. polysorbate 20), sodium hydroxide, succinic acid, and sucrose. In an embodiment, the antibody and hyaluronidase are mixed with each of polysorbate (e.g. polysorbate 20), sodium hydroxide, succinic acid, and sucrose. In an embodiment, the antibody and hyaluronidase are mixed with an inert additive, carrier, or excipient selected from histidine, histidine hydrochloride monohydrate, polysorbate (e.g. polysorbate 20), and trehalose dihydrate. In an embodiment, the antibody and hyaluronidase are mixed with each of histidine, histidine hydrochloride monohydrate, polysorbate (e.g. polysorbate 20), and trehalose dihydrate. In an embodiment, the antibody and hyaluronidase are mixed with an inert additive, carrier, or excipient selected from histidine (e.g. L- histidine), histidine hydrochloride monohydrate (e.g. L-histidine hydrochloride monohydrate), polysorbate (e.g. polysorbate 80), and sucrose. In an embodiment, the antibody and hyaluronidase are mixed with each of histidine (e.g. L-histidine), histidine hydrochloride monohydrate (e.g. L-histidine hydrochloride monohydrate), polysorbate (e.g. polysorbate 80), and sucrose. In an embodiment, the antibody and hyaluronidase are mixed wi th an inert additive, carrier, or excipient selected from 2-(N-morpholino)ethane sulfonic acid (MES), polysorbate (e.g. polysorbate 80), trehalose dihydrate, and sodium chloride. In an embodiment, the antibody and hyaluronidase are mixed with each of 2-(N- morpholino)ethane sulfonic acid (MES), polysorbate (e g. polysorbate 80), trehalose dihydrate, and sodium chloride. In an embodiment, the antibody and hyaluronidase are mixed with an inert additive, carrier, or excipient selected from histidine (e g. L-histidine), histidine monohydrochloride (e.g. L-histidine monohydrochloride), polysorbate (e.g. polysorbate 20), and sucrose. In an embodiment, the antibody and hy aluronidase are mixed with each of histidine (e.g. L-histidine). histidine monohydrochloride (e.g. L-histidine monohydrochloride), polysorbate (e.g. polysorbate 20), and sucrose. In an embodiment, the antibody and hyaluronidase are mixed with an inert additive, carrier, or excipient selected from mannitol (e.g. d-mannitol), histidine (e.g. L-histidine), histidine monohydrochloride (e.g. L-histidine monohydrochloride), and sucrose. In an embodiment, the antibody and hyaluronidase are mixed with an each of mannitol (e.g. d-mannitol), histidine (e.g. L- histidine), histidine monohydrochloride (e.g. L-histidine monohydrochloride), and sucrose. In an embodiment, the antibody and hyaluronidase are mixed with an inert additive, carrier, or excipient selected from acetic acid (e.g. glacial acetic acid), polysorbate (e.g. polysorbate 20), sodium acetate, and sucrose. In an embodiment, the antibody and hyaluronidase are mixed with each of acetic acid (e.g. glacial acetic acid), polysorbate (e.g. polysorbate 20), sodium acetate, and sucrose.

[0176] In an embodiment, the injection of a high volume of the disclosed formulation in a subject has fewer side effects in the subject when compared to an identical subject administered the same volume of a comparable formulation that does not comprise the hyaluronidase. In an embodiment, the injection of a high volume disclosed elsewhere herein with the disclosed formulation has reduced back leakage when compared to a similar formulation that does not comprise the hyaluronidase. In an embodiment, the back leakage is reduced about 54%, about 56%, about 58%, about 60%, about 62%, about 64%, about 66%, about 68%. about 70%, about 72%, about 74%, about 76%. or about 78% when a high volume of the disclosed formulation is administered to a subject using a HVAI fitted with a 23 gauge needle compared to a similar formulation that does not comprise the hyaluronidase. In an embodiment, the back leakage is reduced about 62%, about 64%, about 68%, about 70%, about 72%, about 74%, about 76%, about 78%, about 80%, about 82%, about 84%, or about 86% when a high volume of the disclosed formulation is administered to a subject using a HVAI fitted with a 25 gauge needle compared to a similar formulation that does not comprise the hyaluronidase. In an embodiment, the swelling (bleb) volume is reduced following the injection of the disclosed formulation into a subject when compared to a similar formulation that does not comprise the hyaluronidase. In an embodiment, the swelling height is reduced following the injection of the disclosed formulation when compared to a similar formulation that does not comprise the hyaluronidase. In an embodiment, the swelling size is reduced following the injection of the disclosed formulation when compared to a similar formulation that does not comprise the hyaluronidase. In an embodiment, the swelling area is reduced following the injection of the disclosed formulation when compared to a similar formulation that does not comprise the hyaluronidase. In an embodiment, the swelling induration following the initial injection of the disclosed formulation is minimized compared to a similar formulation that does not comprise the hyaluronidase. In an embodiment, the swelling resolves quicker when the disclosed formulation is injected compared to a similar formulation that does not comprise the hyaluronidase. In an embodiment, the disclosed formulation permits for more consistent delivery (i.e., time to delivery, reduction in bleb swelling volume, height and induration) from injection to injection, compared to a similar formulation that does not comprise the hyaluronidase. In an embodiment, the disclosed formulation permits for faster delivery of the full volume from a HV Al than a comparable formulation that does not comprise the hyaluronidase which results in less pain and discomfort for the subject.

[0177] EXAMPLES

[0178] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0179] Example 1: Pharmacokinetics of Dupilumab Administration Following Subcutaneous Administration with and without Recombinant Human Hyaluronidase PH20 (rHuPHZO)

[0180] Summary

[0181] The objective of this study was to investigate the pharmacokinetics (PK) of a monoclonal antibody dupilumab (Dupixent®) following subcutaneous (SC) administration alone or co-administered with recombinant human hyaluronidase PH20 (rHuPH20; 5000 U / rnL). This study compared the PK of a standard clinical dose of 2 mL of dupilumab alone (300 mg) versus the PK of a 5 mL bolus of dupilumab (750 mg) co-mixed with rHuPH20. The 2 mL doses were administered using a pre-filled syringe (PFS) while the larger dose volume was administered using a prototype HVAI with a 5 mL fill. Blood was collected prior to the start of the study (pre-dose), and at 6 hours (h), lday(d), 2d, 3d, 4d, 5d and 7d post-injection. Blood was collected via jugular vein stick into serum separation tubes (SSTs) and allowed to clot at room temperature for thirty minutes whereupon they were then placed on ice until processed for serum collection. Samples were divided into duplicates and stored at -80 °C until used for bioanalysis.

[0182] The concentration of functional dupilumab of individual serum sample was quantitatively detected using a dupilumab ELISA kit. Basic pharmacokinetic parameters, AUCO-168 and Cmax were determined by non-compartmental analyses in Microsoft Excel. The pharmacokinetic parameters derived from the two dose groups indicated similar dose- normalized PK profiles, with no significant differences in dose-normalized AUCO-168 and Cmax observed.

[0183] Introduction and Objectives

[0184] Rapid subcutaneous administration of large volumes of antibodies has been shown to be feasible when the antibody solution is co-formulated with recombinant human hyaluronidase PH20 (rHuPH20). rHuPH20 has been shown to facilitate SC administration of fluids and drugs by transiently and locally depolymerizing hyaluronan (HA) in the extracellular matrix thereby reducing tissue backpressure in the SC space permitting rapid, large volume administration of fluid. Using this technology, subcutaneous administration of large volumes of antibody has become possible and is replacing intravenous administration as a treatment paradigm.

[0185] The minipig model has been selected due to the high degree of similarity of the subcutaneous space to that of humans. Previous studies using a minipig model have demonstrated the translatability of the model for use in pre-clinical and auto-injector studies. The ability to deliver large volumes of biotherapeutics using rHuPH20 enables treatment paradigms can potentially change from weekly or bi-weekly dosing to monthly, bi-monthly or longer intervals. This study explored the feasibility of using a larger dose volume to achieve serum levels that support an extended clinical dosing period. Five animals were administered a single injection of dupilumab alone (2 mL; 300 mg) or with dupilumab + rHuPH20 (5 mL; 750 mg) and blood samples collected at 6h post-injection and daily afterwards to 7d. The concentration of functional dupilumab in these samples was quantitatively detected using a competitive dupilumab ELISA kit (abx395100, Abbexa LLC).

[0186] Experimental Design and Methods

[0187] Test and Control Articles

[0188] 1 . Dupilumab (Dupixent®) a. Description: Clear colorless liquid b. Lot number: 3L2232 c. Concentration: 150 mg / mL d. Formulation: 30 mM L-arginine hydrochloride, 20 mM L-histidine, 0.4% polysorbate 80, 12.2 mM sodium acetate, 5% sucrose and water for injection, pH 5.9. e. Storage Conditions: 2-8°C f. Handling Conditions: Standard laboratory precautions g. Supplier: Pharmaceutical Buyers, Inc. Recombinant Human Hyaluronidase (rHuPH20) a. Description: Clear colorless liquid b. Lot number: l-FIN-3928 c. Concentration: 1,039,763 U / mL; 10 mg / mL d. Formulation: 10 mM Histidine, 130 mM Sodium Chloride, pH 6.5 e. Storage Conditions: <70°C f. Handling Conditions: Standard laboratory precautions g. Supplier: Halozyme, Inc. Animal Description a. Species: Pig (Sus scrofa domestica) b. Strain: Yucatan miniature c. Sex: Female d. Age: 20-22 weeks e. Quantity: 6 f. Source: Premier BioSource (Ramona, CA)

[0189] Table 1: Summary of Bodyweights and Dates of Birth

[0190] Formulation and Device Preparation

[0191] Preparation of Test Solutions

[0192] The two test solutions administered in this study were dupilumab alone and dupilumab co-mixed with rHuPH20. Dupilumab alone was administered commercially packaged in a 2 mL PFS. To prepare the co-mix, the antibody solution was pooled from dupilumab syringes (28 mL). To the pooled dupilumab, 0.157 mL of rHuPH20 (10 mg / mL) was added to yield 28. 157 mL of test solution. A sample of the co-mix was used for enzymatic activity testing.

[0193] HVAI Device Syringe Filling

[0194] All device syringes were filled in a sterile fill and finish hood under aseptic conditions. Cyclic olefin copolymer (COC) syringes were filled with 5. 1 mL of the co-mix of dupilumab + rHuPH20. To fill the syringes a sterile rubber plunger was initially inserted into the sterile syringe barrel and pushed to a preset depth for filling, The co-mix of dupilumab + rHuPH20 was then pumped into the syringe at a flow rate of 2 mL / min. After adding the test solution to the syringe, the rubber plunger was brought to the final loading position (to remove residual air in the syringe). Syringes were then capped and stored at 2-8°C until removed and brought to room temperature prior to use in device assembly.

[0195] Device Assembly

[0196] Assembly of all devices occurred in a sterile fill and finish hood under aseptic conditions. The syringe cap was removed and replaced using a 25G x 1-inch Becton Dickinson (BD) needle. The needle remained capped until immediately prior to use. The needle and syringe were placed in a proprietary assembly jig with the HVAI for final assembly, which included the placement of a needle guard that brought the exposed needle depth to 10 mm. The length of each needle in the completed device was measured prior to use and all were found to be 10.0 mm. Devices were discarded after use.

[0197] Test Methods

[0198] 1. Hyaluronidase activity testing was performed using an in vitro activity assay (VV- QWUAL-006751 formerly TM010) to confirm the concentration of rHuPH20 in the co-mix drug solution.

[0199] 2. Dupilumab concentrations were measured using a Dupilumab ELISA kit, abx395100, Abbexa LLC. 3. Basic pharmacokinetic parameters, AUCo-168 and Cmax were determined by noncompartmental analyses using Microsoft Excel 365.

[0200] Test Materials

[0201] The following test materials were used in the study. Table 2: Test Materials

[0202] Experimental Design

[0203] This study measured the pharmacokinetics of dupilumab alone or dupilumab comixed with rHuPEI20 following subcutaneous administration. The dose volume for dupilumab alone was 2 mL (300 mg) while the dose volume for dupilumab + rHuPH20 was 5 mL (750 mg). Five animals w ere injected with dupilumab alone and five animals were injected with dupilumab + rEIuPEI20. The treatment groups are shown in Table .

[0204] Table 3: Description of Treatments

[0205] Each animal received a single SC injection to their left lower abdominal region. For Cohort #1, injections were administered using the manufacturer’s pre-filled syringe (2 mL; 300 mg). For Cohort #2, injections of dupilumab + rHuPH20 were delivered using an HVAI device (5 mL; 750 mg). The duration of all injections was timed using a hand-held stopw atch. Following administration any back-leakage was collected for a period of 30 seconds and weighed using an analytical balance. Approximately 3 mL of blood was collected from each animal at 6, 24, 48, 72, 96, 120 and 148h post injection via jugular venipuncture. After the final blood collection timepoint the animal was humanely euthanized.

[0206] Study Procedure

[0207] Prior to the start of study, animals were assessed for general health, body weight recorded, and a pre-treatment baseline blood sample obtained and collected into serum separating tubes (SSTs). Additionally, blood collections were obtained from each animal at 6h post injection, and at 24, 48, 72, 96, 120 and 148h. Blood samples were processed according to manufacturer's instructions and serum removed and stored at -80°C until used for bioanalysis.

[0208] One day prior to the study, syringes were filled with appropriate test solutions and assembled into HVAIs then stored at 2-8°C. Devices were allowed to acclimate to room temperature for at least 30 minutes prior to use and used within 2 hours.

[0209] On the day of the procedure, animals were anesthetized and placed in dorsal recumbence on a heated surgical table and maintained under isoflurane gas for the entire duration of the procedure. Following anesthetization, the abdominal region was cleaned with Nolvasan followed by wiping the injection site with gauze containing 70% isopropanol and wiped dry with sterile gauze. Injection sites were located on the lower left abdominal regions, ~5 cm cranially from the inguinal fold tow ards the midline and ~3 cm towards the midline of the animal. Each injection site was marked with a permanent marker and then photographed prior to needle insertion and immediately post-injection.

[0210] Animals in Cohort #1 received a single SC injection of dupilumab alone administered via a PFS using a standard skin pinch method. Animals in Cohort #2 received a single SC injection of dupilumab + rHuPH20 administered via an HVAI. For HVAI injections, the skin was briefly tented (pinched) for vertical needle insertion and allowed to relax while holding the device in place against the skin prior to activating device.

[0211] Injections were timed using a stopwatch. Upon completion of the injection, the needle and HVAI were removed and discarded. Test solution back-leakage was then absorbed to a tared eye-spear for 30 seconds on the injection site and the weight of the eye spear was measured using an analytical balance. The margins of the injection site bleb were marked with a permanent marker and measured for length, width, and height using a digital caliper and recorded then photographed with the standard camera. Caliper measurements and photographs were taken only immediately post-injection. Local injection sites were qualitatively assessed and graded immediately post-injection (TO) for erythema severity, swelling size appearance, and firmness, using a scoring system described in Table Table 5, and Table 6 respectively.

[0212] Table 4: Grading Scale for Erythema Formation

[0213] Table 5: Grading Scale for Swelling Size Formation Table 6: Grading Scale for Swelling Firmness (Induration)

[0214] Following the blood sampling obtained on d7. the animal was humanely euthanized using an injectable euthanasia drug provided by the vivarium staff.

[0215] Calculations and Statistical Methods

[0216] Assessment of Injection Time The duration of the injection was measured using a digital stopwatch with resolution of 0. 1 seconds.

[0217] Assessment of Local Swelling Volume and Area Using Caliper Measurement

[0218] Volume and area of post-injection swelling were measured using a digital caliper to measure length, width and height of the bleb that formed post-injection. The length and width are defined as the edge to edge measurements of the bleb (i.e., diameter) along their longest axes. These values were manually recorded, and the volume determined using the formula for half of an ellipsoid Vol = (2 / 3)*n*A*B*C where A = Length / 2, B = Width / 2 and C = Height. es

[0219] Approximately 3 mL of whole blood was collected at each time point into serum separating tubes (SST™) and allowed to clot at room temperature for 30 minutes after collection. Tubes w ere then placed on ice until processed by centrifugation (10 min at 1250 ref). Serum was removed from each sample, divided into tw o tubes and stored at -80 °C until thawed for bioanalysis.

[0220] Five animals received a 2 mL injection (300 mg) of dupilumab (Cohort #1) and five animals received a 5 mL injection (750 mg) of dupilumab + rHuPH20 (Cohort #2). The serum concentrations of dupilumab were measured using dupilumab ELISA, abx395100, Lot E2402922A, Exp. Sept. 2024. Briefly, dupilumab in the serum sample was bound to its antigen immobilized on a 96-w ell plate, which then competes with the binding of a detection reagent, biotin labeled dupilumab.

[0221] After incubation and washing to remove unbound dupilumab from the 96-well plate, the bound biotin labeled dupilumab was visualized by horseradish peroxidase substate. Dupilumab concentration was calculated using a four parameter logistic (4PL) fitted standard curve (SoftMax Pro, Molecule Devices). Pharmacokinetic (PK) analysis for dupilumab and dupilumab + rHuPH20 w as performed using Microsoft Excel.

[0222] Summan' of Post-iniection Study Endpoints

[0223] 1. Duration of PFS and HVAI injections: All PFS and HVAI injections were timed.

[0224] 2. Post-injection back-leakage: For all injections the test solution back-leakage was absorbed onto an eye-spear for 30 then weighed using an analytical balance.

[0225] 3. Post-injection bleb swelling size: The margins of the injection site bleb w ere measured for length, width, and height using a digital caliper and then photographed.

[0226] 4. Post-injection scoring: Blebs were qualitatively scored for ery thema, swelling size and induration at TO post-injection.

[0227] 5. Pharmacokinetic sampling: Blood samples (~3 mL) were taken as described in Table .

[0228] 6. Pharmacokinetic modeling: PK modeling was performed on the assayed bioanalytical samples and used to calculate Cmax, AUCo-i48h, and normalized AUC. RESULTS

[0229] Pre-study hyaluronidase activity testing of dupilumab + rHuPPI20 Co-mix

[0230] The enzymatic activity of the co-mix of dupilumab + rHuPh20 was tested using VV- QWUAL-006751 (formerly TM010) The pre-study testing results are shown in Table .

[0231] Table 7: Summary of Pre-study Enzymatic Activity Testing

[0232] As the target concentration of 5000 U / mL was reached, the co-mix solution of dupilumab + rHuPH20 was considered suitable for in vivo use.

[0233] Duration of Injection

[0234] The duration of each injection was measured using a hand-held stopwatch with a precision of 0.1 seconds. The duration of the injections for each test solution is shown in Table 8 and individual animal data shown in Error! Reference source not found.1.

[0235] Table 8: Measurement of Injection Time

[0236] Post-injection Back-leakage

[0237] The back-leakage was collected using a pre-weighed eye spear for 30 seconds postinjection. The eye spear was then re- weighed, and the weight of the back-leakage recorded. The amount of back-leakage for each test solution is show n in Table 9 and individual animal data shown in Figure 2.

[0238] Table 9: Measurement of Back-Leakage

[0239] Assessment of Post-Injection Bleb Volume. Area and Height (Caliper Measurements)

[0240] The local injection site swelling (bleb) was marked and measured using a digital caliper. Bleb volume dispersion area and sw elling height of each bleb was determined as described in Section 6.2 and are summarized in Table 10. Mean and individual post-injection bleb volume, area and height values are shown in Figure 3, Figure 4, and Figure 5, respectively. Table 10: Post-Injection Bleb Volume, Area and Height Caliper Measurements (Mean ± SEM)

[0241] Post-Injection Erythema

[0242] No post-injection erythema was observed for any injection.

[0243] Post-Injection Swelling Size

[0244] Scoring by three evaluators for swelling size using the modified Draize scoring system are summarized in Table 11 and shown in Figure 6.

[0245] Table 11: Post-Injection Swelling After SC Administration of dupilumab and dupilumab + rHuPH20 (Mean ± SEM)

[0246] Post-Injection Induration

[0247] Scoring by three evaluators for bleb induration was performed and is summarized in Table 12 and shown in Figure 7.

[0248] Table 12: Post-Injection Induration After SC Administration of dupilumab and dupilumab + rHuPH20 (Mean ± SEM)

[0249] Notably, despite dupilumab + rHuPH20 having injection volumes 2.5 times that of the dupilumab alone, the induration of the larger volume appeared to be markedly reduced.

[0250] Bioanalytical and Pharmacokinetic Analysis

[0251] The serum values for individual animals at each blood collection timepoint is shown in Table 13 for Cohort #1 (dupilumab) and in Table 14 for Cohort #2 (dupilumab + rHuPH20). Table 13: Concentrations of dupilumab in Individual Pig Serum - Cohort #1: dupilumab

[0252] Table 14: Concentrations of dupilumab in Individual Pig Serum - Cohort #2: dupilumab + rHuPH20 The concentration / time profiles of dupilumab and dupilumab + rHuPH20 are shown in Figure 8.

[0253] The concentration of dupilumab at each timepoint was dose-normalized for both treatment groups. To obtain dose-normalized values, the serum values for dupilumab concentration were divided by 2 (dose volume for Cohort #1) or by 5 (dose volume for Cohort #2). A similar calculation was performed for standard deviation values. The dose- normalized concentration versus time profiles for the two treatment groups are shown in Figure 9. Pharmacokinetic Parameters for Individual Animals

[0254] Individual pharmacokinetic parameters were determined by non-compartmental analyses in Excel and are presented for Cohort #1 (dupilumab) in Table 15 and for Cohort #2 (dupilumab + rHuPH20) in Table 16. Table 15: Pharmacokinetic Parameters for Individual Animals - Cohort #1: dupilumab

[0255] Table 16: Pharmacokinetic Parameters for Individual Animals - Cohort #2: dupilumab + rHuPH20

[0256] Summary and Conclusions • A prototype HVAI was able to successfully deliver 5 mL of dupilumab in approximately 55 seconds using rHuPH20.

[0257] • Despite having a dose volume 2.5x greater than that of dupilumab alone, dupilumab + rHuPH20 resulted in similar post-injection bleb size with less back-leakage. • Post-injection induration was reduced by the addition of rHuPH20 compared to dupilumab alone.

[0258] • Basic pharmacokinetic parameters derived from the two dose groups indicate similar dose-normalized PK profiles, with no significant differences in dose-normalized AUCO-168 or C max observed.

[0259] Example 2; Modeling and Simulation of Dupilumab with Recombinant Human Hyaluronidase PH20 (rHuPH20)

[0260] Summary

[0261] The commercially -approved dosing regimen was defined, along with PK parameters (e.g., volume of distribution and clearance), based on publicly available information (e.g., package insert at https: / / www.accessdata.fda.gov / drugsatfda_docs / label / 2017 / 7610551bl.pdf. See also). Two-compartment PK models were constructed based on this information. Simulations were then generated to compare the PK profiles of the current regimen and SC regimen(s) when co-administered with rHuPH20. Doses administered SC with rHuPH20 were chosen to match the minimum concentration (the driver of efficacy for dupilumab (Kamal, et al.. Pharmacokinetics, pharmacodynamics, and exposure-efficacy of dupilumab in adults with atopic dermatitis. Clin Transl Sci. 2022 Oct;15(10):2342-2354. doi: 10.1111 / cts. 13363. Epub 2022 Aug 20. PMID: 35986664; PMCID: PMC9579381) of the current regimen. Simulations SC with rHuPH20 were generated for the current schedule (Q2W), as well as less frequent (e.g, Q4W and Q8W) schedules.

[0262] Model Definition

[0263] The model leveraged a two-compartment model structure for linear PK, and, when appropriate, accounted for nonlinear kinetics by introducing saturable clearance expressions, for example, Michaelis-Menten, to the central compartment. While the same linear compartmental structure was used in this analysis, the nonlinear approach was replaced with a more mechanistic, streamlined format. Specifically, PK nonlinearity, often characterized as target-mediated drug disposition, was accounted for via incorporation of antibody -target binding kinetics and an antibody-target complex elimination pathway; such an approach allows for the antibody affinity (i.e., equilibrium binding constant, Kd), intrinsic target (steady -state) concentration, and target kinetics (i.e., target half-life) to drive the total (free and bound) antibody PK with one universally applicable framework. If the target is not expressed at significant levels or its turnover is of long enough duration, its impact on the antibody PK will be negligible, with the kinetics following that of a two-compartment model. If the target is highly expressed and / or has a short half-life, this will introduce an additional saturable, nonlinear clearance pathway for the antibody that provides a similar kinetic influence as introducing a Michaelis-Menten expression. The additional benefit of leveraging such a target-binding mechanism is that the associated parameters can be defined a priori based on literature data, as described below. A schematic of the model structure, differential equations, and description of the parameters are in Figure 20.

[0264] Model variables: Ao, Ai, and A2 = antibody in the subcutaneous, central (plasma), and peripheral (tissue interstitial) compartments, respectively; T = target; AT = antibody -target complex. Model parameters: V 1 and V2 = volumes of the central and peripheral compartments, respectively; kei = central compartment elimination rate; ki2 and k2i = distribution rates for central^peripheral and peripheral^central compartments, respectively; kaand kaPH2o = absorption rates for subcutaneous^central compartments, without and with rHuPH20, respectively; F and FPH2O = subcutaneous bioavailability, without and with rHuPH20, respectively; Tss= target steady -state concentration; ksynand kdeg = target synthesis and degradation rates, respectively; konand koff = antibody association and dissociation rates, respectively. kdeg2 = antibody -target complex degradation / elimination rate.

[0265] Initial estimates for the two-compartment parameters, that is, V 1 and V2 (volumes of the central (plasma) and peripheral (tissue interstitial) compartments, respectively), kei (central compartment elimination rate), ki2 and k2i (distribution rates for central -^peripheral and penpheral^central compartments, respectively), and F (bioavailability for SC administration without rHuPH20). w ere defined from the previously published PK models for each of the respective antibodies. For antibodies where published data were not available, generalized population PK parameters derived from Dirks et al. (Dirks NL, Meibohm B. Population pharmacokinetics of therapeutic monoclonal antibodies. Clin Pharmacokinet. 2010;49(10):633-659) were used.

[0266] For target and antibody-target kinetic parameters, that is, Tss(target steady-state concentration). ksyn and kdeg (target synthesis and degradation rates, respectively), kOn and koff (antibody association and dissociation rates, respectively), and kdeg2 (antibody -target complex degradation / elimination rate), data from published literature w ere used. Once defined, these parameters were fixed and not considered for subsequent optimization.

[0267] Since two types of targets exist, soluble or cell-surface, slight differences in the parameterization were considered for each. For soluble targets, steady-state concentrations were readily measurable, and clearance for the antibody -target complex was assumed to follow that of the antibody, that is, kaeg2 = kei. For cell-surface targets, steady-state concentrations (in units of nM) w ere derived from receptor (target) density (XR, in units of receptors / cell) and cell density (Xc, in units of cells / L) according to: T ss = XR-XC / N A- 109, where NA is Avogadro's Number (i.e., 6.02e23), and clearance for the antibody -target complex was assumed to follow that of the target, that is, kdeg2 = kdeg. Once defined, target concentrations were fixed and not considered for subsequent optimization.

[0268] Parameter fl tting

[0269] Parameter fitting was conducted for the collective datasets (i.e., all dose levels and routes of administration) as a Population PK (PopPK) assessment. Covariates were not incorporated, as the average representation of the respective population is sufficient for this application. Initial simulations were conducted using the initial parameter estimates for the respective antibody and target to assess goodness of fit across the collective datasets, that is, all concentration versus time profiles. The default preference was to keep the previously defined, published parameters (i.e., clearances and volumes) unchanged, but for some antibodies re-fitting was necessary. If the simulated curves deviated from the observed data by more than 10% for a given timepoint, then parameter optimization was conducted on the necessary parameters only. For example, if the initial parameters accurately captured the IV curves but not the SC curves, then only the SC relevant parameters (e.g., F and kabs) were redefined using a least-sum-of-squares optimization routine. All simulations were performed using MATLAB® (Mathworks, Natick, MA).

[0270] All simulations, demonstrated the ability to convert the current dosing regimen to a subcutaneous (SC) regimen with rHuPH20, with equivalent Cmin. Additionally, a SC regimen with rHuPH20 can be administered at less frequent dosing schedules, enabling more options for optimizing the safety and / or efficacy profile of the drug, as can be seen in Figures 21A and 2 IB, which show that rHuPH20 can decrease dose frequency. Such regimens could not be achieved without rHuPH20 due to the volume requirements of typical antibody formulations.

[0271] In Figure 21 A, the red curves represent dupilumab concentration after IV administration at 300 mg Q2W dosage (benchmark), and blue curves represent dupilumab concentration after SC administration with rHuPH20 for different antibody dosage.

[0272] In Figure 21B, the red curves represent dupilumab concentration after IV administration at 1000 mg Q4W (benchmark), and blue curves represent dupilumab concentration after SC administration with rHuPH20 for different antibody dosage.

[0273] In this example, a model to characterize the human PK profiles of dupilumab coadministered SC with rHuPH20. The linear PK aspects driven by two-compartment model kinetics were consistent across antibodies in this analysis and aligned with previous works. The nonlinear PK contributions were captured via a mechanistic target engagement framework and a priori-defined values for target and antibody parameters. For dupilumab coadministered with rHuPH20, the human SC PK parameters are quite consistent across a range of dupilumab and target properties. Consequently, the average values in this work can be applied with a high degree of confidence in model simulations for a new antibody prior to entering the clinic to evaluate potential dosing and schedule options for SC co-administration with rHuPH20.

[0274] The SC PK parameterization can be leveraged via a few angles in Phase I as well. If an adaptive design is used, the parameter values can serve as priors. Additionally, when considering the ability of rHuPH20 to accommodate any dose level or volume, Phase I dose escalation could occur exclusively SC, that is. without IV data. While there has been a precedent to date for IV studies preceding SC, this is not an explicit requirement. In fact, for oral drug delivery, IV PK studies are not always required in humans and for many molecules are never executed.

[0275] When similar simulations are run for other antibodies with publicly available data, it is observed that combining the administration of antibodies with rHuPH20 can increase subcutaneous bioavailability. See. e.g., Table 17 showing relative difference between SC bioavailability for adalimumab, bococizumab, crenezumab and tocilizumab.

[0276] Table 17: Publicly available data showing increased SC bioavailability when administered with rHuPH20

[0277] Publicly available data further shows that administration of an antibody with rHuPH20 can increase plasma concentration and Cmax of the antibody. See, e.g., Figure 22 showing increased plasma concentration and Cmax for SC administration of bococizumab with and without rHuPH20; and Figure 23 showing increased plasma concentration and Cmax for SC administration of with and without rHuPH20.

[0278] Administration of antibodies with rHuPH20 can also increase Cmin for SC administration. See, e.g., Figure 24 showing a higher Ctrough when amivantamab was administered SC (1600 mg) with rHuPH20 relative to IV administration (1050 mg) without rHuPH20 at the same schedule. In Figure 24, the upper and lower end of the boxes indicate the 25th and 75th quartiles, respectively, the triangles indicate the means, the horizontal lines within the boxes indicate the medians, and the error bars indicate 95% Cis. Table 18 shows that the total drug dose for different antibodies can also be increased when administered SC with rHuPH20.

[0279] Table 18: Increased dose when administered SC with rHuPH20

Claims

CLAIMS1. A combination dosing regimen, comprising administering a soluble hyaluronidase; and administering dupilumab, wherein the hyaluronidase is administered in an amount effective to increase bioavailability of dupilumab.

2. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered in an amount effective to reduce the variability of distribution of dupilumab compared to dupilumab administered without hyaluronidase.

3. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered in an amount effective to increase plasma concentration of dupilumab compared to dupilumab administered without hyaluronidase.

4. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered in an amount effective to increase the maximum plasma concentration of dupilumab compared to dupilumab administered without hyaluronidase.

5. The combination dosing regimen of claim 1 , wherein the hyaluronidase is administered in an amount effective to increase the minimum plasma concentration of dupilumab compared to dupilumab administered without hyaluronidase.

6. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered in an amount effective to increase the amount of dupilumab administered compared to dupilumab administered without hyaluronidase.

7. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered in an amount effective to decrease the frequency of administration of dupilumab administered compared to dupilumab administered without hyaluronidase.

8. The combination dosing regimen of claim 1, wherein the hyaluronidase is a soluble PH20 hyaluronidase.

9. The combination dosing regimen of claim 8, wherein the soluble hyaluronidase is the composition designated rHuPH20.

10. The combination dosing regimen of claim 1, wherein the hyaluronidase has the sequence set forth as residues 36-482 or 36-483 or has at least 98% sequence identity to the sequence set forth as residues 36-482 or 36-483 of SEQ ID NOT.

11. The combination dosing regimen of claim 1, wherein: the soluble hyaluronidase comprises amino acids 36-464 of SEQ ID NO: 1 or comprises a sequence of amino acids that has at least 85% sequence identity to a sequence of amino acids that contains at least amino acids 36-464 of SEQ ID NOT, and retainshyaluronidase activity.

12. The combination dosing regimen of claim 1, wherein the soluble hyaluronidase comprises a sequence of amino acids that has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence of amino acids that contains at least amino acids 36-464 of SEQ ID NO: 1 and retains hyaluronidase activity.

13. The combination dosing regimen of claim 1, wherein:(a) the soluble hyaluronidase has of amino acids set forth as residues 36-465, 36- 466, 36-467, 36-468, 36-469, 35-470, 36-471, 36-472, 36-474, 36-475, 36-476, 35- 477, 36-478, 36-479. 36-480, 36-481, 36-482, 36-483 35-484, 36-485, 36-486, 36- 487, 36-488, 36-489, 36-490, 35-491, 36-492, 36-493, 36-494, 36-495, 36-496, 36- 497, 35-498, 36-499, and 36-500 of SEQ ID NO: 1, or an N-terminally truncated variant thereof lacking residues 36, 36-37, 36-38, 36-39, or 36-40; or(b) a variant soluble hyaluronidase that has at least 91% sequence identity’ to a soluble hyaluronidase of a).

14. The combination dosing regimen of claim 13, wherein the soluble hyaluronidase has at least 95% sequence identity to the soluble hyaluronidase of claim 13.

15. The combination dosing regimen of claim 13, wherein: the soluble hyaluronidase comprises the replacement F204P, and has increased stability relative the unmodified PH20 that does not comprise F204P; and(a) T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A. L3541. D355K, N356E, E359D and I361T;(b) L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L3541, D355K, N356E, E359D and I361T;(c) M345T, S347T. M348K, K349E. L352Q, L353A, L354I, D355K, N356E, E359D. I361T and N363G;(d) T341G. L342W, S343E, I344N, M345T. S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T;(e) T341A. L342W, S343E, I344N, M345T. S347T, M348K. K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T;(f) T341C, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T;(g) T341D, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q,L353A, L354I, D355K, N356E, E359D and I361T;(h) I344N, M345T. S347T. M348K, K349E. L352Q, L353A, L354 D355K. N356E, E359D and 1361 T, and(i) S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T. increased stability is reflected as increased stability in denaturing condition or at elevated temperatures.1 . The combination dosing regimen of claim 13, wherein: the soluble hyaluronidase is a variant modified polypeptide or catalytically active portion thereof that comprises one or more amino acid residue substitutions selected from among T341A, T341C, T341D, T341G, T341S, L342W, S343E, I344N, M348K, and N363G; numbering is with reference to SEQ ID NO:1; modifications comprise insertions, deletions, and replacements of amino acids; the polypeptides have an N-terminus, at residue 36, 37, 38, 39, or 40, and a C-terminus at a residue corresponding to residues 465 to 500.

17. The combination dosing regimen of claim 13, wherein soluble hyaluronidase comprises amino acid modifications selected from one or more up to all of the follow ing T341S, L342W, S343E, I344N, M345T, S347T. M348K, K349E. L352Q, L353A, L354I, D355K, N356E, E359D and 1361T.

18. The combination dosing regimen of claim 13, wherein the C-terminus of variant PH20 polypeptide is at a residue corresponding to amino acid 467, 468, 469, 470, or 471 with reference to SEQ ID NO: 1.

19. The combination dosing regimen of claim 1 , wherein the soluble hyaluronidase comprises the sequence of amino acids set forth in SEQ ID NO:2 or is a catalytically active fragment thereof.

20. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered at a dose of 2000 to 15,000 U.

21. The combination dosing regimen of claim 20, wherein the hyaluronidase is administered at a dose of 10.000 U.

22. The combination dosing regimen of claim 20, wherein the dupilumab is administered at a dose of 10 mg / kg.

23. The combination dosing regimen of claim 22, wherein the dupilumab is administered at adose of at least or at 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, and 100 mg / kg.

24. The combination dosing regimen of claim 1, wherein the hyaluronidase and dupilumab are administered subcutaneously.

25. The combination dosing regimen of claim 1, wherein: in a first step the soluble hyaluronidase is administered to a patient; and in a second step the dupilumab is administered to the patient.

26. The combination dosing regimen of claim 1, wherein the soluble hyaluronidase and dupilumab are administered in the same composition.

27. The combination dosing regimen of claim 1, wherein the soluble hyaluronidase and dupilumab are administered once a day.

28. The combination dosing regimen of claim 22, wherein hyaluronidase and dupilumab are administered on day one and day eight of a twenty-one-day treatment cycle for three cycles.

29. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered at a rate of about 0.05 mL / sec to about 1.0 mL / sec.

30. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered at a rate of about 0.05 mL / sec to about 0. 10 mL / sec.

31. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered at a rate of about 0. 10 mL / sec to about 0.20 mL / sec.

32. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered at a rate of about 0.20 mL / sec to about 0.30 mL / sec.

33. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered at a rate of about 0.30 mL / sec to about 0.40 mL / sec.

34. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered at a rate of about 0.40 mL / sec to about 0.50 mL / sec.

35. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered at a rate of about 0.50 mL / sec to about 0.60 mL / sec.

36. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered at a rate of about 0.60 mL / sec to about 0.70 mL / sec.

37. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered at a rate of about 0.70 rnL / sec to about 0.80 mL / sec.

38. The combination dosing regimen of claim 1 , wherein the hyaluronidase is administered at a rate of about 0.80 rnL / sec to about 0.90 mL / sec.

39. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered at a rate of about 0.90 mL / sec to about 1.00 mL / sec.

40. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered at a rate of about 0. 10 mL / sec to about 0.90 mL / sec.

41. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered at a rate of about 0.20 mL / sec to about 0.80 mL / sec.

42. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered at a rate of about 0.30 mL / sec to about 0.70 mL / sec.

43. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered at a rate of about 0.40 mL / sec to about 0.60 mL / sec.

44. The combination dosing regimen of claim 1, wherein the hyaluronidase is administered at a rate of about 0.45 mL / sec to about 0.55 mL / sec.

45. The combination dosing regimen of claim 1, wherein the administration takes about 10 seconds to about 60 seconds.

46. The combination dosing regimen of claim 1, wherein the administration takes about 10 seconds to about 20 seconds.

47. The combination dosing regimen of claim 1, wherein the administration takes about 20 seconds to about 30 seconds.

48. The combination dosing regimen of claim 1 , wherein the administration takes about 30 seconds to about 40 seconds.

49. The combination dosing regimen of claim 1, wherein the administration takes about 40 seconds to about 50 seconds.

50. The combination dosing regimen of claim 1, wherein the administration takes about 50 seconds to about 60 seconds.

51. The combination dosing regimen of claim 1, wherein the administration takes about 20 seconds to about 50 seconds.

52. The combination dosing regimen of claim 1, wherein the administration takes about 30 seconds to about 40 seconds.

53. The combination dosing regimen of claim 1, wherein the administration takes at least or less than about 10 seconds to about 60 seconds.

54. The combination dosing regimen of claim 1 , wherein the administration takes at least or less than about 10 seconds to about 20 seconds.

55. The combination dosing regimen of claim 1, wherein the administration takes at least or less than about 20 seconds to about 30 seconds.

56. The combination dosing regimen of claim 1, wherein the administration takes at least or less than about 30 seconds to about 40 seconds.

57. The combination dosing regimen of claim 1, wherein the administration takes at least or less than about 40 seconds to about 50 seconds.

58. The combination dosing regimen of claim 1, wherein the administration takes at least or less than about 50 seconds to about 60 seconds.

59. The combination dosing regimen of claim 1, wherein the administration takes at least or less than about 20 seconds to about 50 seconds.

60. The combination dosing regimen of claim 1, wherein the administration takes at least or less than about 30 seconds to about 40 seconds.

61. The combination dosing regimen of claim 1, wherein swelling (bleb) volume is reduced following the administration into a subject when compared to a formulation that does not comprise the hyaluronidase.

62. The combination dosing regimen of claim 1, wherein administration of a high volume has reduced back leakage compared to a formulation that does not comprise the hyaluronidase.

63. The combination dosing regimen of any preceding claim, wherein the administration is done with a high- volume auto injector.

64. A method of treating atopic dermatitis comprising administering to a patient in need of treatment the combination dosing regimen of claim 1.

65. The method of claim 64, wherein the atopic dermatitis is eczema.

66. A combination, comprising a soluble hyaluronidase and dupilumab.

67. A kit, comprising the combination of claim 66.

68. The combination of claim 66, wherein the hyaluronidase and dupilumab are in separate compositions.

69. The combination of claim 66, wherein the hyaluronidase and dupilumab are coformulated.

70. The combination of claim 66, wherein the hyaluronidase and dupilumab are in separate compositions in a container with at least two compartments.

71. A composition, comprising a soluble hyaluronidase and dupilumab.

72. A multi-compartment container, comprising a suspension comprising dupilumab in one compartment, and a soluble hyaluronidase in a second compartment.

73. The multi-compartment container of claim 72, that is a syringe, comprising two compartments.

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