WO2014135984A2 - Glycosylated mucin-immunoglobulin fusion protein coated device - Google Patents

Abstract

The invention features a device comprising a polymer surface coated with a fusion protein comprising a first polypeptide covalently conjugated to a second polypeptide, the first polypeptide being negatively charged and the second polypeptide being an immunoglobulin polypeptide or a fragment thereof. The device of the current invention is semi-rigid or soft, and is suitable for custom fitting with the curvature of an ocular surface.

Description

GLYCOSYLATED MUCIN-IMMUNOGLOBULIN FUSION PROTEIN COATED

DEVICE

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.

61/774,220, filed March 7, 2013, contents of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] This invention relates generally to contact lens coated with fusion polypeptide comprising mucin.

BACKGROUND OF THE INVENTION

[0003] Mucins are glyco conjugated proteins which are secreted by vesicles and discharged on the surface of the conjunctival epithelium of the eye. Mucins are found on moist, mucosal epithelia, and are thought to combine mechanical protection of eye tissue as well as chemical and immune protection of mucosal tissue. The surface of the eye is kept moist and lubricated by tear film. Mucins anchor this tear film to the epithelium and protect the eye surface from bacterial, chemical and physical invasion of foreign bodies.

[0004] Contact lenses applied to the eye can become coated with "biofilm," made up of many components, including mucus. Lens wear can also lead to ocular surface damage with resultant alterations in the mucin component. Mucins promote surface wettability and eliminate contact-angle hysteresis. Increase or decrease in mucus production with contact lens wear directly influences the tear film characteristics, lens surface coating, and the ultimate wettability and tolerability of lenses themselves.

SUMMARY OF THE INVENTION

[0005] The invention provides a device comprising a polymer surface coated with a fusion protein comprising a first polypeptide covalently conjugated to a second polypeptide, wherein the first polypeptide is negatively charged and the second polypeptide is

immunoglobulin polypeptide or a fragment thereof, wherein the polymer surface is semi-rigid or soft, suitable for custom fitting with the curvature of an ocular surface.

[0006] The current invention provides coating a device comprising a polymer surface with a fusion protein, where the first polypeptide is a mucin polypeptide or a fragment thereof. The mucin polypeptide is at least a region of a P-selectin glycoprotein ligand-1 (PSGL-1). The first polypeptide is an extracellular portion of a P-selectin glycoprotein ligand-1.

[0007] The mucin polypeptide of the fusion protein for coating a polymer surface of the current invention is chosen from PSGL-1, CD34, CD43, CD45, CD96, GlyCAM-1, MAdCAM-1, and fragment thereof. The mucin polypeptide is a secreted mucin or a membrane associated mucin. Secreted mucin is chosen from MUC2, MUC5AC, MUC5B, MUC6, MUC7, and MUC9, and the membrane associated mucin is chosen from MUC1, MUC3A, MUC3B, MUC4, and MUC16.

[0008] An embodiment of the current invention provides a polymer surface coated with a fusion protein comprising a sialylated mucin polypeptide.

[0009] The embodiments of the invention provide a polymer surface coated with a fusion protein, where the second polypeptide is a region of a heavy chain immunoglobulin polypeptide, e.g., the Fc region of an immunoglobulin heavy chain.

[0010] In some embodiments of the current invention, the polymer surface is a contact lens. The fusion protein increases the wettability of the polymer surface.

[0011] The device of the current invention is a surface of polymer selected from unplasticized polyvinyl chloride (UPVC), polycarbonate (PC), polyethylene terephthalate (PET), polystyrene (PS), poly(methylmethacrylate) (PMMA), polytetrafluoroethylene (PTEE), silicone and silicone hydrogel. Further, in some embodiments the device is any of the common contact lens material groups PMMA, hybrid, gas permeable, hydrogel, silicone hydrogel and derivatives thereof.

[0012] The one embodiment, the current invention provides a device comprising a polymer surface coated on the inner layer, an outer layer, or both layers of the polymer surface. The polymer surface is coated with PSGL-l/mIgG2b fusion protein. In one embodiment, the polymer surface is coated with PSGL-1 /hIgG4 fusion protein.

[0013] The current invention provides a polymer surface coated with a mucin polypeptide, which is a mono or disialylated core 1 polypeptide, for example, PSGL- l/mIgG2b glycosylated with mono and disialylated core 1 structures. In yet another embodiment, the polymer surface is coated with PSGL-l/hIgG4 glycosylated with mono and disialylated core 1 structures.

[0014] The embodiments of this invention include an ophthalmic device, for example, a contact lens, coated with a fusion protein. The fusion protein coating the contact lens is PSGL-1 /mIgG2b, PSGL-l/hIgG4, any variations of mucin/IgG, or any combination thereof. [0015] In yet another embodiment, the invention provides an ophthalmic device, for example, a contact lens, comprising a surface covalently linked to a cationic co-polymer. The copolymer comprises hydrophilic, cationic monomeric units and/or hydrophobic, cationic monomeric units derived from a non-cationic, ethylenically unsaturated hydrophilic monomer and/or monomeric units derived from an ethylenically unsaturated monomer containing a moiety reactive with complementary reactive functionalities at the lens surface.

[0016] The copolymer is covalently linked directly to the device surface with the moiety reactive with the complementary functionality at the device surface, or covalently linked with an intermediate polymer reactive with both the device surface and the copolymer.

[0017] According to some embodiments of the current invention, the cationic copolymer on the device surface complexes with mucin or a mucin fusion polypeptide. The copolymer is complexed with a mucin fusion polypeptide, for example, with PSGL- l/mIgG2b and PSGL-l/hIgG4. The mucin fusion polypeptide complexed with the copolymer on the device is glycosylated with mono and/or disialylated core 1 and/or core 2 structures.

[0018] In one aspect, the invention provides a fusion polypeptide that includes a first polypeptide that carry one or more of the following carbohydrate epitopes

Siaa3Galp3GalNAca, Siaa3Gal 4GlcNAc , Siaa3Galp3GlcNAcp, Siaa6Galp3GalNAca, Siaa6Gal 4GlcNAc , and/or Siaa6Galp3GlcNAcp, operably linked to a second polypeptide. The first polypeptide is multivalent for these epitopes. The first polypeptide is, for example, a mucin polypeptide such as PSGL-1 or portion thereof. In some embodiments of the present disclosure the mucin polypeptide is the extracellular portion of PSGL-1.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0020] Other features and advantages of the invention will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to understand the invention and to demonstrate how it may be carried out in practice, embodiments now described, by way of non-limiting example only, with reference to the accompanying drawings in which:

[0022] Figure 1 shows images of Contact angle (CA) measurements (static)

Water on PMMA surface.

[0023] Figure 2 shows images of measurement of values of Advancing (Ad) CA of

Water on PMMA surface.

[0024] Figure 3 shows images of measurement of Ad and Re angles of water on

PMMA surface.

[0025] Figure 4 shows images of CA measurement of 50 ppm BSM in 155 mM NaCl solution on PMMA surface.

[0026] Figure 5 shows images of CA measurement of BSM on PMMA surface.

[0027] Figure 6 shows images of CA measurement of BSM on PMMA surface.

[0028] Figure 7 shows images of CA measurement of 50 ppm PSGL-l/mIgG₂b in 155 mM NaCl solution on PMMA surface.

[0029] Figure 8 shows images CA measurement of PSGL-l/mIgG_2b on PMMA surface.

[0030] Figure 9 shows images of CA measurement of CA with water on BSM coated

PMMA surface.

[0031] Figure 10 shows images of CA measurement of CA with water on BSM coated PMMA surface.

[0032] Figure 11 shows images of CA measurement of CA with water on PSGL- l/mIgG₂b coated PMMA surface.

[0033] Figure 12 shows images of CA measurement with water on PSGL-l/mIgG₂b coated PMMA surface.

[0034] Figure 13 shows a graph of QCM measurement of adsorption of BSM on

PMMA surface. The frequency and dissipation change after introduction of 2 ppm BSM solution were negligible. This was likely due to the adsorption of BSM onto the tubing, so very little BSM existed in the solution when it entered the QCM cell. Rinsing after adsorption from 25 and 50 ppm solution resulted in an abrupt increase of f and decrease of D because of rinsing away weakly bound protein on the adsorbed layer.

[0035] Figure 14 shows a graph of adsorption of PSGL-l/mIgG_2b coated PMMA surface. Rinsing after 2 and 25 ppm solution lead to smaller increase of f and decrease of D compared to BSM, indicating smaller desorption. Rinsing after 50 ppm and 100 ppm solution resulted in an increase of D.

[0036] Figures 15 A-B show frequency and dissipation change as a function of concentration. The adsorption of BSM reached saturation at the concentration of 50 ppm; PSGL-l/mIgG_2b possibly reached saturation at 100 ppm.

[0037] Figures 16A-B show frequency and dissipation change as a function of concentration. The frequency change after adsorption of PSGL-l/mIgG_2b was larger than that of BSM, indicating more adsorption of purified PSGL- l/mIgG_2b on PMMA surface. The dissipation change of PSGL-l/mIgG_2b was also larger than for BSM. The data indicated that the adsorbed PSGL-l/mIgG_2b layer was less rigid than the BSM layer, possibly due to more extended polymer structure or a more viscous adsorbed layer.

[0038] Figures 17A-C show the stepwise adsorption of mucin on PMMA surface.

Figure 17A shows sensed mass of BSM as a function of concentration. Figure 17B shows sensed mass of PSGL-l/mIgG_2b as a function of concentration. The adsorbed mass of PSGL- l /mIgG_2b including water trapped in the layer is significantly larger, close to a factor of 2, than the mass of adsorbed BSM with associated water. The PSGL- l/mIgG_2b molecules have higher affinity towards the PMMA surface than BSM molecules. Figure 17C shows that sensed (Voigt) mass of BSM is less than of C-PSL_ex PSGL-l/mIgG_2b and C-P₅₅ PSGL- l/mIgG_2b; sensed mass of C-P55 PSGL- l/mIgG_2b (shorter side chains; less branched side chains) is less than that of C-PSL_ex PSGL- l/mIgG_2b (longer side chains; more branched side chains); adsorption difference between C-PSL_ex PSGL-l/mIgG_2b and C-P₅₅ PSGL-l/mIgG_2b is due to the PSGL-1 part. Mucin part in C-P₅₅ PSGL-l/mIgG_2b is shorter and less branched than those in C-PSL_ex PSGL-1 /mIgG_2b.

[0039] Figure 18 shows the contact angle of water on BSM coated PMMA surface

(black curve) and PSGL-1 /mIgG_2b coated PMMA surface (red curve) as a function of time. The data suggests that water is transported from the droplet to within the PSGL-l/mIgG_2b film, and results in a force that spreads the water droplet over the surface to achieve close to complete wetting. Water penetration into the BSM film is less, and results in insignificant water spreading on the BSM coated PMMA surface.

[0040] Figures 19A-E show normal force-distance curves between PMMA surfaces across 155 mM NaCl solution (Figure 19A), BSM layers adsorbed on PMMA surface across 100 ppm BSM solution (Figure 19B), and PSGL- l/mIgG_2b layers adsorbed on PMMA surface across 100 ppm PSGL-l/mIgG_2b solution (Figure 19C). The force is normalized by the probe radius. The black curve is measured on approach and the red one on separation. The (close to) lack of adhesion between the PSGL-l/mIgG₂b layers suggests a more complete coverage of the PMMA than what was achieved for BSM. Figures 19D and 19E shows longer ranger repulsion for C-PSL_ex PSGL-l/mIgG₂b compared to C-P55 PSGL-l /mIgG₂b but similar very week adhesion on separation.

[0041] Figures 20A-B show normal force-distance curves during compression (Figure

20A) and decompression (Figure 20B). The force is normalized by the probe radius. Figure 20A shows that the steric repulsion between PSGL- l/mIgG₂b layers is higher than that between BSM layers, which is attributed to larger sensed mass and larger layer thickness of the PSGL- l/mIgG₂b layers. Figure 20B shows that there is a significantly stronger steric repulsion between PSGL-l/mIgG₂b layers on retraction compared to that for BSM layers. This is due to a more rapid recovery of the layer structure after compression of the PSGL- 1 /mIgG₂b layers.

[0042] Figures 21 A-B shows friction force Ff vs. load, F_n and F R between two bare

PMMA surfaces (black), BSM coated PMMA surfaces (circles) and PSGL- l/mIgG₂b coated PMMA surfaces (triangles) measured across 100 ppm mucin solution in 155 mM NaCl.

Filled and unfilled symbols represent data obtained on loading and on unloading,

respectively. The effective friction coefficient versus load for BSM (circles) and PSGL- l /mIgG₂b (triangles). The friction is very high between PMMA coated surfaces across 155 mM NaCl, significantly smaller between BSM coated PMMA, and even much smaller between PSGL-l/mIgG_2b coated PMMA surfaces. At pressures below 1 MPa the friction coefficient for PMMA coated with PSGL-l/mIgG_2b is 0.06, for PMMA coated with BSM μ = 0.7 and for bare PMMA surfaces μ = 1.7. Thus, PSGL- l/mIgG₂b layers provide superior lubrication in aqueous environment compared to BSM.

[0043] Figure 22 shows Voigt mass of BSM (triangles) and PSGL- l/mIgG_2b (circles) as a function of concentration of the respective mucin.

[0044] Figure 23 shows the data obtained when first adsorbing BSM to PMMA and then allowing PSGL- l/mIgG₂b to interact with the BSM-coated surface. Frequency (filled circles) and dissipation (open circles) changes as a function of time during adsorption of an initial BSM layer followed by rinsing and PSGL- l/mIgG₂b adsorption (a) and the reverse (b) (PSGL-l/mIgG₂b - rinsing - BSM adsorption). The concentration of all mucin solutions was 100 ppm in 155 mM NaCl. The unlabeled arrows mark the starting points of mucin injection and the arrows labeled with "R" mark the starting point for rinsing with 155 mM NaCl solution. [0045] Figure 24 shows Voigt mass (circles) and thickness of IgG-Fc on PMMA as a function of IgG-Fc concentration in 155 mM NaCl.

[0046] Figure 25 shows AD - Δ/plot of BSM (circles), PSGL-l/mIgG_2b (squares) and

IgG-Fc (triangles) adsorbed on PMMA. The plot shows structural transitions as the adsorption proceeds. The AD - Af plot for IgG-Fc display a linear relation between AD and Af. In contrast, the AD - Af plot for BSM consists of two regions, a first linear region up to 30 Hz and a second region with decreasing dissipation, suggesting that after the initial adsorption the adsorbed BSM molecules slowly change their conformation to form a thinner layer to maximize the favorable interaction with the surface. The AD - Af plot for PSGL- l/mIgG2b mucin has a first linear region (up to 30 Hz), decreasing slope in the second region (30 to 45 Hz) indicating that the energy dissipated per unit sensed mass decreases, and increasing slope in the last region (>45 Hz), suggesting a structural change towards a more extended layer conformation.

[0047] Figure 26 shows force-separation curves (approach: filled circle; separation: open circles) between PMMA surfaces across 155 mM NaCl solution. The force is normalized by the particle radius.

[0048] Figures 27A-B show force-separation curves (approach: filled circles;

separation: open circles) between BSM-coated PMMA (Fig. 27A) and PSGL-l/mIgG_2b - coated PMMA (Fig. 27B) across 100 ppm solution of the respective mucin in 155 mM NaCl solution. The force is normalized by the particle radius.

[0049] Figure 28 shows force-separation curves measured on approach for BSM

(open circles) and PSGL-l/mIgG_2b (filled circles) on a logarithmic scale. The mucin concentration is 100 ppm and the NaCl concentration was 155 mM. The force is normalized by the particle radius.

[0050] Figures 29A-B show force-separation curves of C-P55 PSGL-l/mIgG_2b and C-

PSL_ex PSGL-l/mIgG_2b. Pure repulsion is observed between the C-P55 PSGL-l/mIgG₂b layers (Fig. 29A), but smaller repulsive force is observed between C-P55 PSGL-l/mIgG_2b layers than between C-PSL_ex PSGL-l/mIgG_2b layers on both compression and decompression (Fig. 29B).

[0051] Figure 30 shows lubrication properties of uncoated PMMA, BSM coated

PMMA, C-PSL_ex PSGL-l/mIgG_2b coated PMMA, and C-P55 PSGL-l/mIgG_2b coated PMMA. Superior lubrication was observed between C-PSL_ex PSGL-l/mIgG_2b layers at load <20 nN. [0052] Figures 31 A-B shows adsorption of mucins on PMMA surfaces. Adsorption from mucin solutions of increasing concentration: 2 ppm, 25 ppm, 50 ppm, and 100 ppm. The frequency drop for adsorption of C-P55 PSGL-l/mIgG_2b (45 Hz) from 2 ppm solution is much bigger than that for C-PSLex PSGL-l/mIgG_2b (20 Hz). The adsorption of C-P55 PSGL-l/mIgG₂b is less concentration dependent.

[0053] Figure 32 shows structure of BSM (train-of-brushes) and C-PSL_ex PSGL- l/mIgG_2b (brush-with-anchor) mucins.

DETAILED DESCRIPTION

[0054] The present invention is directed to a device comprising a polymer surface coated with a fusion protein comprising a first polypeptide covalently conjugated to a second polypeptide, wherein the first polypeptide is negatively charged and the second polypeptide is immunoglobulin polypeptide or a fragment thereof, wherein the polymer surface is semi-rigid or soft, suitable for custom fitting with the curvature of an ocular surface.

[0055] The present invention is directed to a device comprising a polymer surface, such as a contact lens, coated with a fusion protein for maintaining wetting of the polymer surface. The fusion protein, for example, a mucin/IgG fusion protein, coating of the contact lens of the current invention maintains wetting of the surface of the contact lens and ensures clear vision. The fusion protein for coating of a contact lens is a high molecular weight protein, having a high carbohydrate-to-protein ratio. Alternatively, the fusion protein coating a contact lens has a low carbohydrate-to-protein ratio. In some embodiments, the fusion protein is glycosylated with O-glycosidic linkages.

[0056] The fusion protein covered contact lens of the current invention is used in a method of treating dry eye or dry eye related disease or disorder or of reducing dry eye or dry eye related disease or disorder symptoms. The fusion protein covered contact lens of the current invention treats or prevents dry eye or dry eye related disease or disorder by maintaining hydrophilicity of the corneal and contact lens surfaces by trapping contaminants that might interfere with wettability and create dry spots on the cornea and contact lens.

[0057] The current invention provides coating a device comprising a polymer surface with a fusion protein, where the first polypeptide is a mucin polypeptide or a fragment thereof. The present disclosure provides a mucin polypeptide of at least a region of a P- selectin glycoprotein ligand-1 (PSGL-1). In some embodiments, the first polypeptide is an extracellular portion of a P-selectin glycoprotein ligand-1. PSGL-1 is a mucin-type glycoprotein expressed on the surface of myeloid cells. It is the high-affinity receptor for P- selectin which is expressed on activated endothelial cells and platelets. The interaction between PSGL-1 and its receptor P-selectin mediates tethering and rolling of leukocytes along the vascular endothelium at sites of inflammation. It is a membrane bound protein with an extracellular domain rich in serines, threonines, and prolines. It has a highly extended structure with an extracellular domain that is about 50 nm long and it has 53 potential O- glycosylation and 3 potential N-glycosylation sites.

[0058] In some embodiments, the mucin polypeptide of the fusion protein for coating a polymer surface of the current invention is chosen from PSGL-1 , CD34, CD43, CD45, CD96, GlyCAM-1 , MAdCAM-1, and fragment thereof. The present disclosure provides, a mucin polypeptide, which is a secreted mucin or a membrane associated mucin. Secreted mucin is chosen from MUC2, MUC5AC, MUC5B, MUC6, MUC7, and MUC9, and the membrane associated mucin is chosen from MUC1 , MUC3A, MUC3B, MUC4, and MUC16.

[0059] An embodiment of the current invention provides a polymer surface coated with a fusion protein comprising a sialylated mucin polypeptide.

[0060] The embodiments of the invention provide a polymer surface coated with a fusion protein, where the second polypeptide is a region of a heavy chain immunoglobulin polypeptide, e.g. , the Fc region of an immunoglobulin heavy chain.

[0061] PSGL-l/mIgG_2b is a recombinant mucin-type fusion protein consisting of the extracellular part of P-selectin glycoprotein ligand-1 (PSGL-1) fused to the Fc part of mouse IgG_2b. PSGL-l/mIgG_2b is mainly expressed as a dimer when produced in CHO cells and the molecular weight is around 300 kDa. The present disclosure provides PSGL-1 /mIgG₂b produced in non-glycoengineered CHO cells (C-P55) and/or glycoengineered CHO cells co- expressing the core 2 N-acetylglycosyl transferase I (C2 GnT-I) and the al,3- fucosyltransferase VII (FUT-VII) (C-PSLex).

[0062] The fusion polypeptide of the present disclosure has a higher affinity towards

Poly(methylmethacrylate) (PMMA) surface compare to Bovine Submaxillary Mucin (BSM). BSM consists of alternating glycosylated and non-glycosylated regions. A fusion polypeptide of the present disclosure, e.g., PSGL-l/mIgG_2b, has a higher affinity towards a surface polymer, e.g. , PMMA surface, than BSM molecules and binds to the surface permanently. The PSGL-l/mIgG_2b also is adsorbed more on the surface polymer, e.g., PMMA surface, and forms a more extended layer, e.g., about 9 nm, about 10 nm, about 1 1 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, or about 20 nm, compared to BSM molecules, which bind to a surface polymer, e.g. , PMMA surface, with a layer between about 8 nm or less.

[0063] Some key features of PSGL-l/mIgG₂b is summarized in Table 1. [0064] Table 1. Some data for Bovine Submaxillary Mucin (BSM) and PSGL- l/mIgG₂b mucins

Mucin Overall structure Molecular weight Carbohydrate

(kDa) content (wt%)

BSM Train-of-brushes 7000 61-69

PSGL-l/mIgG_2b Brush- with-anchor 300 43^3S

[0065] In some embodiments of the current invention, the polymer surface is a contact lens. The fusion protein(s) of the present disclosure increases the wettability of the polymer surface. Wettability can be defined as the tendency for a liquid to spread over a solid surface, which is commonly characterized by measuring the contact angle at the liquid and solid interface. It is particularly of relevance to contact lenses because the lens surface needs to support a stable ocular tear film. PMMA is widely used in production of rigid gas permeable contact lens. It contains both hydrophobic (methylene) and hydrophilic (carbonyl) groups in each repeating unit, and the contact angle (68 °) shows (Table 2) that it is weakly hydrophilic in nature. In addition, mucin solutions showed similar contact angle on PMMA surfaces. This means that neither of the two mucins spreads on the PMMA surface outside the droplet and they therefore does not promote water spreading on the uncoated PMMA. In contrast, after mucin adsorption on the PMMA surface, the contact angles were decreased to 52 °for BSM coated surface and 5.0 ° for C-PSLex PSGL-l/mIgG_2b coated surface respectively, which means that adsorbed layers of both mucins increase the wettability of the PMMA surface, but the C-PSLex PSGL-l/mIgG_2b layer promotes wetting significantly more than the BSM layer. The variation of the contact angle of water on mucin coated PMMA surfaces during as a function of time is shown in Figure 18. The contact angle for BSM coated surface almost remained constant, while the value for the C-PSLex PSGL-l /mIgG₂b coated surface decreased around 30 ⁰ within 25 seconds. Water is transported from the droplet to within the C-PSLex PSGL-l/mIgG_2b film, and results in a force that spreads the water droplet over the surface to achieve close to complete wetting. Water penetration into the BSM film is less, and results in insignificant water spreading on the surface.

[0066] The device of the present disclosure is a surface of polymer selected from unplasticized polyvinyl chloride (UPVC), polycarbonate (PC), polyethylene terephthalate (PET), polystyrene (PS), poly(methyl methacrylate) (PMMA), polytetrafluoroethylene (PTEE), hydrogel (e.g. Poly-2-hydroxyethyl methacrylate (pHEMA) which belongs to the FDA Group I materials; Methacrylic acid (MAA) commonly used in combination with pHEMA and found in some FDA Group III materials and almost always present in FDA Group IV lens materials; N-vinylpyrrolidone (NVP), a hydrophilic monomer used to increase water content of pHEMA or polymethyl methacrylate (pMMA), also exist as polymer (PVP) and is present in most FDA Group II lenses), silicone and silicone hydrogel (components that are commonly seen in silicone hydrogel contact lenses are DMA (N,N-dimethylacrylamide), PDMS (polydimethylsiloxane), TPVC (tris-(trimethylsiloxysilyl) propylvinyl carbamate), TRIS (trimethylsiloxy silane), proplvinyl carbamate, PVP and other siloxane macromers).

[0067] The one embodiment, the current invention provides a device comprising a polymer surface coated on the inner layer, an outer layer, or both layers of the polymer surface. The polymer surface is coated with PSGL-l/mIgG2b fusion protein. In one embodiment, the polymer surface is coated with PSGL-l/hIgG4 fusion protein.

[0068] The current invention provides a polymer surface coated with a mucin polypeptide, which is a mono or disialylated core 1 polypeptide, for example, PSGL- l/mIgG2b glycosylated with mono and disialylated core 1 structures. In yet another embodiment, the polymer surface is coated with PSGL-l/hIgG4 glycosylated with mono and disialylated core 1 structures.

[0069] The embodiments of this invention include a contact lens coated with a fusion protein. The fusion protein coating the contact lens is PSGL-l/mIgG2b, PSGL-l/hIgG4, any variations of mucin/IgG, or any combination thereof.

[0070] In yet another embodiment, the invention provides a contact lens comprising a surface covalently linked to a cationic co-polymer. The copolymer comprises hydrophilic, cationic monomeric units and/or hydrophobic, cationic monomeric units derived from a non- cationic, ethylenically unsaturated hydrophilic monomer and/or monomeric units derived from an ethylenically unsaturated monomer containing a moiety reactive with complementary reactive functionalities at the lens surface.

[0071] The copolymer is covalently linked directly to the device surface with the moiety reactive with the complementary functionality at the device surface, or covalently linked with an intermediate polymer reactive with both the device surface and the copolymer.

[0072] According to some embodiments of the current disclosure, the cationic copolymer on the device surface complexes with mucin or a mucin fusion polypeptide. The present disclosure provides a copolymer complexed with a mucin fusion polypeptide, for example, with PSGL-l/mIgG2b and PSGL-l/hIgG4. The mucin fusion polypeptide 00866 complexed with the copolymer on the device is glycosylated with mono and/or disialylated core 1 structures.

[0073] In particular, the present invention relates to an ophthalmic pharmaceutical composition for treating and/or preventing the ophthalmologic clinical symptoms and/or signs in keratoconjunctivitis sicca, or dry eye syndrome, which comprises a recombinant mucin polypeptide as an effective ingredient.

[0074] In an embodiment, the fusion protein is incubated for 1 -4 hours with the polymeric surface in 50 mM carbonate buffer, pH 9.6. In yet another embodiment, the fusion protein is incubated for 1 -2 hours with the polymeric surface in 50 mM carbonate buffer, pH 9.6. The buffer used during incubation is any buffer well known in the art, with the pH between pH 7.0-9.9.

[0075] In some embodiments, the fusion proteins of the present disclosure are bound to the ophthalmic lens by reversible and/or irreversible interactions (e.g. , covalent bonds, non-covalent interactions, or the like). In certain embodiments, the present disclosure provides that the surface bound fusion proteins are adhered to the ophthalmic lens surface by direct adsorption, hydrophobic ionic, or covalent binding or by linker chemistries selected from the group consisting of homo- or hetero-bifunctional linkers, N-hydroxy succinimidyl esters, biotin, avidin, streptavidin, maleimide, thiol bonding, amines, hydrazones, dendrimers, and carbodiimides.

[0076] Provided in certain embodiments herein is an ophthalmic device, comprising an ophthalmic lens with an outer surface and an inner surface and fusion proteins of this invention associated with at least a portion of the outer or inner surface in an amount effective to provide ocular boundary lubrication in an ocular environment of an individual wearing the ophthalmic lens. In certain embodiments, the fusion proteins are bound to the surface of the ophthalmic lens. In some embodiments, a device described herein comprises a lubricating composition disposed on the surface of the ophthalmic lens, the lubricating composition comprising (i) a gel-forming agent, a surfactant, or a combination thereof; and (ii) fusion proteins of the invention.

[0077] In some embodiments, lubricating, gel forming or surfactant composition further comprises one or more ophthalmically acceptable agents selected from the group consisting of an ophthalmically acceptable demulcent, ophthalmically acceptable excipient, ophthalmically acceptable astringent, ophthalmically acceptable vasoconstrictor, and ophthalmically acceptable emollient. Exemplary ophthalmically acceptable demulcents contemplated in the present invention include, but are not limited to, carboxymethylcellulose sodium (e.g., about 0.2 to about 2.5% w/v), hydroxyethyl cellulose (e.g., about 0.2 to about 2.5% w/v), hypromellose (e.g. , about 0.2 to about 2.5% w/v), methylcellulose (e.g., about 0.2 to about 2.5% w/v), dextran 70 (e.g., about 0.1% w/v), gelatin (e.g. , about 0.01 % w/v), glycerin (e.g. , about 0.2 to about 1 % w/v), polyethylene glycol 300 (e.g. , about 0.2 to about 1 % w/v), polyethylene glycol 400 (e.g. , about 0.2 to about 1 % w/v), polysorbate 80 (e.g., about 0.2 to about 1% w/v), propylene glycol (e.g., about 0.2 to about 1% w/v), polyvinyl alcohol (e.g., about 0.1 to about 4% w/v), povidone (e.g. , about 0.1 to about 2% w/v).

[0078] Exemplary ophthalmically acceptable excipients/emollients contemplated in the present invention include, but are not limited to, anhydrous lanolin (e.g. , up to or equal to about 1 up to or equal to about 10% w/v), lanolin (e.g. , up to or equal to about 1 to up to or equal to about 10% w/v), light mineral oil (e.g. , up to or equal to about 50% w/v), mineral oil (e.g. , up to or equal to about 50% w/v), paraffin (e.g. , up to or equal to about 5% w/v), petrolatum (e.g., up to or equal to about 100% w/v), white ointment (e.g. , up to or equal to about 100%) w/v), white petrolatum (e.g. , up to or equal to about 100% w/v), white wax (e.g. , up to or equal to about 5% w/v), yellow wax (e.g. , up to or equal to about 5% w/v). An exemplary ophthalmically acceptable astringent contemplated in the present invention includes, but is not limited, to, zinc sulfate (e.g. , about 0.25% w/v). Exemplary

ophthalmically acceptable vasoconstrictors contemplated in the present invention include, but are not limited to, ephedrine hydrochloride (e.g. , about 0. 123% w/v), naphazoline

hydrochloride (e.g., about 0.01 to about 0.03% w/v), phenylephrine hydrochloride (e.g. , about 0.08 to about 0.2% w/v), and tetrahydrozoline hydrochloride (e.g. , about 0.01 to about 0.05% w/v).

[0079] In some of these embodiments, the demulcents, excipients, astringents, vasoconstrictors, emollients and electrolytes provide a means to deliver the boundary lubricant molecules in an ophthalmically acceptable manner. Ophthalmically acceptable compositions are suitable for topical application to the ocular surface if they lack

unacceptable eye toxicity, burning, itchiness, viscosity, blurred vision, etc. upon application.

[0080] In certain embodiments, the gel forming or surfactant composition further comprises other ophthalmic lens care compounds that may be suspended in a phosphate buffered saline or an osmotically balanced salt solution of tear electrolytes, including one or more of sodium chloride (e.g. , about 44% to about 54% mole fraction), potassium chloride (e.g. , about 8% to about 14% mole fraction), sodium bicarbonate (e.g. , about 8% to about 18%) mole fraction), potassium bicarbonate (e.g. , about 0% to about 4% mole fraction), calcium chloride (e.g., about 0% to about 4% mole fraction), magnesium chloride (e.g., about 4 000866

0% to about 4% mole fraction), trisodium citrate (e.g. about 0% to about 4% mole fraction), and hydrochloric acid (e.g. , about 0% to about 20% mole fraction) or sodium hydroxide (e.g. , about 0% to about 20% mole fraction). In one embodiment, the carrier could be formulated to generate an aqueous electrolyte solution in the about 150-200 mM range.

[0081] In certain embodiments, the ophthalmic lens care compounds are suspended in an ophthalmically acceptable balanced salt solution comprising at least three electrolytes, including but not limited to, sodium chloride (NaCl) about 0.64%, potassium chloride (KC1) about 0.075%), calcium chloride dihydrate (CaCl₂.2H₂0) about 0.048%, magnesium chloride hexahydrate (MgCl₂.6H₂0) about 0.03%, sodium acetate trihydrate (C2H₃Na0₂.3H₂0) about 0.39%, sodium citrate dehydrate (C₆H₅Na₃0₇.2H₂0) about 0.17%, sodium hydroxide and/or hydrochloric acid (to adjust pH to approximately 7.5) with an osmolarity of approximately 300 mOsms/L.

[0082] In certain embodiments, the ophthalmic lens Care compounds are suspended in an ophthalmically acceptable balanced salt solution, comprised of sodium (Na⁺) of approximately 128 mM, potassium (K⁺) of approximately 24 mM, chloride (CI^") of approximately 1 13 mM, calcium (Ca²⁺) of approximately 0.4 mM, magnesium (Mg²⁺) of approximately 0.3 mM, HC03^" of approximately 5 mM, citrate of approximately 1 mM, phosphate of approximately 14 mM, acetate of approximately 15 mM, and sodium hydroxide and/or hydrochloric acid (to adjust pH to approximately 7.5) with an osmolarity of approximately 300 mOsms/L.

[0083] The preparations are useful for the treatment of disorders such as

keratoconjunctivitis sicca or dry eye syndrome. In general, the preparations are also effective for the relief of symptoms of eye irritation, such as those caused by dry environmental conditions or by contact lens wear.

[0084] The embodiments of the current invention may use plasmid expression systems for enhanced productivity.

MUCIN POLYPEPTIDES

[0085] The mucins are the product of corneal and conjunctival cells and they contribute to the epithelial cell surface structure and anchor the overlying aqueous

component. The mucins are primarily produced by goblet cells of the conjunctiva. This gel helps cleanse the eye surface by removing debris and bacteria, and by exfoliating cells. In various aspects, the invention provides composition containing a recombinant mucin polypeptide useful for coating devices placed into contact with mucosal tissue, e.g. , 2014/000866 ophthalmic device, intended for placement in contact with epithelial tissue, especially corneal onlays and contact lenses.

[0086] A "mucin polypeptide" refers to a polypeptide having a mucin domain. The mucin polypeptide has one, two, three, five, ten, twenty or more mucin domains. The mucin polypeptide is any glycoprotein characterized by an amino acid sequence substituted with O- glycans. For example, a mucin polypeptide has every second or third amino acid being a serine or threonine. The mucin polypeptide is a secreted protein. Alternatively, the mucin polypeptide is a cell surface protein.

[0087] Mucin domains are rich in the amino acids threonine, serine and proline, where the oligosaccharides are linked via N-acetylgalactosamine to the hydroxy amino acids (O-glycans). A mucin domain comprises or alternatively consists of an O-linked

glycosylation site. A mucin domain has 1 , 2, 3, 5, 10, 20, 50, 100 or more O-linked glycosylation sites. Alternatively, the mucin domain comprises an N-linked glycosylation site. A mucin polypeptide has about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 100% of its mass due to the glycan. A mucin polypeptide is any polypeptide encoded for by a MUC gene (i. e. , MUC1 , MUC2, MUC3, MUC4, MUC5a, MUC5b, MUC5c, MUC6, MUCH , MUC 12, etc.). In some embodiments, a mucin polypeptide is P-selectin glycoprotein ligand 1 (PSGL- 1), CD34, CD43, CD45, CD96, GlyCAM- 1 , MAdCAM- 1 , red blood cell glycophorins, glycocalicin, glycophorin, sialophorin, leukosialin, LDL-R, ZP3, and epiglycanin. The present disclosure provides PSGL- 1 mucin polypeptide fused to second polypeptide or at least a 3 or more amino acid fragment thereof. PSGL- 1 is a homodimeric glycoprotein with two disulfide-bonded 120 kDa subunits of type 1 transmembrane topology, each containing 402 amino acids. In the extracellular domain there are 15 repeats of a 1 0-amino acid consensus sequence that contains 3 or 4 potential sites for addition of O-linked oligosaccharides. PSGL- 1 is predicted to have more than 53 sites for O-linked glycosylation and 3 sites for N-linked glycosylation in each monomer.

[0088] The mucin polypeptide contains all or a portion of the mucin protein.

Alternatively, the mucin protein includes the extracellular portion of the polypeptide. For example, the mucin polypeptide includes the extracellular portion of PSGL-1 or a portion thereof (e.g., amino acids 19-319 disclosed in GenBank Accession No. A57468). The mucin polypeptide also includes the signal sequence portion of PSGL- 1 (e.g., amino acids 1 -18), the transmembrane domain (e.g. , amino acids 320-343), and the cytoplasmic domain (e.g. , amino acids 344-412). [0089] The recombinant mucin polypeptides may exist as oligomers, such as dimers, trimers or pentamers. In some embodiments, the fusion polypeptide is a dimer.

[0090] A "non-mucin polypeptide" refers to a polypeptide of which at least less than

40% of its mass is due to glycans.

[0091] The mucin polypeptide corresponds to all or a portion of a mucin protein. For example, the recombinant mucin polypeptide comprises at least a portion of a mucin protein. "At least a portion" is meant that the mucin polypeptide contains at least one mucin domain (e.g., an O-linked glycosylation site). The mucin protein comprises the extracellular portion of the polypeptide. For example, the mucin polypeptide comprises the extracellular portion of PSGL-1.

[0092] The recombinant mucin polypeptide is glycosylated by one or more glycosyltransferases. The first polypeptide is glycosylated by 2, 3, 5 or more

glycosyltransferases. Glycosylation is sequential or consecutive. Alternatively glycosylation is concurrent or random, i.e., in no particular order. The first polypeptide is glycosylated by any enzyme capable of adding or producing N-linked or O-linked glycans to or on a protein backbone. For example the first polypeptide is glycosylated by a.2,3- and/or a2,6- sialyltransferase. The first polypeptide contains equal to or greater than about 40%, equal to or greater than about 50%, equal to or greater than about 60%, equal to or greater than about 70%, equal to or greater than about 80%, equal to or greater than about 90% or equal to or greater than about 95% of its mass due to carbohydrate.

[0093] The mucin polypeptide, and/or nucleic acids encoding the mucin polypeptide, is constructed using mucin encoding sequences are known in the art.

[0094] The mucin polypeptide moiety is provided as a variant mucin polypeptide having an alteration in the naturally-occurring mucin sequence (wild type) that results in increased carbohydrate content (relative to the non-mutated sequence). As used herein, an alteration in the naturally-occurring (wild type) mucin sequence includes one or more one or more substitutions, additions or deletions into the nucleotide and/or amino acid sequence such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Alterations can be introduced into the naturally-occurring mucin sequence by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0095] For example, the variant mucin polypeptide comprised additional O-linked glycosylation sites compared to the wild-type mucin. Alternatively, the variant mucin polypeptide comprises an amino acid sequence alteration that results in an increased number of serine, threonine or proline residues as compared to a wild type mucin polypeptide. This 4 000866 increased carbohydrate content can be assessed by determining the protein to carbohydrate ratio of the mucin by methods known to those skilled in the art.

[0096] Alternatively, the mucin polypeptide moiety is provided as a variant mucin polypeptide having alterations in the naturally-occurring mucin sequence (wild type) that results in a mucin sequence with more O-glycosylation sites or a mucin sequence preferably recognized by peptide N-acetylgalactosaminyltransferases resulting in a higher degree of glycosylation.

[0097] In some embodiments, the mucin polypeptide moiety is provided as a variant mucin polypeptide having alterations in the naturally-occurring mucin sequence (wild type) that results in a mucin sequence more resistant to proteolysis (relative to the non-mutated sequence).

[0098] The mucin polypeptide includes full-length PSGL-1. Alternatively, the first polypeptide comprise less than full-length PSGL-1 polypeptide, e.g. , a functional fragment of a PSGL-1 polypeptide. For example the first polypeptide is less than 400 contiguous amino acids in length of a PSGL-1 polypeptide, e.g., less than or equal to 300, 250, 150, 100, or 50, contiguous amino acids in length of a PSGL-1 polypeptide, and at least 25 contiguous amino acids in length (i.e., 25-300 amino acids in length, 25-250 amino acids in length, 25-150 amino acids in length, 25-100 amino acids in length, or 25-50 amino acids in length) of a PSGL-1 polypeptide. The first polypeptide is, for example, the extracellular portion of PSGL- 1 , or includes a portion thereof.

SECOND POLYPEPTIDES

[0099] In some embodiments, the second polypeptide of the present disclosure is soluble. In some embodiments, the second polypeptide includes a sequence that facilitates association of the fusion polypeptide with a second mucin polypeptide. The second polypeptide includes at least a region of an immunoglobulin polypeptide. "At least a region" is meant to include any portion of an immunoglobulin molecule, such as the light chain, heavy chain, Fc region, Fab region, Fv region or any fragment thereof. Immunoglobulin fusion polypeptides are known in the art and are described in e.g. , US Patent Nos. 5,516,964; 5,225,538; 5,428,130; 5,514,582; 5,714,147; and 5,455,165.

[00100] The second polypeptide comprises a full-length immunoglobulin polypeptide. Alternatively, the second polypeptide comprises less than full-length immunoglobulin polypeptide, e.g., a heavy chain, light chain, Fab, Fab₂, Fv, or Fc. In some embodiments, the second polypeptide includes the heavy chain of an immunoglobulin polypeptide. More preferably the second polypeptide includes the Fc region of an immunoglobulin polypeptide. 6

[00101] The second polypeptide has less effector function than the effector function of an Fc region of a wild-type immunoglobulin heavy chain. Alternatively, the second polypeptide has similar or greater effector function of an Fc region of a wild-type

immunoglobulin heavy chain. An Fc effector function includes for example, Fc receptor binding, complement fixation and T cell depleting activity (see for example, US Patent No. 6,136,310). Methods of assaying T cell depleting activity, Fc effector function, and antibody stability are known in the art. In one embodiment the second polypeptide has low or no affinity for the Fc receptor. Alternatively, the second polypeptide has low or no affinity for complement protein Clq.

FUSION POLYPEPTIDES

[00102] In various aspects the invention provides fusion proteins that include a first polypeptide containing at least a portion of a glycoprotein, e.g., a mucin polypeptide or an alpha-globulin polypeptide, operatively linked to a second polypeptide. As used herein, a "fusion protein" or "chimeric protein" includes at least a portion of a glycoprotein polypeptide operatively linked to a non-mucin polypeptide.

[00103] A "mucin polypeptide" refers to a polypeptide having a mucin domain. The mucin polypeptide has one, two, three, five, ten, twenty or more mucin domains. The mucin polypeptide is any glycoprotein characterized by an amino acid sequence substituted with O- glycans. For example, a mucin polypeptide has every second or third amino acid being a serine or threonine. The mucin polypeptide is a secreted protein. Alternatively, the mucin polypeptide is a cell surface protein.

[00104] In some aspect the recombinant mucin polypeptide is operatively linked to a second polypeptide. As used herein, a "fusion protein" or "chimeric protein" includes at least a portion of a mucin polypeptide operatively linked to a non-mucin polypeptide.

[00105] Within the fusion protein, the term "operatively linked" is intended to indicate that the mucin polypeptide and second polypeptides are chemically linked (most typically via a covalent bond such as a peptide bond) in a manner that allows for O-linked and/or N-linked glycosylation of the mucin polypeptide. When used to refer to nucleic acids encoding a fusion polypeptide, the term operatively linked means that a nucleic acid encoding the mucin polypeptide and the non-mucin polypeptide are fused in-frame to each other. The non-mucin polypeptide can be fused to the N-terminus or C-terminus of the mucin polypeptide.

[00106] Optionally, the mucin fusion polypeptide is linked to one or more additional moieties. For example, in one embodiment the fusion protein is additionally (i.e. , in addition to be conjugation between a mucin or at least a 3 amino acid fragment thereof and an immunoglobulin or at least a 3 amino acid fragment thereof) linked to a GST fusion protein in which the fusion protein sequences are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences. Such fusion proteins facilitate the purification of the fusion protein. Alternatively, in one embodiment, the fusion protein is additionally (i.e., in addition to be conjugation between a mucin or at least a 3 amino acid fragment thereof and an immunoglobulin or at least a 3 amino acid fragment thereof) linked to a solid support.

Various solid supports are known to those skilled in the art. For example, the fusion protein is linked to a particle made of, e.g., metal compounds, silica, latex, polymeric material; a microtiter plate; nitrocellulose, or nylon or a combination thereof.

[00107] The fusion protein includes a heterologous signal sequence (i.e. , a polypeptide sequence that is not present in a polypeptide encoded by a mucin nucleic acid) at its

N-terminus. For example, the native mucin glycoprotein signal sequence can be removed and replaced with a signal sequence from another protein. In certain host cells (e.g., mammalian host cells), expression and/or secretion of polypeptide can be increased through use of a heterologous signal sequence.

[00108] A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as

appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. The fusion gene is synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments is carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that encode a fusion moiety (e.g. , an Fc region of an immunoglobulin heavy chain). A mucin encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the immunoglobulin protein.

[00109] The fusion polypeptides may exist as oligomers, such as dimers, trimers or pentamers. In some embodiments the present disclosure provides a fusion polypeptide, which is a dimer. 2014/000866

[00110] Mucin domains are rich in the amino acids threonine, serine and proline, where the oligosaccharides are linked via N-acetylgalactosamine to the hydroxy amino acids (O-glycans). A mucin domain comprises or alternatively consists of an O-linked

glycosylation site. A mucin domain has about 1 , about 2, about 3, about 5, about 10, about 20, about 50, about 100 or more O-linked glycosylation sites. The mucin domain also has one or more N-linked glycosylation site(s). In some embodiments, mucin polypeptide is any polypeptide encoded for by a MUC gene (e.g., MUC 1 , MUC2, MUC3). The present disclosure also provides mucin polypeptides, for example, mucin polypeptide P-selectin glycoprotein ligand 1 (PSGL-1 ), CD34, CD43, CD45, CD96, GlyCAM-1 , MAdCAM or red blood cell glycophorins. In some embodiments, the mucin of the present disclosure is PSGL- 1.

[00111] In additional embodiments a mucin polypeptide, for example, CD34, CD43, CD45, CD96, GlyCAM- 1 , MAdCAM- 1 , fused to an immunoglobulin polypeptide or a fragment thereof may be glycosylated with mono- and/or disialylated core 1 and/or core 2 structures.

[00112] The second polypeptide comprises at least a region of an immunoglobulin polypeptide. For example, the second polypeptide comprises a region of a heavy chain immunoglobulin polypeptide. Alternatively, the second polypeptide comprises the FC region of an immunoglobulin heavy chain.

[00113] Also included in the invention is an AV (avidin) fusion polypeptide in which a first polypeptide is fused to AV and/or a second polypeptide, e.g. , an immunoglobulin polypeptide. The AV fusion polypeptide is expressed from a vector containing AV fusion polypeptide-encoding nucleic acids described herein, and a cell containing the vectors or nucleic acids described herein. Optionally, the vector further comprises a nucleic acid encoding one or more glycosyltransferases necessary for the synthesis of the desired carbohydrate epitope. For example, the vector contains a nucleic acid encoding an a2,6- sialyltransferase. In some embodiments, the AV fusion polypeptide of the present disclosure is a multimer. The present disclosure provides an AV fusion polypeptide which is a dimer.

[00114] Within an AV fusion protein of the invention the mucin polypeptide corresponds to all or a portion of a mucin protein. An AV fusion protein comprises at least a portion of a mucin protein. "At least a portion" is meant that the mucin polypeptide contains at least one mucin domain (e.g. , an O-linked glycosylation site). The mucin protein comprises the extracellular portion of the polypeptide. For example, the mucin polypeptide comprises the extracellular portion of PSGL-1 . [00115] The first polypeptide is glycosylated by one or more glycosyltransferases. The first polypeptide is glycosylated by 2, 3, 5 or more glycosyltransferases. Glycosylation is sequential or consecutive. Alternatively glycosylation is concurrent or random, i.e., in no particular order. The first polypeptide is glycosylated by any enzyme capable of adding N- linked or O-linked sialic acid determinants to a protein backbone. For example the first polypeptide is glycosylated by one or more of the following: a core 2 βό-N- acetylglucosaminyltransferase, a core 3 p3-N-acetylglucosaminyltransferase, a β4- galactosyltransferase, a 3-galactosyltransferase, an a3-sialyltransferase, an a6- sialyltransferase, and/or an a3-N-acetylgalactosaminyltransferase. The first polypeptide is more heavily glycosylated than the native {i.e., wild-type) glycoprotein. For example, the first polypeptide may have 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold or more glycans than a native glycoprotein. The first polypeptide may contain greater than or equal to about 40%, greater than or equal to about 50%, greater than or equal to about 60%, greater than or equal to about 70%, greater than or equal to about 80%, greater than or equal to about 90% or greater than or equal to about 95% of its mass due to carbohydrate.

[00116] The carbohydrate epitopes on the fusion polypeptide of the current invention are Siaa3Galp3GalNAca, Siaa3Galp4GlcNAcp, Siaa3Gal 3GlcNAc ,

Siaa6Gal 3GalNAca, Siaa6Gal 4GlcNAc , and/or Siact6Galp3GlcNAcp.

[00117] Within the fusion protein, the term "operatively linked" is intended to indicate that the first and second polypeptides are chemically linked (most typically via a covalent bond such as a peptide bond) in a manner that allows for O-linked and/or N-linked glycosylation of the first polypeptide. When used to refer to nucleic acids encoding a fusion polypeptide, the term operatively linked means that a nucleic acid encoding the mucin or alpha globulin polypeptide and the non-mucin polypeptide are fused in-frame to each other. The non-mucin polypeptide can be fused to the N-terminus or C-terminus of the mucin or alpha globulin polypeptide.

[00118] The present disclosure provides AV fusion protein linked to one or more additional moieties. For example, the AV fusion protein is additionally linked to a GST fusion protein in which the AV fusion protein sequences are fused to the C-terminus of the GST {i.e. , glutathione S-transferase) sequences. Such fusion proteins may facilitate the purification of the AV fusion protein. Alternatively, the AV fusion protein may additionally be linked to a solid support. Various solid supports are known to those skilled in the art. Such compositions can facilitate removal of anti-blood group antibodies. For example, the AV 0866 fusion protein may be linked to a particle made of, e.g., metal compounds, silica, latex, polymeric material; a microtiter plate; nitrocellulose, or nylon or a combination thereof.

[00119] The fusion protein may include a heterologous signal sequence ( . e. , a polypeptide sequence that is not present in a polypeptide encoded by a mucin or a globulin nucleic acid) at its N-terminus. For example, the native mucin or alpha-glycoprotein signal sequence may be removed and replaced with a signal sequence from another protein. In certain host cells (e.g. , mammalian host cells), expression and/or secretion of polypeptide may be increased through use of a heterologous signal sequence.

[00120] AV fusion polypeptides may exist as oligomers, such as dimers, trimers or pentamers. Preferably, the AV fusion polypeptide is a dimer.

[00121] The first polypeptide, and/or nucleic acids encoding the first polypeptide, is constructed using mucin or an alpha-globulin encoding sequences known in the art. Suitable sources for mucin polypeptides and nucleic acids encoding mucin polypeptides include GenBank Accession Nos. NP663625 and NM145650, CAD10625 and AJ417815, XP 140694 and XM140694, XP006867 and XM006867 and NP00331777 and NM009151 respectively, and are incorporated herein by reference in their entirety. Suitable sources for alpha-globulin polypeptides and nucleic acids encoding alpha-globulin polypeptides include GenBank Accession Nos. AAH26238 and BC026238; NP000598; and BC012725, AAH12725 and BC012725, and NP44570 and NM053288 respectively, and are incorporated herein by reference in their entirety.

[00122] The mucin polypeptide moiety is provided as a variant mucin polypeptide having a mutation in the naturally-occurring mucin sequence (wild type) that results in increased carbohydrate content (relative to the non-mutated sequence). For example, the variant mucin polypeptide comprised additional O-linked glycosylation sites compared to the wild-type mucin. Alternatively, the variant mucin polypeptide comprises an amino acid sequence mutations that results in an increased number of serine, threonine or proline residues as compared to a wild type mucin polypeptide. This increased carbohydrate content can be assessed by determining the protein to carbohydrate ratio of the mucin by methods known to those skilled in the art.

[00123] Similarly, the alpha-globulin polypeptide moiety is provided as a variant alpha-globulin polypeptide having a mutation in the naturally-occurring alpha-globulin sequence (wild type) that results in increased carbohydrate content (relative to the non- mutated sequence). For example, the variant alpha-globulin polypeptide comprised additional N-linked glycosylation sites compared to the wild-type alpha-globulin. [00124] Alternatively, the mucin or alpha-globulin polypeptide moiety is provided as a variant mucin or alpha-globulin polypeptide having mutations in the naturally-occurring mucin or alpha-globulin sequence (wild type) that results in a mucin or alpha-globulin sequence more resistant to proteolysis (relative to the non-mutated sequence).

[00125] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms

"transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

[00126] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the fusion polypeptides or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[00127] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) fusion polypeptides. Accordingly, the invention further provides methods for producing using polypeptides using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which recombinant expression vector encoding fusion polypeptides has been

introduced) in a suitable medium such that fusion polypeptides is produced. In another embodiment, the method further comprises isolating polypeptide from the medium or the host cell.

[00128] The fusion polypeptides may be isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis or the like. For example, the immunoglobulin fusion proteins may be purified by passing a solution through a column which contains immobilized protein A or protein G which selectively binds the Fc portion of the fusion protein. See, e.g., Reis, K. J., et al. , J. Immunol. 132:3098-3102 (1984); PCT Application, Publication No. WO87/00329. The fusion polypeptide may then be eluted by treatment with a chaotropic salt or by elution with aqueous acetic acid (1 M).

[00129] Alternatively, the mucin polypeptide and or the fusion polypeptides according to the invention can be chemically synthesized using methods known in the art. Chemical synthesis of polypeptides is described in, e.g., Peptide Chemistry, A Practical Textbook, Bodasnsky, Ed. Springer-Verlag, 1988; Merrifield, Science 232: 241 -247 (1986); Barany, et al, Intl. J. Peptide Protein Res. 30: 705-739 (1987); Kent, Ann. Rev. Biochem. 57:957-989 (1988), and Kaiser, et al, Science 243: 187-198 (1989). The polypeptides are purified so that they are substantially free of chemical precursors or other chemicals using standard peptide purification techniques. The language "substantially free of chemical precursors or other chemicals" includes preparations of peptide in which the peptide is separated from chemical precursors or other chemicals that are involved in the synthesis of the peptide. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of peptide having less than about 30% (by dry weight) of chemical precursors or non-peptide chemicals, more preferably less than about 20% chemical precursors or non-peptide chemicals, still more preferably less than about 10% chemical precursors or non-peptide chemicals, and most preferably less than about 5% chemical precursors or non-peptide chemicals.

[00130] Chemical synthesis of polypeptides facilitates the incorporation of modified or unnatural amino acids, including D-amino acids and other small organic molecules.

Replacement of one or more L-amino acids in a peptide with the corresponding D-amino acid isoforms can be used to increase the resistance of peptides to enzymatic hydrolysis, and to enhance one or more properties of biologically active peptides, i.e., receptor binding, functional potency or duration of action. See, e.g. , Doherty, et al, 1993. J. Med. Chem. 36: 2585-2594; Kirby, et al., 1993. J. Med. Chem. 36:3802-3808; Morita, et al, 1994. FEBS Lett. 353: 84-88; Wang, et al , 1993. Int. J. Pept. Protein Res. 42: 392-399; Fauchere and

Thiunieau, 1992. Adv. Drug Res. 23: 127-159.

[00131] Introduction of covalent cross-links into a peptide sequence can

conformationally and topographically constrain the polypeptide backbone. This strategy can be used to develop peptide analogs of the fusion polypeptides with increased potency, selectivity and stability. Because the conformational entropy of a cyclic peptide is lower than T IB2014/000866 its linear counterpart, adoption of a specific conformation may occur with a smaller decrease in entropy for a cyclic analog than for an acyclic analog, thereby making the free energy for binding more favorable. Macro-cyclization is often accomplished by forming an amide bond between the peptide N- and C-termini, between a side chain and the N- or C-terminus [e.g., with K₃Fe(CN)₆ at pH 8.5] (Samson et al, Endocrinology, 137: 5182-5185 (1996)), or between two amino acid side chains. See, e.g., DeGrado, Adv Protein Chem, 59: 51- 124 (1988). Disulfide bridges are also introduced into linear sequences to reduce their flexibility. See, e.g., Rose, et al, Adv Protein Chem, 37: 1-109 (1985); Mosberg et al, Biochem Biophys Res Commun, 106: 505-512 (1982). Furthermore, the replacement of cysteine residues with penicillamine (Pen, 3-mercapto-(D) valine) has been used to increase the selectivity of some opioid-receptor interactions. Lipkowski and Carr, Peptides: Synthesis, Structures, and Applications, Gutte, ed., Academic Press pp. 287-320 (1995).

PROTEIN EXPRESSION

[00132] Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding mucin polypeptides, or derivatives, fragments, analogs or homologs thereof. The vector contains a nucleic acid encoding a mucin

polypeptide operably linked to a nucleic acid encoding an immunoglobulin polypeptide, or derivatives, fragments analogs or homologs thereof. Additionally, the vector comprises a nucleic acid encoding a glycosyltransferase such as an a2,3- and/or a2,6-sialyltransferase. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced {e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of 4 000866 expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[00133] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector,

"operably-Iinked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

[00134] The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:

METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.

[00135] The recombinant expression vectors of the invention can be designed for expression of fusion polypeptides in prokaryotic or eukaryotic cells. For example, fusion polypeptides can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculo virus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. ( 1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[00136] Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non- fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (z) to increase expression of recombinant protein; (/ ) to increase the solubility of the recombinant protein; and (in) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[00137] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al, (1988) Gene 69:301 -315) and pET 1 Id (Studier et al, GENE

EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[00138] One strategy to maximize recombinant protein expression in E. coli is to express the protein in host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 1 19-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et al, 1992. Nucl. Acids Res. 20: 21 1 1 -21 18). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[00139] The fusion polypeptide expression vector is a yeast expression vector.

Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (Baldari, et al, 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al, 1987. Gene 54: 1 13-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

[00140] Alternatively, fusion polypeptide can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Mamestra brassicae cells or SF9 cells) include the pAc series (Smith, et al, 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and

Summers, 1989. Virology 170: 31-39). 2014/000866

[00141] A nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g. , Chapters 16 and 17 of Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[00142] Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and

"recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[00143] A host cell can be any prokaryotic or eukaryotic cell. For example, fusion polypeptides can be expressed in bacterial cells such as E. coli, insect cells such as M brassicae, yeast or mammalian cells (such as human, Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art. The CHO cells may co-express, for example only and without being limited to the example, PSGL-l/mIgG2b, C2 GnT-1 (Core 2 pi ,6-N-acetylglycosyltransferase I) and FUT-VII (a 1, 3 -fucosy transferase VII). The CHO cells of the current invention co-expressing PSGL-l/mIgG2b, C2 GnT-1 , and FUT-VII are defined as C-PSLex. The CHO cells of the current invention may also co- express PSGL-l/hIgG4 and only, C2 GnT-1 , or co-express any mucin/IgG fusion polypeptide of the current invention, C2 GnT-1, and sialyltransferase.

[00144] The host cells of the current invention may be non-glycoengineered CHO cells. Non-glycoengineered CHO cells may be used to produce any mucin/IgG fusion polypeptide of the current invention. For example, the fusion polypeptide is PSGL- l/mIgG2b, PSGL-l/hIgG4, or any mammalian mucin protein fused to a suitable fragment of a mammalian immunoglobulin protein. In some embodiments of the current invention, the fusion polypeptide may be sialylated. For example, the fusion peptide may be glycosylated with mono- and/or disialylated core 1 structures and/or core 2 structures. For example, the PSGL-l/mIgG2b is glycosylated with mono- and/or disialylated core 1 and/or core 2 IB2014/000866 structures. For example, PSGL-l/mIgG2b or PSGL-l/hIgG4 may be glycosylated with mono- and/or disialylated core 1 and/or core 2 structures.

[00145] Fusion polypeptides of the current invention can be expressed in yeast such as Pichia pastoris. Mucin/IgG fusion polypeptides of the current invention can be expressed in yeast such as Pichia pastoris. For example, the fusion polypeptide is PSGL-l/mIgG2b, PSGL-l/hIgG4, or any mammalian mucin protein fused to a suitable fragment of a mammalian immunoglobulin protein. In some embodiments of the current invention, mucin/IgG fusion polypeptides produced from yeast such as Pichia pastoris may be mannosylated. For example, PSGL-l/mIgG2b or PSGL-l/hIgG4 is mannosylated.

[00146] PSGLl/mIgG2b of the present disclosure is produced in CHO cells co- expressing the fusion protein and the genes coding for βΙ,ό-Ν-acetylglucosaminyltransferase I (C2 GnT-I) and l ,3fucosyltransferase VII (FucT-VII). This cell line is produced to express the carbohydrate epitope sialyl Lewis x (Siaa2,3Gaipi ,4(Fucal ,3)GlcNAc). The CHO cells co-expressing PSGL-l/mIgG2b, C2 GnT-1 , and FUT-VII are defined as C-PSLex. Fusion protein produced in this cell line mainly expresses mono- and/or disialylated core 1

(Siaa2,3Gaipi,3(Siaa2,6)GalNAc), as well as sialylated type 2 on a core 2 chain

(Siaa2,3Galpl ,4GlcNAcpl,6(Galpl ,3)GalNAc) as well as small amounts of sialyl Lewis x (SLex). Likewise, PSGL-l/hIgG4 is produced in non-glycoengineered CHO cells. The PSGL-l/hIgG4 expressed in these cells carries mainly mono- and disialylated core 1 glycans.

[00147] PSGL-l/hIgG4 of the present disclosure is produced in non-glycoengineered CHO cells or CHO cells co-expressing the fusion protein and the gene coding for β1 ,6-Ν- acetylglucosaminyltransferase I (C2 GnT-I). This cell line is produced to express the carbohydrate epitope mono- or disialylated Core 2 (Siaa2,3Gaipi,4GlcNAc). The CHO cells of the current invention co-expressing PSGL-l/hIgG4 and C2 GnT-1 are defined as C-PSC2 (sialylated Core 2). Fusion protein expresses mono- and/or disialylated core 1

(Siaa2,3Gaipi ,3(Siaa2,6)GalNAc), as well as sialylated type 2 on a core 2 chain

(Siaa2,3Galpl,4GlcNAcpl,6(Gal l ,3)GaINAc). In some embodiments, the CHO cells of the current invention also express an alpha2,3- or alpha2,6-sialyltransferase in order to increase the sialylation of the glycans.

[00148] In some embodiments, the present disclosure provides PSGL-l/hIgG4 and PSGLl/mIgG2b expressed in Pichia pastoris. In some embodiments, the fusion proteins are mannosylated. 4 000866

DEVICES

[00149] The invention is useful for devices, e.g., a biomedical device. The invention is useful for devices placed into contact with mucosal tissue, e.g. , ophthalmic device, intended for placement in contact with epithelial tissue, especially corneal onlays and contact lenses. The following disclosure references contact lenses, but is applicable to various other devices, e.g., biomedical devices. The current invention relates to a device comprising a polymer surface with a fusion protein coating the surface. The invention is useful for all known types of contact lenses, including both soft and rigid lens materials.

[00150] The device may comprise copolymers prepared from hydrophilic monomers. Examples of useful lens-forming hydrophilic monomers include, for example, without being limiting: amides such as N-dimethylacrylamide and N, N-dimethylacrylaminde; cyclic lactams such as N-vinyl-2-pyrrolidone; meth(acrylated) alcohols, such as 2-hydroxyethyl methacrylate and 2-hydroethylacrylate; and meth(acrylated) poly(ethyleneglycol)s. In one embodiment, the lens forming material of the current invention is poly(methyl methacrylate) (PMMA).

[00151] Contact lenses have specific effects on the tear film. They disrupt the film and increase the rate of tear evaporation. In patients with adequate tear volume, the impact of contact lenses on tear film is tolerable, but in those with inadequate tear volume, dry eye can result, which is estimated to occur in up to 30% of those who wear soft contact lenses and more than 80% of those wearing rigid contact lenses. See Tomlinson A. Epidemiology of Dry Eye Disease. In: Asbell PA, Lemp MA, eds. Dry Eye Disease, 1^st Ed. New York; Theime; 2006, ppl-15. The current invention provides coating of contact lens with fusion protein carrying carbohydrate moieties in order to improve wettability and tolerability of the contact lens.

[00152] The present disclosure provides a device, e.g. , a biomedical device, coated with a fusion protein carrying carbohydrate moieties. The fusion protein comprises a mucin polypeptide or a fragment thereof and is conjugated to an immunoglobulin or a fragment thereof. The mucin polypeptide (or a fragment thereof) and the immunoglobulin (or a fragment thereof) is glycosylated with one or more sialylated glycans. The carbohydrate composition of mucins improves characteristics of the medical device such as the wetting, adsorption, surface forces and friction of the surface. In some embodiments, the fragment of a mucin polypeptide is the extracellular domain of the polypeptide, which is at least three amino acids in length. 17IB2014/000866

[00153] The mucin polypeptide used as a first polypeptide of a fusion polypeptide of the present disclosure to coat a device, e.g., a medical device, e.g. , a contact lens, includes full-length PSGL-1. In some embodiments, the first polypeptide of a fusion polypeptide used for coating a device, e.g. , a medical device, e.g. , a contact lens, comprises less than full- length PSGL-1 polypeptide, e.g. , Ά functional fragment of a PSGL-1 polypeptide. For example the first polypeptide of a fusion polypeptide used for coating a device, e.g. , a medical device, e.g., a contact lens, is less than 400 contiguous amino acids in length of a PSGL-1 polypeptide, e.g., less than or equal to 300, 250, 150, 100, or 50, contiguous amino acids in length of a PSGL-1 polypeptide, and at least 25 contiguous amino acids in length (i.e. , 25-300 amino acids in length, 25-250 amino acids in length, 25-150 amino acids in length, 25-100 amino acids in length, or 25-50 amino acids in length) of a PSGL-1 polypeptide. The first polypeptide of a fusion polypeptide used for coating a device, e.g., a medical device, e.g. , a contact lens, is, for example, the extracellular portion of PSGL-1 , or includes a portion thereof.

[00154] In some embodiments, the second polypeptide of a fusion polypeptide used for coating a device, e.g. , a medical device, e.g. , a contact lens, is a fragment of the

immunoglobulin polypeptide. In some embodiments that second polypeptide of a fusion polypeptide used for coating a device, e.g., a medical device, e.g. , a contact lens, is the γ heavy chains or light chains of an IgG. In some embodiments, the immunoglobulin fragment is the Fc region of IgG 1 , IgG2, IgG3, or IgG4 for use in coating a medical device; and the medical device is coated as-such.

[00155] The present disclosure provides the mucin polypeptide or a fragment larger than 3 amino acids thereof, conjugated to IgA, IgD, IgE, IgM, or a domain fragment (at least 3 amino acids long) thereof, for use in coating a medical device; and the medical device is coated as-such. In some embodiments, the mucin polypeptide or a fragment at least 3 amino acids of a mucin polypeptide is conjugated to α, γ, δ, ε, or μ heavy chains of an

immunoglobulin for use in coating a medical device; and the medical device is coated as- such. The present disclosure provides an extracellular domain of a mucin conjugated to α, γ, δ, ε, or μ heavy chains of an immunoglobulin for use in coating a medical device; and the medical device is coated as-such. In some embodiments, PSGL-1 , CD34, CD43, CD45, CD96, GlyCAM-1 , MAdCAM- 1 , or at least 3 amino acid long fragment thereof. The present disclosure provides a secreted mucin, a membrane associated mucin, or at least a 3 amino acids long fragment thereof conjugated to α, γ, δ, ε, or μ heavy chains of an immunoglobulin for use in coating a medical device; and the medical device is coated as-such. Secreted 2014/000866 mucin is chosen from MUC2, MUC5AC, MUC5B, MUC6, MUC7, and MUC9, and the membrane associated mucin is chosen from MUCl , MUC3A, MUC3B, MUC4, and MUCl 6, and is conjugated to α, γ, δ, ε, or μ heavy chains of an immunoglobulin for use in coating a medical device; and the medical device is coated as-such. In some embodiments, at least a 3 amino acid fragment of a secreted mucin chosen from MUC2, MUC5AC, MUC5B, MUC6, MUC7, and MUC9, or at least a 3 amino acid fragment of a membrane associated mucin chosen from MUCl, MUC3A, MUC3B, MUC4, and MUCl 6, is conjugated to α, γ, δ, ε, or μ heavy chains of an immunoglobulin for use in coating a medical device; and the medical device is coated as-such.

[00156] The mucin polypeptide for use in coating a device, e.g. , a medical device, is full-length PSGL-1. In some embodiments, the first polypeptide, for use in coating a device, e.g., a medical device, is not the full-length PSGL-1 polypeptide, e.g. , a functional fragment of a PSGL-1 polypeptide. For example, the first polypeptide, for use in coating a device, e.g., a medical device, is less than 400 contiguous amino acids in length of a PSGL-1 polypeptide, e.g., less than or equal to 300, 250, 150, 100, or 50, contiguous amino acids in length of a PSGL-1 polypeptide, and at least 25 contiguous amino acids in length of a PSGL-1 polypeptide. The first polypeptide, for use in coating a device, e.g. , a medical device, is, for example, the extracellular portion of PSGL-1, or includes a portion thereof.

[00157] The present disclosure provides a fusion polypeptide for use in coating a device, e.g. , a medical device, in which the first polypeptide is glycosylated by one or more glycosyltransferases. In some embodiments, the first polypeptide, for use in coating a device, e.g., a medical device, is glycosylated by 2, 3, 5 or more glycosyltransferases. In some embodiments, glycosylation is sequential or consecutive. In additional embodiments, glycosylation is concurrent or random, i.e., in no particular order. The first polypeptide is glycosylated by any enzyme capable of adding N-linked or O-linked sialic acid determinants to a protein backbone. For example the first polypeptide is glycosylated by one or more of the following: a core 2 p6-N-acetylglucosaminyltransferase, a core 3 p3-N- acetylglucosaminyltransferase, a p4-galactosyltransferase, a p3-galactosyltransferase, an a3- sialyltransferase, an a6-sialyltransferase, and/or an a3-N-acetylgalactosaminyltransferase. The first polypeptide is more heavily glycosylated than the native (i.e. wild-type)

glycoprotein. For example, the first polypeptide may have about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 fold, or more glycans than the native first polypeptide or a native glycoprotein. The first polypeptide may contain equal to or greater that about 40%, equal to or greater that about 50%, equal to or greater that about 60%, equal 14 000866 to or greater that about 70%, equal to or greater that about 80%, equal to or greater that about 90% or equal to or greater that about 95% of its mass due to carbohydrate.

[00158] The present disclosure provides, a fusion polypeptide for use in coating a device, e.g. , a medical device, e.g. , a contact lens, in which the first polypeptide contains equal to or greater that about 40%, equal to or greater that about 50%, equal to or greater that about 60%, equal to or greater that about 70%, equal to or greater that about 80%, equal to or greater that about 90% or equal to or greater that about 95% of its mass due to carbohydrate.

[00159] For example, fusion polypeptide for use in coating a device, e.g. , a medical device, e.g. , a contact lens, is a fusion of PSGL-1 with a second polypeptide, for example, the Fc region of IgG. In some embodiments, the fusion protein is PSGL-l/hIgG4

[00160] The fusion proteins of the present disclosure are produced in C-PSLex (e.g.,

CHO cells co-expressing PSGL-1 /mIgG_2b, C2 GnT-I (core 2 β Ι ,ό-iV- acetylglycosyltransferase I) and FUT-VII (al ,3-fucosyltransferase VII)) or in C-PSC2 (e.g. , CHO cells co-expressing PSGL-l/mIgG_2b or PSGL-l/hIgG4 and C2 GnT-I (core 2 βΙ ,ό-iV- acetylglycosyltransferase I with or without co-expression of an alphal ,3- or an alphal ,6- sialyl transferase). The fusion proteins produced in C-PSLex or C-PSC2 may carry multiple negative charges. The wetting and adsorption properties of PSGL-l/mIgG₂b or PSGL- l/hIgG4, produced may be measured. PMMA may be used, as an exemplary contact lens material, for coating the surface substrate for wetting and adsorption measurements.

[00161] The present disclosure provides a device, e.g. , contact lens, coated with PSGL- l/mIgG_2b or PSGL-l/hIgG4 under standard conditions of the art. For example, the lens is coated following the method described by Baines et al. , or variation thereof. See Baines et al. , Adsorption and Removal of Protein Bound to Hydrogel Contact Lenses, Optom Vis. Sci (1990), 67(1 1): 807-10. In some embodiments, the coating is performed under conditions to prevent removal of all proteins from the lens. For example, the coating is carried out under conditions to prevent denaturation of proteins. In one embodiment, the coating of the lens with the fusion protein is carried out in the absence of lysozyme or other protein denaturing enzymes, buffers, or pharmaceutical excipients.

[00162] The present disclosure provides contact lens coated with a fusion polypeptide, e.g. , PSGL-l/mIgG_2b or PSGL-l/hIgG4, in which the contact angles are measured to investigate the wetting properties of protein solutions. Contact angles on bare PMMA surface are measured using, e.g. , water, about 50 ppm BSM solution, about 50 ppm PSGL-l/mIgG_2b or about 50 ppm PSGL-l/hIgG4 solution. [00163] COATING LENS WITH FUSION POLYPEPTIDE

[00164] Mucin solutions of different concentrations e.g. , between about 0.5 ppm - 500 ppm in a solution containing salt (e.g., NaCl) are introduced sequentially to the device (e.g. , a contact lens) and allowed to adsorb for about several minutes. The present disclosure provides mucins (e.g. , PSGL-l/mIgG_2b , C-P55 PSLG-l/hIgG4, C-PSL_ex PSGL-l/mIgG_2b, C- PSLex PSGL- l/hIgG4, C-PSC2 PSGL- l/mIgG_2b or C-PSC2 PSGL- l/hIgG4) of

concentrations of about 1 -5 ppm, about 2 -10 ppm, about 3 -15 ppm, about 4 -20 ppm, about 5 -25 ppm, about 6 -30 ppm, about 7 - 35 ppm, about 8 - 40 ppm, about 9 - 45 ppm, about 10 - 50 ppm, about 1 1 - 55 ppm, about 12 - 60 ppm, about 13 - 65 ppm, about 14 - 70 ppm, about 15 - 75 ppm, about 16 - 80 ppm, about 17 - 85 ppm, about 18 - 90 ppm, about 19 - 95 ppm, about 20 - 100 ppm, about 21 - 105 ppm, about 22 - 1 10 ppm, about 23 - 1 15 ppm, about 24 - 120 ppm, about 25 - 125 ppm, about 26 - 130 ppm, about 27 - 135 ppm, about 28

- 140 ppm, about 29 - 145 ppm, about 30 - 150 ppm, about 35 - 155 ppm, about 40 - 160 ppm, about 45 - 165 ppm, about 50 - 170 ppm, about 55 - 175 ppm, about 60 - 180 ppm, about 65 - 185 ppm, about 70 - 190 ppm, about 75 - 195 ppm, about 80 - 200 ppm, about 85

- 205 ppm, about 90 - 210 ppm, about 95 - 215 ppm, about 100 - 220 ppm, or between 100

- 500 ppm. In some embodiments, about 2 ppm, about 25 ppm, about 50 ppm, or about 100 ppm mucins (e.g., C-P55 PSGL-l/mIgG_2b, C-P55 PSGL-l/hIgG4, C-PSL_ex PSGL-l/mIgG_2b, C-PSLex PSGL-l/hIgG4, C-PSC2 PSGL- l/mIgG_2b or C-PSC2 PSGL- l/hIgG4) are in a salt solution. Mucins of about 2 ppm, about 25 ppm, about 50 ppm, or about 100 ppm (e.g. , C- P55 PSGL-l/mIgG_2b, C-P55 PSGL-l/hIgG4, C-PSL_ex PSGL-l/mIgG_2b, C-PSLex PSGL- l/hIgG4, C-PSC2 PSGL-l/mIgG_2b or C-PSC2 PSGL- l/hIgG4) are in 50 - 200 mM NaCl solution. In some embodiments, about 2 ppm, about 25 ppm, about 50 ppm, or about 100 ppm mucins (e.g., C-P55 PSGL-l/mIgG_2b, C-P55 PSGL-l/hIgG4, C-PSL_ex PSGL-l/mIgG_2b, C-PSLex PSGL-l/hIgG4, C-PSC2 PSGL-l/mIgG_2b or C-PSC2 PSGL-l/hIgG4) are in a 50 - 200 mM salt solution. Mucins of about 2 ppm, about 25 ppm, about 50 ppm, or about 100 ppm (e.g. , C-P55 PSGL- l/mIgG_2b, C-P55 PSGL- l/hIgG4, C-PSL_ex PSGL- l/mIgG_2b, C- PSLex PSGL-l/hIgG4, C-PSC2 PSGL-l/mIgG_2b or C-PSC2 PSGL-l/hIgG4) are in 155 mM NaCl solution.

[00165] The present disclosure provides coating a device, e.g. , contact lens, with a fusion polypeptide in about 1 mM - about 300 mM salt solution. In some embodiments, the salt solution is about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, about 195 mM, about 200 mM, about 205 mM, about 210 mM, about 215 mM, about 220 mM, about 225 mM, about 230 mM, about 235 mM, about 240 mM, about 245 mM, about 250 mM, about 255 mM, about 260 mM, about 265 mM, about 270 mM, about 275 mM, about 280 mM, about 285 mM, about 290 mM, about 295 mM, or about 300 mM.

[00166] The present disclosure provides mucin fusion polypeptide (e.g. , C-P55 PSGL- l/mIgG_2b, C-P55 PSGL-l/hIgG4, C-PSL_ex PSGL-l/mIgG_2b, C-PSLex PSGL-l/hIgG4, C- PSC2 PSGL-l/mIgG_2b or C-PSC2 PSGL-l/hIgG4) of concentrations of about 1 -5 ppm, about 2-10 ppm, about 3-15 ppm, about 4 -20 ppm, about 5 -25 ppm, about 6 -30 ppm, about 7-35 ppm, about 8-40 ppm, about 9-45 ppm, about 10-50 ppm, about 11 -55 ppm, about 12-60 ppm, about 13-65 ppm, about 14-70 ppm, about 15-75 ppm, about 16-80 ppm, about 17-85 ppm, about 18-90 ppm, about 19-95 ppm, about 20-100 ppm, about 21-105 ppm, about 22 - 110 ppm, about 23 - 115 ppm, about 24- 120 ppm, about 25- 125 ppm, about 26- 130 ppm, about 27- 135 ppm, about 28- 140 ppm, about 29

- 145 ppm, about 30- 150 ppm, about 35 - 155 ppm, about 40- 160 ppm, about 45-165 ppm, about 50- 170 ppm, about 55 - 175 ppm, about 60- 180 ppm, about 65 - 185 ppm, about 70- 190 ppm, about 75- 195 ppm, about 80 - 200 ppm, about 85 - 205 ppm, about 90

- 210 ppm, about 95-215 ppm, about 100 - 220 ppm, or between 100-500 ppm. In some embodiments, about 2 ppm, about 25 ppm, about 50 ppm, or about 100 ppm mucin fusion polypeptides (e.g., C-P55 PSGL-l/mIgG_2b, C-P55 PSGL-l/hIgG4, C-PSL_ex PSGL-l/mIgG_2b, C-PSLex PSGL-l/hIgG4, C-PSC2 PSGL-l/mIgG_2b or C-PSC2 PSGL-l/hIgG4) are in a NaCl solution of about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM, about 175 mM, about 180 mM, about 185 mM, about 190 mM, about 195 mM, about 200 mM, about 205 mM, about 210 mM, about 215 mM, about 220 mM, about 225 mM, about 230 mM, about 235 mM, about 240 mM, about 245 mM, about 250 mM, about 255 mM, about 260 mM, about 265 4 000866 mM, about 270 mM, about 275 mM, about 280 mM, about 285 mM, about 290 niM, about 295 mM, or about 300 mM.

[00167] The present disclosure provides adsorption frequency of mucins on a polymer surface between 5 Hz to about 45 minutes, with rinsing by about 155 mM NaCl solution for about 20 minutes in between each mucin solution as well as after the about 100 ppm mucin solution.

PROPERTIES OF FUSION POLYPEPTIDE COATED LENS

[00168] The present disclosure provides force and friction measurements in a fused silica liquid cell (volume ~ 0.1 mL), using a Nanoscope Multimode III Pico Force system by employing an AFM (Veeco Instruments Inc.).

[00169] The present disclosure provides AFM experiments by measuring the normal forces between the PMMA surface and the PMMA probe across about a 155 mM NaCl solution. An about 100 ppm solution of mucin (BSM or PSGL-l/mIgG₂b) in about 155 mM NaCl is introduced to the fused silica liquid cell and the polymer is allowed to adsorb for about 45 minutes. The normal forces are measured at 25 °C, followed by friction

measurements. The normal forces are measured with a constant approach and 821 nm/second retraction speed. The friction forces are determined by sliding the surfaces backwards and forwards about 10 times at each normal load using a scanning angle perpendicular to the cantilever at a 2 μη /second sliding speed.

[00170] In single asperity contacts the friction vs. load relationship can often be analyzed using the relation:

F_f = C + F„ where the constant C is known as the critical shear stress and accounts for the adhesion between the surfaces, and μ is the friction coefficient. The present disclosure provides adhesion observed only between the bare PMMA surfaces, having a non-zero critical shear stress value. When discussing friction data it is sometimes better to report contact pressure than load, since the pressure values can be compared between experiments using different probe radius and materials with different Young's modulus (stiffness). The mean pressure that corresponds to a given load is calculated using JKR theory for bare PMMA surfaces where the adhesion contribution is significant, and the Hertz model for PMMA surfaces with mucin films where adhesion effects can be ignored. See Johnson et. al., Surface Energy and the Contact of Elastic Solids. Proceedings of the Royal Society of London. A. Mathematical and Physical Sciences 1971 , 324 (1558), 301-313 and Hertz, Angew. Math. 1881 , 92, 156- 171.

[00171] In some embodiments dry eye in a subject is confirmed by Schirmer test. In some embodiments, after ocular surface staining, a formulation comprising a fusion polypeptide of the present disclosure is instilled as a solution at concentrations of about 0.01 , about 0.1 , about 1.0%, about 5%, or about 10% in neutral, isotonic buffered aqueous solution. The formulation is administered in about one 50 microliter drop to the ocular surface up to 1 - 5 times a day, every day for 2-10 weeks. The symptoms of dry eye are monitored once a week for 2- 10 weeks and an increase in Schirmer scores and/or a decrease in the amount of ocular surface staining indicates the efficacy of the formulation of the current invention in the treatment of dry eye disease.

[00172] The present disclosure provides a measurement of the effects of carbohydrate composition on mucins independently or as part of the fusion polypeptide, with regard to wetting, adsorption, surface forces and friction. In some embodiments, PSGL-l/mIgG₂b produced in C-PSLex (CHO cells co-expressing PSGL-l/mIgG_2b, C2 GnT-I (core 2 p l ,6-N- acetylglycosyltransferase I) and FUT-VII (al ,3-fucosyltransferase VII)), produces a fusion protein carrying multiple negative charges. The present disclosure provides measurements of the wetting and adsorption properties of PSGL-l/mIgG₂b, produced in C-PSLex. PMMA is used, as an exemplary contact lens material, as the coated surface substrate for wetting and adsorption measurements. Addition materials used in the measurements are PMMA coated gold QCM crystal; BSM; and PSGL-l/mIgG_2b produced in C-PSLex.

[00173] Contact angles ("CA") on bare PMMA surface are measured using: Water, 50 ppm BSM solution, 50 ppm a fusion polypeptide, e.g. , PSGL-l/mIgG₂b, solution. The present disclosure provides measurement of water contact angles on BSM or PSGL-l/mIgG₂b coated PMMA surface.

[00174] In some embodiments, the amount of protein with coupled and trapped solvent adsorbed on PMMA surface is measured. BSM and a fusion polypeptide, e.g., PSGL- l/mIgG_2b, solution have similar CA on PMMA surface to water, but much lower the Re CA than for water, indicating superior wetting property due was to adsorption of the mucins. After coating by BSM and a fusion polypeptide, e.g. , PSGL-l/mIgG_2b, the CA of the PMMA surface decreases by 20° and the Ad CA of a fusion polypeptide, e.g. , PSGL-l/mIgG_2b, is lowered even more, indicating that these mucins made the PMMA surface more hydrophilic. With prolonged time, a fusion polypeptide, e.g. , PSGL-l/mIgG_2b, solution gives smaller CA than BSM, showing superior wetting ability, and indicating water penetration into the layers. PC17IB2014/000866

[00175] In some embodiments, adsorption of a fusion polypeptide, e.g. , PSGL- l/mIgG_2b, on PMMA surfaces is measured. BSM adsorption on PMMA surface is Af=-30 Hz, and a fusion polypeptide, e.g. , PSGL-l/mIgG_2b, adsorption on PMMA surface is Af=-67 Hz. The larger negative shift in Δί, the larger mass of the layer (mucin+water) incorporated in the layer. The frequency change after adsorption of a fusion polypeptide, e.g. , PSGL-l/mIgG_2b, is larger than that of BSM, indicating more adsorption of purified fusion polypeptide, e.g. , PSGL-l/mIgG_2b, on PMMA surface. The dissipation change of a fusion polypeptide, e.g. , PSGL-l/mIgG2b, is also larger than that of BSM. The adsorbed fusion polypeptide, e.g. , PSGL-l/mIgG₂b, layer is less rigid than the BSM layer, possibly due to more extended polymer structure or a more viscous adsorbed layer. The present disclosure provides that a fusion polypeptide, e.g., PSGL-l/mIgG_2b, has a higher affinity for PMMA surface and a better wetting property than BSM.

[00176] The embodiments of the present disclosure provide CA value calculation and showing that water wets a PMMA surface coated with a fusion polypeptide, e.g. , PSGL- l/mIgG_2b, is better than a PMMA surface coated with BSM.

[00177] Table 2

[00178] The present disclosure provides measurement of stepwise adsorption of a fusion polypeptide, e.g., PSGL-l/mIgG_2b and BSM on PMMA surface. The stepwise adsorption is quantified as the sensed mass of a fusion polypeptide, e.g. , PSGL-l/mIgG₂b or BSM as a function of concentration which is adsorbed on the PMMA surface. Sensed mass refers to adsorbed mass including coupled water derived from Voigt modeling. This model is based on the assumption that the adsorbed layer has a homogeneous thickness; and it takes into account the viscoelastic properties of the system. In this experiment mucin solutions of different concentrations (2, 25, 50, and 100 ppm in about 155 mM NaCl) are introduced sequentially to the cell and allowed to adsorb for about 45 minutes, with rinsing by about 155 mM NaCl solution for about 20 minutes in between each mucin solution as well as after the aboutl OO ppm mucin solution.

[00179] The present disclosure provides that the sensed mass of BSM increased from

2 2

about 0.5 mg/m to about 10 mg/m when the concentration is increased from about 2 ppm to about 50 ppm before rinsing and remained at the same value as the concentration is increased further to about 100 ppm. This suggests that the adsorption reaches saturation at about 50 ppm. In all cases the mass on the surface decreases by rinsing with about 155 mM NaCl. This reveals that some of the adsorbed BSM molecules are rinsed away, and also implies that the binding strength between BSM molecules and the PMMA surface is not strong enough to resist rinsing.

[00180] The present disclosure provides that the sensed mass of a fusion polypeptide, e.g. , PSGL-l/mIgG₂b increases constantly from about 6 mg/m² (at about 2 ppm) to about 16.4 mg/m² (at about 100 ppm) before rinsing. The present disclosure provides that the adsorbed mass of a fusion polypeptide, e.g., PSGL-l/mIgG₂b and water trapped in the layer is significantly larger, close to a factor of 2, than the mass of adsorbed BSM with associated water. Furthermore, the difference between the sensed mass before and after rinsing is negligible for a fusion polypeptide, e.g. , PSGL-l/mIgG_2b, which contrasts to the significant desorption observed upon rinsing the PMMA surface coated with BSM. The present disclosure provides that the fusion polypeptide, e.g. , PSGL-l/mIgG₂b molecules have higher affinity towards the PMMA surface compared to the affinity of BSM molecules and most fusion polypeptide, e.g. , PSGL-l/mIgG₂b, molecules are permanently bound to the PMMA surface.

[00181] Table 3 shows the frequency and dissipation change for the 3^rd overtone when adsorbed from BSM and PSGL-l/mIgG_2b solution of 100 ppm, as well as the sensed mass and thickness extracted using the Voigt extended viscoelastic modeling of data obtained from the 3^rd and 7^th overtones. The frequency change after adsorption of PSGL-l/mIgG₂ is about twice the value of BSM, indicating more adsorption of PSGL- l/mIgG₂b on PMMA surface. The dissipation change of PSGL-l/mIgG₂b is also larger than that of BSM, suggesting that the adsorbed PSGL-l/mIgG_2b layer is more extended than the BSM layer. The modeling result further confirmed that PSGL-l/mIgG_2b molecules adsorbed more on the PMMA surface and formed a more extended layer, about 15 nm, than BSM molecules, about 8 nm. 14 000866

[00182] Table 3

[00183] The present disclosure provides measurement of contact angle (CA) measurements of water, BSM, or PSGL-l/mIgG₂b liquid phases on uncoated or BSM or PSGL-l/mIgG₂b coated PMMA surfaces. Mucin solutions show similar contact angles on PMMA surfaces. This means that neither of the two mucins spreads on the PMMA surface outside the droplet and they therefore do not promote water spreading on the uncoated PMMA. In contrast, after mucin adsorption on the PMMA surface, the contact angles are decreased to about 52° for BSM coated surface and about 5.0° for PSGL-l/mIgG b coated surface respectively, suggesting that adsorbed layers of both mucins increase the wettability of the PMMA surface, but the PSGL-l/mIgG₂b layer promotes wetting significantly more than the BSM layer.

[00184] In some embodiments, CA measurements of PSGL-l/hIgG4 liquid phases on uncoated or PSGL- l/hIgG4 coated PMMA surfaces are performed. Adsorbed layers of PSGL-l/hIgG4 increase the wettability of the PMMA surface.

[00185] Table 4

[00186] The present disclosure provides contact angle measurements of water on BSM coated PMMA surface and PSGL-l/mIgG₂b coated PMMA surface as a function of time. The contact angle for BSM coated surface almost remained constant, while the value for the PSGL-l/mIgG₂b coated surface decreases around 30° within 25 seconds. The present disclosure provides that water is transported from a droplet to within the PSGL-l/mIgG_2b film, resulting in a force that spreads the water droplet over the surface to achieve close to complete wetting. Water penetration into the BSM film is less, and results in insignificant water spreading on the surface.

[00187] The present disclosure provides contact angle measurements of water on PSGL-l/hIgG4 coated PMMA surface as a function of time. The contact angle for the PSGL- 1/ hIgG4 coated surface decreases around 30° within 25 seconds. Water is transported from the droplet to within the PSGL- l/hIgG4 film, resulting in a force that spreads the water droplet over the surface to achieve close to complete wetting.

[00188] In some embodiments, the normal force-distance curve between PMMA surfaces across about 155 mM NaCl solution shows an attraction from about 40 to about 50 nm. On separation, an adhesion force of around -3 mN/m is observed between the PMMA probe and PMMA surface. The large range of the attraction suggests that some PMMA chains extend from the surfaces and these bridges over to the opposing surface at small enough separations (e.g. , less than or equal to about 50 nm), giving rise to a so-called bridging attraction.

[00189] The present disclosure provides the normal force-distance curve after adsorption of BSM on PMMA probe and PMMA surface are repulsive and of steric origin, i.e., due to compression of the BSM layer. On separation, a small adhesion force is observed due to the presence of small patches on the PMMA surface that are not covered by BSM molecules.

[00190] The present disclosure demonstrates a strong steric repulsion on approach and no or insignificant adhesion on separation observed from the normal force-distance curve between PSGL-l/mIgG_2b layers adsorbed on PMMA surfaces. The (close to) lack of adhesion between the PSGL- l/mIgG_2b layers suggests a more complete coverage than is achieved for BSM. The hysteresis between trace and retrace force curves is small for PSGL- l/mIgG₂b layers, suggesting a rapid recovery of the initial conformation as the pressure is released. No new and long-lived PSGL-l/mIgG_2b -surface bonds are formed due to compression. In some embodiments, one part of the PSGL-l/mIgG_2b molecule is strongly anchored to the surface, whereas the other part prefers contact with bulk solution.

[00191] Normal force-distance curves during compression and decompression of PMMA surfaces are provided in these disclosures. A comparison of normal forces during compression demonstrates long-range attraction between uncoated PMMA and PMMA 4 000866 surfaces. The present disclosure provides that the steric repulsion between PSGL-l/mIgG_2b layers is higher than that between BSM layers, due to larger sensed mass and larger layer thickness of the PSGL-l/mIgG₂b layers.

[00192] Introduction of 100 ppm BSM solution into the silica cell resulted in the adsorption of polymer to both PMMA probe and PMMA surface. Figure 19B shows the normal force curve after adsorption of BSM on PMMA probe and PMMA surface. The forces experienced on approach were purely repulsive and of steric origin, i.e. due to compression of the BSM layer. On separation, a small adhesion force was observed, which may be due to the presence of small patches on the PMMA surface that are not covered by BSM molecules.

[00193] The normal force curve between C-PSLex PSGL- l/mIgG₂b layers adsorbed on PMMA surfaces is shown in Figure 19C. A strong steric repulsion was observed on approach and no or insignificant adhesion was observed on separation. The (close to) lack of adhesion between the C-PSLex PSGL- l/mIgG_2b layers suggests a more complete coverage than was achieved for BSM. The hysteresis between trace and retrace force curves is small for C- PSLex PSGL-l/mIgG_2b layers, which suggests a rapid recovery of the initial conformation as the pressure is released. This, in turn, suggests that no new and long-lived C-PSLex PSGL- l/mIgG₂b -surface bonds are formed due to compression. This can be understood if one part of the C-PSLex PSGL- l/mIgG₂b molecule is strongly anchored to the surface, whereas the other part prefers contact with bulk solution.

[00194] A comparison of normal forces during compression is shown in Figure 20A. It clearly demonstrates that a long-range attraction only exists between uncoated PMMA and PMMA surfaces. It also shows that the steric repulsion between C-PSLex PSGL-l/mIgG₂b layers is higher than that between BSM layers, which we attribute to larger sensed mass and larger layer thickness of the C-PSLex PSGL- l/mIgG_2b layers. Figure 20B shows the comparison of normal forces when the probe was withdrawn from the flat surface. A large pull-off force (adhesion, -3 mN/m) was found between PMMA and PMMA surfaces and it was reduced to around -0.2 mN/m by adsorption of BSM molecules, and no or insignificant attraction was observed between C-PSLex PSGL- l/mIgG_2b layers. Significantly stronger steric repulsion between C-PSLex PSGL- l/mIgG₂b layers on retraction compared to for BSM layers was observed. This is due to a more rapid recovery of the layer structure after compression of the C-PSLex PSGL-l/mIgG_2b layers.

[00195] The present disclosure provides measurements across about 155 mM NaCl of friction force Ff versus load, F„ and F R between two bare PMMA surfaces, BSM coated PMMA surfaces, and PSGL-l/mIgG_2b coated PMMA surfaces. In some embodiments, very high friction is observed between PMMA coated surfaces across about 155 mM NaCl, significantly smaller between BSM coated PMMA, and even smaller between PSGL- l/mIgG_2b coated PMMA surfaces. The friction coefficient between PMMA surfaces in about 155 mM NaCl is very high, μ=1.2 was obtained up to a load of 22.1 nN, corresponding to a pressure of 0.5 MPa. The friction coefficient is around 0.7 for PMMA coated with BSM as measured up to the maximum load of 68.9 nN (pressure= 1.8 MPa). The friction versus load curve for PSGL-l/mIgG_2b and PSGL-l/mIgG_2b is not linear with applied load, so the friction coefficients are calculated for different load regions. When the load was smaller than 1 1.6 nN (corresponding to a pressure of 1.0 MPa), a very small friction coefficient was obtained, about 0.06. The friction coefficient increased to 0.1 when the load was between 1 1.6 nN and 23.2 nN (1.3 MPa); and at even higher loads, up to 50.2 nN (1.6 MPa), another Amnotons' law like behavior was recovered and here μ= 0.37.

[00196] At pressures below 1 MPa the friction coefficient for PMMA coated with PSGL-l/mIgG_2b or PSGL-l/hIgG4 is 0.06, for PMMA coated with BSM 0.7 and for bare PMMA surfaces 1.7. PSGL-l/mIgG_2b or PSGL-l/hIgG4 layers provide superior lubrication in aqueous environment compared to BSM.

[00197] PMMA coated gold sensors (AT cut quartz crystals) with a diameter of 14 mm, QSX 999 (Q-sense, Vastra Frolunda, Sweden), having a nominal resonance frequency of about 5 MHz, are used as the substrate in the present disclosure. The PMMA layer is spin- coated on the gold crystal surface and has a thickness of about 40 nm. The crystals are cleaned by rinsing with Milli-Q water and dried with a gentle flow of nitrogen gas before use. The water contact angle on the PMMA surface is determined to be about 68°, as measured with a DataPhysics OCA40 micro (DataPhysics GmbH, Germany) instrument at 23 °C ± 0.5 °C and a humidity of 44%.

[00198] The present disclosure provides use of a Q-sense E4 device (Q-sense, Sweden) for studying adsorption of mucins on PMMA surfaces. This device has the capacity to continuously measure the change in frequency and dissipation at the fundamental frequency as well as at six overtone frequencies (15, 25, 35, 45, 55, 65 MHz). The frequency change observed during adsorption (Af) depends on the total mass added to the crystal, including solvent coupled to the adsorbed layer. The sensed mass is directly proportional to the frequency change according to the Sauerbrey equation, provided the adsorbed layer is thin, rigid and homogeneous. However, in many cases the adsorbed layer is viscoelastic, and this requires more elaborate analysis models. The QCM-D device also measures dissipation changes (AD), which are energy losses in the adsorbed film. This allows a more accurate estimation of the sensed mass by using a viscoelastic model to analyze changes in both frequency and dissipation for several overtones, e.g., using the Voigt representation, which treats the viscoelastic response of the layer as that of a spring and a dashpot coupled in parallel. The current disclosure utilizes the extended viscoelastic model.

[00199] The extended viscoelastic model takes into account the frequency dependence of the viscoelastic properties of the adsorbed layer, and frequency and dissipation data from the 3^rd, 5^th, and 7^th overtones are utilized in the analysis of the present disclosure. The modeling parameters used for analyzing the adsorption of PSGL-l/mIgG₂b on PMMA are provided in the present disclosure.

[00200] The present disclosure provides adsorption of BSM or a fusion polypeptide reaching equilibrium within about 2 hours, about 2-3 hours, about 3-4 hours, about 4-5 hours.

[00201] In some embodiments, exposure of PSGL-l/mIgG_2b layer on PMMA surface to a BSM solution results in minor changes in frequency and dissipation. BSM cannot associate with the preadsorbed PSGL-l/mIgG_2b layer, whereas PSGL-l/mIgG₂b interacts with preadsorbed layers of BSM (Figure 23a). The region of the PSGL-l/mIgG_2b molecule that associates with BSM is buried within the preadsorbed PSGL-l/mIgG_2b layer, and it is the IgG Fc part of PSGL-l/mIgG_2b that provides both the anchoring to PMMA and facilitates association with preadsorbed BSM, presumably via the non-glycosylated regions of BSM.

[00202] The present disclosure provides Voigt mass and Voigt thickness of the PSGL- l/mIgG_2b layer is more than about 2 times higher than those of the BSM layer. The Voigt mass of the BSM layer decreases after rinsing, which can be explained by a limited desorption. In contrast, the Voigt mass increases slightly for the PSGL-l/mIgG₂b layer after rinsing due to the pH difference of the mucin solution (pH of about 4.8) and the buffer solution (pH of about 5.8) that results in higher charge of PSGL-l/mIgG₂b and increases the mass of water associated with the layer.

[00203] Table 5. Characteristics of BSM and PSGL- 1 /mIgG_2b layers on PMMA.

Experiment 1 Voigt Voigt Experiment 2 Voigt Voigt

BSM followed thickness mass PSGL-l/mIgG_2b thickness mass by PSGL- (nm) (mg/m²) followed by (nm) (mg/m²) l/mIgG_2b BSM

BSM 7.3± 0.5 7.9 ± 0.5 PSGL-l/mIgG_2b 18.9 ± 0.2 20.4 ± 0.2

After rinsing 6.6 ± 0.5 7.2 ±0.5 After rinsing 20.4 ± 0.2 22.0 ± 0.2

PSGL-l/mIgG_2b 13.2 14.3 BSM 20.5 22.1 Experiment 1 Voigt Voigt Experiment 2 Voigt Voigt

BSM followed thickness mass PSGL-l/mIgG_2b thickness mass by PSGL- (nm) (mg/m²) followed by (nm) (mg/m²) l/mIgG₂b BSM

After rinsing 9.8 10.6 After rinsing 20.1 21.7

[00204] To better understand the adsorption of P SGL- 1 /mIgG_2b to PMMA, experiments were also carried out with the IgG-Fc part of PSGL-l/mIgG_2b, and the results are shown in Figure 4.

[00205] IgG-Fc adsorption on PMMA results in a Voigt mass of about 6 mg/m at a concentration of about 25 ppm (almost 3 hours was required to reach equilibrium), increasing slightly to 6.5 mg/m at about 50 and about 100 ppm IgG-Fc concentration. The

corresponding Voigt layer thickness is around 5 nm. The weak dependence of the sensed mass on IgG-Fc bulk concentration is a sign of high surface affinity. The current disclosure provides that the IgG-Fc fragment of PSGL-l/mIgG_2b mucin contributes significantly to the anchoring of this mucin to PMMA.

AD vs. Af plots

[00206] The AD - Af plots for adsorption of the two mucins and IgG-Fc on PMMA are shown in Figure 25. This type of plot sheds light on structural transitions occurring as the adsorption proceeds. The AD - Af curve for IgG-Fc display a linear relation between AD and Af In contrast, the AD -Af curve for BSM consist of two regions, a first linear region up to 30 Hz and a second region with decreasing dissipation. This provides that after the initial adsorption the adsorbed BSM molecules slowly change their conformation to form a thinner layer to maximize the favorable interaction with the surface. The relatively small thickness of the BSM layer, around 7 nm, suggests that most molecules are oriented parallel to the surface.

[00207] The AD - Af curve for PSGL-l/mIgG_2b mucin is similar to that of some synthetic bottle-brush polymers. The first linear region (up to 30 Hz) provides that initially most PSGL-l/mIgG_2b molecules interact with the PMMA surface in a similar way as BSM, i.e. , with the chains mainly parallel to the surface. The decreasing slope in the second region (30 to 45 Hz) provides that the energy dissipated per unit sensed mass decreases, which signify that the layer becomes stiffer due to increased interactions between adsorbed polymer chains, as also reported in other studies. The increasing slope in the last region (> 45 Hz) suggests a structural change towards a more extended layer conformation. This feature has T IB2014/000866 also been observed for copolymers having one part that adsorbs stronger than the other, and interpreted as expulsion of the block with less surface affinity from direct contact with the surface by the more strongly bound block. The current disclosure provides block mediating strong adsorption with the IgG-Fc part of the PSGL-l/mIgG2b.

Normal and friction forces - Normal forces between PMMA surfaces

[00208] The forces acting between two PMMA surfaces across about 155 mM NaCl solution are shown in Figure 26. On approach, the attractive force is noted once the separation has decreased to about 40-50 nm, and it gives rise of an adhesion with a magnitude of around 3 mN/m. On separation, the attraction persists until the separation has increased to around 200 nm. The range of the attraction is larger than expected for a van der Waals force, suggesting that it originates from some PMMA polymer chains bridging between the surfaces, giving rise to a bridging attraction. The present disclosure provides that the exact range of the attraction and the adhesion (2 to 6 mN/m) varied somewhat, between

measurements, which is expected if bridging by a few polymer chains is the cause. The absence of any measurable electrostatic double-layer force does not mean that the surfaces must be uncharged, but it is a consequence of the high ionic strength (about 155 mM) that reduces the Debye-screening length to 0.8 nm and thus the range of any possible double-layer force accordingly.

Normal forces between mucin-coated PMMA

[00209] The present disclosure provides force-separation curves for BSM and PSGL- l/mIgG₂b- The forces experienced for BSM on approach are purely repulsive and of steric origin, i.e. , due to compression of the BSM layer. On separation, a small but long range attractive force is observed, which we assign to polymer bridging. The magnitude of the adhesion is reduced by about a factor of 10 compared to that observed before BSM

adsorption. The presence of weak bridging attraction indicates that the surfaces are not fully covered by the adsorbed BSM molecules.

[00210] The normal force curves recorded between PSGL-l/mIgG₂b-coated PMMA are shown in Figure 27b. A strong steric repulsion is observed on approach and no or insignificant adhesion is noted on separation. The (close to) lack of adhesion between the PSGL-l/mIgG₂b layers suggests a more complete coverage than that achieved for BSM, which is consistent with the QCM-D results. The hysteresis between trace and retrace force curves is small for PSGL-l/mIgG₂b layers, indicating rapid recovery of the initial

conformation as the force is released. This, in turn, suggests that no new and long-lived PSGL-l/mIgG₂b - surface bonds were formed due to compression. This can be understood if one part of the PSGL-l/mIgG₂b molecule (the IgG-Fc part) is strongly anchored to the surface, whereas the other part (the glycosylated bottle- brush part) prefers contact with the bulk solution. The present disclosure provides that the adsorbed mucin molecules are not removed by the combined action of shear and load.

[00211] The forces measured on approach between BSM-coated and PSGL-l/mIgG_2b- coated PMMA surfaces are compared in Figure 28, using a logarithmic force scale. Force- separation curves are measured on approach for BSM (open circles) and PSGL-l/mIgG_2b (filled circles) on a logarithmic force scale. The mucin concentration is about 100 ppm and the NaCl concentration is 155 mM. The force is normalized by the particle radius.

[00212] An exponential decay of the steric repulsion is observed at large separations as expected in the dilute tail region, where the repulsion mainly originates from the increase in osmotic pressure caused by the higher segment density between the surfaces compared to in bulk solution. At further compression a more rapid increase in steric repulsion is expected and observed due to compression of the adsorbed polymer chains. The present disclosure provides that the PSGL-l/mIgG_2b layer is significantly thicker than the BSM layer.

Friction forces

[00213] The friction force measured between PMMA surfaces across about 155 mM NaCl are high, significantly smaller between BSM-coated PMMA, and even smaller between PSGL-l/mIgG_2b-coated PMMA, as illustrated in Figure 21a.

The friction vs. load relationship can often be analyzed using the relation:

I UU.-£ J.

F_f= C + F_n [1] where C = SQA, with Sc being the critical shear stress and A the contact area. The quantity C/μ is sometimes referred to as the dynamic adhesion as it accounts for the adhesion between the surfaces during sliding motion, and μ is the friction coefficient. Significant adhesion between the bare PMMA surfaces is observed suggesting that the critical shear stress has a non-zero value in only this case. The friction force and the friction coefficient between PMMA surfaces in 155 mM NaCl is very high. The friction coefficient, μ, equals 1.2 up to the highest load investigated. Thus the friction force law (in nN) for the uncoated PMMA surfaces can be written as:

F_f= 50+\ .2F_n [2]

[00215] For BSM-coated PMMA surface the critical shear stress, and thus the dynamic adhesion, is close to zero and the classical Amontons' rule _f = u-F„ is recovered. The friction coefficient is found to be close to 0.7 up to the maximum load of 69 nN in the present disclosure. Thus, in this case the friction force law is:

F_f = 0 F„ [3]

[00216] The friction versus load curve for PSGL-l/mIgG_2b layers on PMMA is not linear with applied load (see Figure 21a), and in this case it is appropriate to define an effective friction coefficient, μ^, as:

^P [4]

[00217] The effective friction coefficient as a function of load for PSGL-l/mIgG_2b- coated PMMA is shown in Figure 21b. The effective friction coefficient is low; increasing from 0.02 at low loads to 0.24 at the highest load explored, 50 nN. PSGL-l/mIgG_2b layers provide superior lubrication of PMMA surfaces in aqueous environment compared to BSM.

[00218] The mass of the BSM layer, including trapped water, on PMMA is about 7.2 mg/m². This value is higher than for BSM on PMMA (5.1 ± 0.59 mg/m² including trapped water) that was found on hydrophobized silica (prepared by using chloro- (dimethyl)octylsilane) where a sensed mass of about 5.9 mg/m including water was obtained.

[00219] The Voigt thickness of the BSM layer on PMMA found in this disclosure is around 7 nm (see Table 2), which is in the range of other reported values on hydrophobic surfaces. The majority of the material adsorbed is concentrated close to the surface, and that BSM has an orientation preferentially parallel to the surface. This orientation is, based on theoretical considerations, expected for charged polyelectrolytes adsorbed on uncharged surfaces.

[00220] The fact that a relatively small thickness value is obtained from QCM-D and ellipsometric measurements does not preclude the presence of some long tails since these techniques are insensitive to the existence of a dilute tail region. Indeed, some long tails are present for mucin layers on hydrophobic surfaces, and the adsorbed layer consists of an inner compact region with a thickness of 3 - 10 nm and an outer dilute tail region. The present disclosure provides that the compact inner region dominates the QCM-D response, whereas the dilute tail region results in a long-range steric force that clearly is observed in the surface force data (Figures 27-28).

[00221] The driving force for adsorption of mucins on hydrophobic surfaces is the entropic gain due to removal of water from contact with the hydrophobic surface and the non- glycosylated regions of the peptide backbone. The PMMA surface is weakly hydrophobic (contact angle -68°) and the non-glycosylated regions of the BSM mucin backbone is preferentially adsorbed on the surface with the carbohydrate side chains extending towards solution.

[00222] The present disclosure provides that at pressures below 1 Pa, the friction coefficient for PMMA coated with C-PSLex PSGL-l/mIgG_2b is about 0.06, for PMMA coated with BSM about 0.7 and for bare PMMA surfaces about 1.7. In some embodiments, C-PSLex PSGL-l/mIgG₂b layers provide superior lubrication in aqueous environment compared to BSM. The present disclosure provides that the friction is very high between PMMA coated surfaces across about 155 mM NaCl, significantly smaller between BSM coated PMMA, and even smaller between PSGL- l/mIgG₂b coated PMMA surfaces.

Adsorption of PSGL-l/mIgG_2b on PMMA

[00223] Compared to BSM, PSGL-l/mIgG_2b adsorbs significantly more to PMMA, and a more extended inner region of the layer is formed, as evidenced by the QCM-D data. The dilute tail region for BSM and PSGL-l/mIgG₂b on PMMA surface does, on the other hand, appear to be rather similar as judged from the surface force curves (Figure 28). The more extended inner region of the layer for PSGL-l/mIgG₂b is attributed to the strong affinity of the IgG-Fc part of this mucin that displaces the glycosylated region away from the surface, as also suggested by the AD - Δ/ plot (Figure 25).

[00224] Table 6

Interactions between adsorbed mucin layers on non-polar surfaces

[00225] The present disclosure provides an attraction on separation and pronounced relaxation effects between PGM layers, due to slow relaxation, chain entanglement and bridging. The present disclosure provides different behavior of two mucins is related to their different carbohydrate composition since their adsorbed amounts were similar.

[00226] The present disclosure further provides bridging due to the small attraction on separation between BSM-coated PMMA surfaces. In contrast, no significant attractive force between PSGL-l/mIgG₂b-coated PMMA surfaces is observed. The higher layer mass and more extended layer structure (compared to BSM on PMMA surface) effectively prevent bridging and chain entanglement. 2014/000866

Lubrication ability of mucin layers on non-polar surfaces

[00227] The present disclosure provides low effective friction coefficient between PSGL-l/mIgG_2b coated PMMA surfaces at low loads, < 0.05.

[00228] Interfacial properties of a brush-of-trains type mucin, BSM, and a brush-with- anchor type mucin, PSGL-l/mIgG₂b, on PMMA in contact with 155 mM NaCl solution have been investigated using QCM-D and surface force and friction measurements. Large differences between these two types of mucins are observed. The PSGL-l/mIgG₂b mucin layer has a significantly larger mass than the BSM layer (including water in the layer), and the inner region of this layer is more extended than that for BSM as deduced from the QCM- D measurements. Both layers also contain an outer dilute tail region, which generates a long- range steric repulsion that is rather similar for the two types of mucins.

[00229] The IgG-Fc anchor block of the PSGL-l/mIgG₂b mucin replaces the oligosaccharide rich domains in PSGL-l/mIgG₂b at the PMMA surface at the later stage of the adsorption process, as judged from the AD vs. Δ/ plot.

[00230] The present disclosure provides PSGL-l/mIgG₂b layers having superior lubrication of PMMA surfaces in an aqueous environment. The recombinant PSGL-l/mIgG₂b mucin is a superior boundary lubricant on PMMA compared to BSM up to pressures in the 8- 9 MPa regime. The higher layer mass for PSGL-l/mIgG₂b and the higher surface affinity provided by the IgG-Fc anchor block, which counteracts bridging and lateral motion of molecules along the surface during shearing. The favorable boundary lubrication properties of PSGL-l/mIgG_2b layers are found even though the layers are formed by adsorption from dilute solution (100 ppm) within a short adsorption time (45 minutes).

OPHTHALMIC FORMULATIONS

[00231] The invention features novel ophthalmic formulation comprising a

recombinant mucin which is comfortable upon instillation to the ocular surface, and safe for repeated, chronic use. As such, the comfortable ophthalmic formulations described herein will treat signs and symptoms of dry eye and/or ocular irritation, and increase long term patient compliance in the use of such formulations for the treatment and/or prevention of signs and symptoms associated with dry eye disease and/or ocular discomfort.

[00232] The invention is also based, in part, on that a recombinant mucin alone may be effective to improve tear film stability (assessed as an increase in tear film break up time and the Ocular Protection Index) and improve overall ocular surface health (assessed as reduced corneal staining and conjunctival redness, increased corneal sensitivity, decreased blink rate, and improved visual performance).

[00233] As such, the formulations are comfortable upon instillation into the eye, and may be used for relief of acute or chronic dry eye disease, and are particularly suitable for both intermittent and long term use. The formulations of the invention can also be used to treat another eye disorder if it contains a drug for that disorder.

[00234] The amount of mucin in an ophthalmic formulation can vary greatly depending on the product type. For example, in contact lens related solutions the mucin concentration would vary from about 0.001% to about 5.0% by weight. In dry eye

preparations the mucin level could vary from about 0. 1% to about 10.0% by weight. In a solid ocular insert delivery device the mucin level could range to about 90.0% or greater by weight. Within each type of preparation, the concentration might be varied, depending on such factors as the severity of the dry eye condition being treated, to enhance particular properties of the mucin solution. These ranges are for purpose of illustration and are not meant in any manner to limit the scope of the claims.

[00235] The exemplary ophthalmic compositions finds particular utility as lubricating eye drops, i. e. , an artificial tear solution, a tear fluid supplement, a delivery vehicle for topical ophthalmic drug application. In most of these applications, the compositions are provided in a buffered, sterile aqueous solution. Typically, these solutions have a viscosity from about 1 to 100 cps. As a solution the compositions are dispensed in the eye in the form of an eye drop. It should be understood, however, that the compositions described herein may also be formulated as viscous liquids, . e. , viscosities from several hundred to several thousand cps, gels or ointments. In these applications the mucin component would be dispersed or dissolved in an appropriate vehicle such as Lubragel, GRR Lubricating Jelly or Karajel, all trademarked products of United-Guardian, Inc., Hauppauge, N.Y.

[00236] The exemplary compositions may also be formulated as solid ocular inserts that dissolve or erode over time when placed in the cul-de-sac of the eye.

[00237] Swelling-controlled release devices would consist of mucin homogeneously dispersed in a glassy polymer such as a water soluble cellulosic. When the insert is placed in the eye, the tear fluid begins to penetrate the matrix, followed by swelling, and finally dissolution, of the matrix. As this process occurs, mucin is released into the eye to provide relief of dry eye symptoms over a long period of time. [00238] Erodible devices would again consist of mucin homogeneously dispersed in a polymer matrix. In this case, mucin is released by a chemical reaction (hydrolysis) that results in solubilization of the matrix polymer, usually at the surface of the device. Generally, the matrix material is a polyanhydride or a poly(ortho ester).

[00239] In another embodiment the mucin may be chemically modified or crosslinked to act as its own "matrix", where mucin comprises the entire, or nearly entire, device, thus providing the maximum amount of mucin available to the eye.

[00240] Furthermore, in some contact lens related embodiments, the exemplary transmembrane or surface mucin disclosed herein may be incorporated into contact lens soaking and conditioning solutions as well as lubricating eye drops for contact lens wearers.

[00241] In another embodiment the mucin may be utilized in drug delivery. The most common and convenient method for delivery of ocular drugs is by way of topical eye drops. Generally, the solution vehicles employed are quickly diluted by the tear fluid and drain from the eye in a matter of minutes. This short residence time hinders the absorption and hence the bioavailability of the drug in the eye. Oftentimes the short residence time is overcome by greatly increasing the concentration of the drug to improve bioavailability. This often leads to significant undesirable side effects due to the systemic actions of many of the ocular drugs currently prescribed.

[00242] Much research has been done to improve the residence time of the drug vehicle at the ocular surface and also to promote interaction or association of the drug with the vehicle. One approach that has been commercialized is to utilize a cross-linked carboxy- functional polymer such as CARBOPOL®, supplied by B.F. Goodrich. The bioadhesive nature of this polymer has been the basis for controlled release ophthalmic formulations as described in U.S. Pat. No. 4,615,697 and U.S. Pat. No. 5, 188,826, both of which are incorporated by reference in their entirety.

[00243] These cross-linked carboxy-functional polymers swell in aqueous solution but remain as micron-size hydrated particles. Furthermore, at neutral pH, they are substantially anionic in nature. Since many ophthalmic drugs, for example timolol and pilocarpine, are positively charged, they will associate with the negatively charged polymer particles through electrostatic interaction. Also, since the hydrated particles are microporous, the drug can be absorbed into the matrix. When an ophthalmic solution of this type is placed in the eye, the hydrated polymer particles adhere to the mucosal surface, providing extended residency time. During this residence the drug is released from the hydrated polymer particles, thus providing for a more efficient local delivery to the eye. [00244] The mucins, used in the exemplary compositions are by definition

"bioadhesive" and contain multiple negative charges. Given this information one would expect the mucins of this invention to act in a similar manner to the cross-linked carboxy- functional polymers as an ophthalmic drug delivery vehicle. In practice, these transmembrane or surface mucins provide superior retention time due to their ability to interact not only with the epithelial surface but also with the natural mucins in the tear film.

[00245] Exemplary ophthalmic formulations include recombinant mucins from any number of the exemplary sources described herein. In addition, other solution components may be employed as required:

EXCIPIENTS

[00246] In some embodiments, the mucin formulations of the invention comprise one or more pharmaceutically acceptable excipients. The term excipient as used herein broadly refers to a biologically inactive substance used in combination with the active agents of the formulation. An excipient can be used, for example, as a solubilizing agent, a stabilizing agent, a surfactant, a demulcent, a viscosity agent, a diluent, an inert carrier, a preservative, a binder, a disintegrant, a coating agent, a flavoring agent, or a coloring agent. For example, at least one excipient is chosen to provide one or more beneficial physical properties to the formulation, such as increased stability and/or solubility of the active agent(s). A

"pharmaceutically acceptable" excipient is one that has been approved by a state or federal regulatory agency for use in animals, and preferably for use in humans, or is listed in the U.S. Pharmacopia, the European Pharmacopia or another generally recognized pharmacopia for use in animals, and preferably for use in humans.

[00247] Further examples of excipients include certain inert proteins such as albumins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as aspartic acid (which may alternatively be referred to as aspartate), glutamic acid (which may alternatively be referred to as glutamate), lysine, arginine, glycine, and histidine; fatty acids and

phospholipids such as alkyl sulfonates and caprylate; surfactants such as sodium dodecyl sulphate and polysorbate; nonionic surfactants such as such as TWEEN®, PLURONICS®, or a polyethylene glycol (PEG) designated 200, 300, 400, or 600; a Carbowax designated 1000, 1500, 4000, 6000, and 10000; carbohydrates such as glucose, sucrose, mannose, maltose, trehalose, and dextrins, including cyclodextrins; polyols such as mannitol and sorbitol;

chelating agents such as EDTA; and salt-forming counter-ions such as sodium. [00248] Examples of carriers that may be used in the formulations of the present invention include water, mixtures of water and water-miscible solvents, such as Cp to C₇- alkanols, vegetable oils or mineral oils comprising from 0.5 to 5% non-toxic water-soluble polymers, natural products, such as gelatin, alginates, pectins, tragacanth, karaya gum, xanthan gum, carrageenan, agar and acacia, starch derivatives, such as starch acetate and hydroxypropyl starch, and also other synthetic products, such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxide, preferably cross-linked polyacrylic acid, such as neutral Carbopol, or mixtures of those polymers. The concentration of the carrier is, typically, from 1 to 100000 times the concentration of the active ingredient.

[00249] In a particular embodiment, the carrier is a polymeric, mucoadhesive vehicle.

Examples of mucoadhesive vehicles suitable for use in the methods or formulations of the invention include but are not limited to aqueous polymeric suspensions comprising one or more polymeric suspending agents including without limitation dextrans, polyethylene glycol, polyvinylpyrolidone, polysaccharide gels, GELRITE®, cellulosic polymers, and carboxy-containing polymer systems. In a particular embodiment, the polymeric suspending agent comprises a crosslinked carboxy-containing polymer (e.g. , polycarbophil). In another particular embodiment, the polymeric suspending agent comprises polyethylene glycol (PEG). Examples of cross-linked carboxy-containing polymer systems suitable for use in the stable ophthalmic mucin formulations of the invention include but are not limited to Noveon AA- 1, CARBOPOL®, and/or DURASITE® (InSite Vision).

[00250] In particular embodiments, the mucin formulations of the invention comprise one or more excipients selected from among the following: a tear substitute, a tonicity enhancer, a preservative, a solubilizer, a viscosity enhancing agent, a demulcent, an emulsifier, a wetting agent, a sequestering agent, and a filler. The amount and type of excipient added is in accordance with the particular requirements of the formulation and is generally in the range of from about 0.0001% to 90% by weight.

TEAR SUBSTITUTES

[00251] The present disclosure provides use of fusion polypeptides as a tear substitute.

The present also provides use of fusion polypeptide coated medical device, e.g. , contact lens, in conjunction with a tear substitute. The term "tear substitute" refers to molecules or compositions which lubricate, "wet," approximate the consistency of endogenous tears, aid in natural tear build-up, or otherwise provide temporary relief of dry eye signs or symptoms and conditions upon ocular administration. A variety of tear substitutes are known in the art and include, but are not limited to: monomeric polyols, such as, glycerol, propylene glycol, and ethylene glycol; polymeric polyols such as polyethylene glycol; cellulose esters such hydroxypropylmethyl cellulose, carboxymethyl cellulose sodium and hydroxy

propylcellulose; dextrans such as dextran 70; water soluble proteins such as gelatin; vinyl polymers, such as polyvinyl alcohol, polyvinylpyrrolidone, and povidone; and carbomers, such as carbomer 934P, carbomer 941 , carbomer 940 and carbomer 974P. Many such tear substitutes are commercially available, which include, but are not limited to cellulose esters such as BION TEARS®, CELLUVISC®, GENTEAL®, OCCUCOAT®, REFRESH®, SYSTANE®, TEARGEN II®, TEARS NATURALE®, TEARS NATURAL II®, TEARS NATURALE FREE®, and THERATEARS®; and polyvinyl alcohols such as AKWA TEARS®, HYPOTEARS®, MOISTURE EYES®, MURINE LUBRICATING®, and VISINE TEARS®, SOOTHE®. Tear substitutes may also be comprised of paraffins, such as the commercially available LACRI-LUBE® ointments. Other commercially available ointments that are used as tear substitutes include LUBRIFRESH PM®, MOISTURE EYES PM® and REFRESH PM®.

[00252] In one preferred embodiment of the invention, the tear substitute comprises hydroxypropylmethyl cellulose (Hypromellose or HPMC). According to some embodiments, the concentration of HPMC ranges from about 0.1% to about 2% w/v, or any specific value within said range. According to some embodiments, the concentration of HPMC ranges from about 0.5% to about 1.5% w/v, or any specific value within said range. According to some embodiments, the concentration of HPMC ranges from about 0.1% to about 1% w/v, or any specific value within said range. According to some embodiments, the concentration of HPMC ranges from about 0.6%) to about 1% w/v, or any specific value within said range.

[00253] In some embodiments, the concentration of HPMC ranges from about 0.1% to about 1.0%) w/v, or any specific value within said range (i. e., equal to or about 0.1- equal to or about 0.2%, equal to or about 0.2 - equal to or about 0.3%, 0.3 equal to or about - equal to or about 0.4%, equal to or about 0.4 - equal to or about 0.5%), equal to or about 0.5 - equal to or about 0.6%, equal to or about 0.6 - equal to or about 0.7%>, equal to or about 0.7 - equal to or about 0.8%o, equal to or about 0.8 - equal to or about 0.9%), equal to or about 0.9 - equal to or about 1.0%; equal to or about 0.2%, equal to or about 0.21%, equal to or about 0.22%, equal to or about 0.23%, equal to or about 0.24%), equal to or about 0.25%, equal to or about 0.26%, equal to or about 0.27%, equal to or about 0.28%), equal to or about 0.29%, equal to or about 0.30%), equal to or about 0.70%), equal to or about 0.71%, equal to or about 0.72%, equal to or about 0.73%, equal to or about 0.74%, equal to or about 0.75%, equal to or about 00866

0.76%, equal to or about 0.77%, equal to or about 0.78%, equal to or about 0.79%, equal to or about 0.80%, equal to or about 0.81%, equal to or about 0.82%, equal to or about 0.83%, equal to or about 0.84%o, equal to or about 0.85%, equal to or about 0.86%, equal to or about 0.87%, equal to or about 0.88%, equal to or about 0.89%, or equal to or about 0.90%).

[00254] For example, without limitation, a tear substitute which comprises

hydroxypropyl methyl cellulose is GENTEAL® lubricating eye drops. GENTEAL®

(CibaVision— Novartis) is a sterile lubricant eye drop containing hydroxypropylmethyl cellulose 3 mg/g and preserved with sodium perborate. Other examples of an HPMC-based tear are provided.

[00255] In another preferred embodiment, the tear substitute comprises carboxymethyl cellulose sodium. For example, without limitation, the tear substitute which comprises carboxymethyl cellulose sodium is REFRESH® Tears. REFRESH® Tears is a lubricating formulation similar to normal tears, containing a, mild non-sensitizing preservative, stabilised oxychloro complex (PURITE®)), that ultimately changes into components of natural tears when used.

[00256] In a preferred embodiment, the tear substitute, or one or more components thereof, is an aqueous solution having a viscosity in a range which optimizes efficacy of supporting the tear film while minimizing blurring, lid caking, etc. Preferably, the viscosity of the tear substitute, or one or more components thereof, ranges from 1-150 centipoise (cpi), e.g. , 5-150 cpi, 5-130 cpi, 30-130 cpi, 50-120 cpi, 60-115 cpi (or any specific value within said ranges). In a particular embodiment, the viscosity of the tear substitute, or one or more components thereof, is about 70-90 cpi, or any specific value within said range (for example without limitation, 85 cpi).

[00257] Viscosity may be measured at a temperature of 20° C.+/-1 ° C. using a

Brookfield Cone and Plate Viscometer Model VDV-III Ultra⁺ with a CP40 or equivalent Spindle with a shear rate of approximately 22.50+/-approximately 10 (1/sec), or a Brookfield Viscometer Model LVDV-E with a SC4-1 or equivalent Spindle with a shear rate of approximately 26+/-approximately 10 (1/sec). Alternatively, viscosity may be measured at 25° C.+/- C. using a Brookfield Cone and Plate Viscometer Model VDV-III Ultra⁺ with a CP40 or equivalent Spindle with a shear rate of approximately 22.50+/-approximately 10 (1/sec), or a Brookfield Viscometer Model LVDV-E with a SC4-18 or equivalent Spindle with a shear rate of approximately 26+/-approximately 10 (1/sec).

[00258] In some embodiments, the tear substitute, or one or more components thereof is buffered to a pH 5.0 to 9.0, preferably pH 5.5 to 7.5, more preferably pH 6.0 to 7.0 (or any 2014/000866 specific value within said ranges), with a suitable salt (e.g., phosphate salts). In some embodiments, the tear substitute further comprises one or more ingredients, including without limitation, glycerol, propyleneglycerol, glycine, sodium borate, magnesium chloride, and zinc chloride.

SALTS, BUFFERS, AND PRESERVATIVES

[00259] The formulations of the present invention may also contain pharmaceutically acceptable salts, buffering agents, or preservatives. Examples of such salts include those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, boric, formic, malonic, succinic, and the like. Such salts can also be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts. Examples of buffering agents include phosphate, citrate, acetate, and 2-(N- morpholino)ethanesulfonic acid (MES).

[00260] For the adjustment of the pH, preferably to a physiological pH, buffers may especially be useful. The pH of the present solutions should be maintained within the range of 4.0 to 8.0, more preferably about 5.5 to 7.5, more preferably about 6.0 to 7.0. Suitable buffers may be added, such as boric acid, sodium borate, potassium citrate, citric acid, sodium bicarbonate, TRIS, and various mixed phosphate buffers (including combinations of

Na₂HP0₄, NaH₂P0₄ and KH₂P0₄) and mixtures thereof. Borate buffers are preferred.

Generally, buffers will be used in amounts ranging from about 0.05 to 2.5 percent by weight, and preferably, from 0.1 to 1.5 percent.

[00261] In certain embodiments, the formulations additionally comprise a preservative. A preservative may typically be selected from a quaternary ammonium compound such as benzalkonium chloride, benzoxonium chloride or the like. Benzalkonium chloride is better described as: N-benzyl-N— (Cg-Ci₈ alkyl)-N,N-dimethylammonium chloride. Further examples of preservatives include antioxidants such as vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium; the amino acids cysteine and methionine; citric acid and sodium citrate; and synthetic preservatives such as thimerosal, and alkyl parabens, including for example, methyl paraben and propyl paraben. Other preservatives include

octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzethonium chloride, phenol, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol, phenylmercuric nitrate, phenylmercuric acetate or phenylmercuric borate, sodium perborate, sodium chlorite, alcohols, such as chlorobutanol, butyl or benzyl alcohol or phenyl ethanol, guanidine derivatives, such as chlorohexidine or polyhexamethylene biguanide, sodium perborate, POLYQUAD®, GERMAL®II, sorbic acid and stabilized oxychloro complexes (e.g. , 0866

PURITE®). Preferred preservatives are quaternary ammonium compounds, in particular benzalkonium chloride or its derivative such as Poly quad {see U.S. Pat. No. 4,407,791), alkyl-mercury salts, parabens and stabilized oxychloro complexes {e.g. , PURITE®). Where appropriate, a sufficient amount of preservative is added to the ophthalmic composition to ensure protection against secondary contaminations during use caused by bacteria and fungi.

[00262] In particular embodiments, the mucin formulations of the invention comprise a preservative selected from among the following: benzalkonium chloride, 0.001% to 0.05%; benzethonium chloride, up to 0.02%; sorbic acid, 0.01 % to 0.5%; polyhexamethylene biguanide, 0.1 ppm to 300 ppm; polyquaternium-1 (Omamer M)— 0.1 ppm to 200 ppm; hypochlorite, perchlorite or chlorite compounds, 500 ppm or less, preferably between 10 and 200 ppm); stabilized hydrogen peroxide solutions, a hydrogen peroxide source resulting in a weight % hydrogen peroxide of 0.0001 to 0.1% along with a suitable stabilizer; alkyl esters of p-hydroxybenzoic acid and mixtures thereof, preferably methyl paraben and propyl paraben, at 0.01% to 0.5%; chlorhexidine, 0.005% to 0.01%; chlorobutanol, up to 0.5%; and stabilized oxychloro complex (PURITE®) 0.001% to 0.5%.

[00263] In another embodiment, the topical formulations of this invention do not include a preservative. Such formulations would be useful for patients who wear contact lenses, or those who use several topical ophthalmic drops and/or those with an already compromised ocular surface {e.g. dry eye) wherein limiting exposure to a preservative may be more desirable.

VISCOSITY ENHANCING AGENTS AND DEMULCENTS

[00264] In certain embodiments, viscosity enhancing agents may be added to the mucin formulations of the invention. Examples of such agents include polysaccharides, such as hyaluronic acid and its salts, chondroitin sulfate and its salts, dextrans, various polymers of the cellulose family, vinyl polymers, and acrylic acid polymers.

[00265] In certain embodiments, the mucin formulations of the invention comprise ophthalmic demulcents and/or viscosity enhancing polymers selected from one or more of the following: cellulose derivatives such as carboxymethycellulose (about 0.01 to about 5%) hydroxyethylcellulose (about 0.01% to about 5%), hydroxypropyl methylcellulose or hypromellose (about 0.01% to about 5%), and methylcelluose (about 0.02% to about 5%); dextran 40/70 (about 0.01% to about 1%); gelatin (about 0.01% to about 0.1%); polyols such as glycerin (about 0.01% to about 5%), polyethylene glycol 300 (about 0.02% to about 5%), polyethylene glycol 400 (about 0.02% to about 5%), polysorbate 80 (about 0.02% to about 3%), propylene glycol (about 0.02% to about 3%), polyvinyl alcohol (about 0.02% to about 4 000866

5%), and povidone (about 0.02% to about 3%); hyaluronic acid (about 0.01% to about 2%); and chondroitin sulfate (about 0.01% to about 2%).

[00266] Viscosity of the stable ophthalmic mucin formulations of the invention may be measured according to standard methods known in the art, such as use of a viscometer or rheometer. One of ordinary skill in the art will recognize that factors such as temperature and shear rate may effect viscosity measurement. In a particular embodiment, viscosity is measured at 20 °C+/-1 °C using a Brookfield Cone and Plate Viscometer Model VDV-III Ultra+ with a CP40 or equivalent Spindle with a shear rate of approximately

22.50+/-approximately 10 (1/sec), or a Brookfield Viscometer Model LVDV-E with a SC4- 18 or equivalent Spindle with a shear rate of approximately 26+/-approximately 10 (1/sec). In another embodiment, viscosity of the ophthalmic formulations of the invention is measured at 25 °C +/-1 °C. using a Brookfield Cone and Plate Viscometer Model VDV-III Ultra+ with a CP40 or equivalent Spindle with a shear rate of approximately

22.50+/-approximately 10 (1/sec), or a Brookfield Viscometer Model LVDV-E with a SC4- 18 or equivalent Spindle with a shear rate of approximately 26+/-approximately 10 (1/sec).

TONICITY ENHANCERS

[00267] Tonicity is adjusted if needed typically by tonicity enhancing agents. Such agents may, for example be of ionic and/or non-ionic type. Examples of ionic tonicity enhancers are alkali metal or earth metal halides, such as, for example, CaCl₂, KBr, KC1, LiCl, Nal, NaBr or NaCl, Na₂S0₄ or boric acid. Non-ionic tonicity enhancing agents are, for example, urea, glycerol, sorbitol, mannitol, propylene glycol, or dextrose. The aqueous solutions of the present invention are typically adjusted with tonicity agents to approximate the osmotic pressure of normal lachrymal fluids which is equivalent to a 0.9% solution of sodium chloride or a 2.5% solution of glycerol. An osmolality of about 225 to 400 mOsm/kg is preferred, more preferably 280 to 320 mOsm.

SOLUBILIZING AGENTS

[00268] The formulation may additionally require the presence of a solubilizer, in particular if one or more of the ingredients tend to form a suspension or an emulsion. Suitable solubilizers include, for example, tyloxapol, fatty acid glycerol polyethylene glycol esters, fatty acid polyethylene glycol esters, polyethylene glycols, glycerol ethers, polysorbate 20, polysorbate 80 or mixtures of those compounds. In a preferred embodiment, the solubilizer is a reaction product of castor oil and ethylene oxide, for example the commercial products CREMOPHOPv EL® or CREMOPHOR RH40®. Reaction products of castor oil and ethylene 4 000866 oxide have proved to be particularly good solubilizers that are tolerated extremely well by the eye. In another embodiment, the solubilizer is tyloxapol or a cyclodextrin. The concentration used depends especially on the concentration of the active ingredient. The amount added is typically sufficient to solubilize the active ingredient. For example, the concentration of the solubilizer is from 0.1 to 5000 times the concentration of the active ingredient. Preferably, the solubilizer is not a cyclodextrin compound (for example alpha-, beta- or gamma-cyclodextrin, e.g. alkylated, hydroxyalkylated, carboxyalkylated or alkyloxycarbonyl-alkylated derivatives, or mono- or diglycosyl-alpha-, beta- or gamma-cyclodextrin, mono- or dimaltosyl-alpha-, beta- or gamma-cyclodextrin or panosyl-cyclodextrin). In one embodiment the fusion polypeptide of the current invention may be in complex, in association, or in formulation with CAPTISOL®.

METHODS OF USE

[00269] The invention further features methods of treating and/or preventing the signs and symptoms associated with dry eye and/or eye irritation in a subject comprising use of the novel NS AID alone formulations or combined tear NS AID formulations described above. For example, a method of treating and/or preventing dry eye and/or eye irritation may comprise administering to the eye surface of the subject in need thereof a formulation comprising a recombinant mucin.

[00270] The present disclosure provides methods of correcting vision with corrective lens, e.g. , contact lens, in which the lens is covered with a fusion polypeptide of the current disclosure.

[00271] The device, e.g. , contact lens of the present disclosure is useful in the treatment of eye diseases or disorders including keratoconjunctivitis sicca (dry eye), allergic conjunctivitis, conjunctivitis, diabetic retinopathy, macular oedema (including wet macular oedema and dry macular oedema), post-operative cataract inflammation or, particularly, uveitis (including posterior, anterior and pan uveitis) (e.g. eye diseases or disorders including allergic conjunctivitis, conjunctivitis, diabetic retinopathy, macular oedema (including wet macular oedema and dry macular oedema), post-operative cataract inflammation or, particularly, uveitis (including posterior, anterior and pan uveitis)).

[00272] The dry eye syndrome (also known as keratitis sicca) is an ocular surface disease which may be due to a reduction of the activity of the lacrimal glands with the consequent lower tear production, in which case it is referred to as "hyposecretory dry eye" 2014/000866 or to an excessive loss of water of the exposed ocular surface in the presence of a normal secretory function, in which case it is known as "evaporative dry eye."

[00273] It is currently defined as a multifactorial disease resulting in ocular discomfort, visual alteration and instability of the tear film, with potential damages of the ocular surface. It is accompanied by an increased osmolarity of the tear film and by inflammation of the ocular surface (DEWS, The Ocular Surface, April 2007, Vol. 5, 2: 69-202).

[00274] There are multiple causes which can cause dry eye, being more common in the elderly. It can be caused, among other factors, by the aging process, use of contact lenses, hormonal changes in women, environmental factors, side effects of diseases/medicinal products, laser surgeries for vision correction, destabilization of the tear composition and other chronic eye diseases. The diseases causing dry eyes include vitamin A deficiency, Sjogren's syndrome, rheumatoid arthritis and other rheumatologic diseases, it can also occur due to chemical or thermal burns, and drugs such as atenolol, chlorpheniramine,

hydrochlorothiazide, isotretinoin, ketorolac, ketotifen, levocabastine, levofloxacin, oxybutynin or tolterodine. As the disease progresses, there is a thickening of the cornea and a reduction of visual acuity. Other symptoms of dry eyes are a stinging or burning sensation in the eye, foreign body sensation, itching or pruritus, rheum and conjunctival reddening.

[00275] Blepharitis is a term which is used to describe the inflammation of the tissue forming the eyelid. Its origin is often due to a malfunctioning of the glands which are located in the eyelid margin. Under normal conditions, these glands produce an oily secretion which aids in lubricating the surface of the eye and the inner side of the eyelids, preventing the evaporation of tears. In subjects with blepharitis, these glands are obstructed, their secretions are stagnant and fatty acids are formed which irritate the ocular surface. The margin of the eyelids is inflamed and reddened in these cases. The eye is irritated and produces secretion of mucus and proteins, and the latter accumulate in the eyelid margin, often creating a crust. The accumulation of these materials provides the optimal conditions for the growth of bacteria, which in turn release toxins which contribute to irritating the eyelids even more and to further aggravating the pathological process. Therefore, in blepharitis there is a chain of events including eyelid gland dysfunction, irritation and formation of small crusts in the eyelid margin, in addition to bacterial infection. The severity of blepharitis varies considerably among individuals. In some cases, it only represents a moderate discomfort, creating a slight, intermittent irritation. In others, it is a more serious disease which can affect vision. PC17IB2014/000866

[00276] Blepharitis is a common process affecting 5% of the population, with a chronic nature and which is presented in outbreaks. This disease occurs both in men and in women without distinction, but it has a slightly greater incidence in men. Nevertheless, associated with other diseases, it can have an incidence of up to 15% as in the case of ocular cicatricial pemphigoid or of up to 19% in the case of its association with dry eyes. There are two types of blepharitis: anterior (seborrheic or staphylococcal) blepharitis and posterior (hypersecretory or obstructive) blepharitis, the latter may occur due to a meibomian gland dysfunction (MGD). In seborrheic anterior blepharitis, the appearance of the edge of the eyelid is very "oily," with soft and "sticky" plaques or flakes, with abundant yellowish-white secretion at the exit of the glands, with the eyelashes adhered to one another by the oil.

Favored by this alteration of glandular secretion, there is bacterial colonization, the most important bacterium causing the infection being a staphylococcus. There is greater reddening due to the direct irritation of the bacteria and their toxins. In staphylococcal posterior blepharitis, there are no oily and sticky flakes as in seborrheic blepharitis, but rather dry, larger crusts which are visible without a microscope. Genuine collarettes are often formed the eyelashes, and it seems like "dandruff in the eyelashes, with reddened skin and irritated eyes. Posterior blepharitis may occur when the meibomian glands are affected between others, the inner part of the eyelashes which is in contact with the eye also being affected. This blepharitis has other names, such as meibomitis, meibomian gland dysfunction (MGD), meibomian foam, etc., and although the symptoms do not seem to be as significant as seborrheic anterior blepharitis, it is easier for the ocular surface to be altered. Due to the fact that the meibomian glands are in charge of producing the lipid component of tears, if meibomitis occurs, this component is altered and the tears are "of a poor quality," they break and do not remain homogeneously distributed over the surface of the eye. Thus, when blepharitis is referred to as the cause of dry eye, reference is almost always made to posterior blepharitis (a meibomian gland problem). Likewise, anterior and posterior blepharitis can occur simultaneously, in fact it is quite usual. For example, in the case of seborrheic blepharitis, there is an alteration of the glands both in the front part (eyelashes "covered" in oil) and in the rear part (meibomitis).

[00277] The diagnosis of dry eye is normally performed with Schirmer's test, which consists of placing a strip of blotting paper hanging from the lower eyelid, keeping the eyes closed for 5 minutes and observing what length of the paper is wet with tears, the normal length being 15 mm. This test has the drawback that tears can be generated due to the irritation with the paper and it is often recommended to use local anesthesia. Therefore, 2014/000866 several new tests have been developed over time, such as the measurement of lactoferrin, since it seems that the amount of this molecule is closely related to tear production. Patients with low tear production and dry eyes have low levels of lactoferrin. There is also another method which consists of measuring the lysozyme concentration in tears. Another test consists of adding drops of fluorescein in the eye of the patient, such that the fluorescein should pass from the tear duct to the nose in 2 minutes. If the patient does not have enough tears to entrain the marker, this time will be longer.

[00278] Both diseases, dry eye syndrome and blepharitis, are closely related and often occur together in the patient (being referred to as mixed dry eyes-blepharitis pathology). It is known that approximately 70% of the patients with dry eyes have associated blepharitis (Lemp MA. Report of the National Eye Institute/Industry workshop on Clinical Trials in Dry Eyes. CLAO J. 1995; 21 :221-32). It is therefore important to differentiate if the patient suffers only from dry eye syndrome, blepharitis or both pathologies.

[00279] The present disclosure provides methods of treating, preventing or alleviating dry eye syndrome or blepharitis with fusion polypeptide coated device, .e.g. , contact lens.

[00280] One standard dry eye severity grading scheme is described in Behrens et al.,

Dysfunctional tear syndrome, A Delphi approach to treatment recommendations, Cornea 2006, (25):90-97. Under this grading scheme, 1 : mild and/or episodic (occurs under environmental stress); 2: moderate episodic or chronic, stress or no stress; 3: severe frequent or constant without stress; and 4: severe and/or disabling and constant. See DEWS Definition and Classification, The Definition and Classification of Dry Eye Disease: Report of the Definition and Classification Subcommittee of the International Dry Eye Workshop (2007).

[00281] Table 7

TBUT: fluorescein tear break-up time.

[00282] Schirmer test which measures aqueous tear production is easy to perform in clinical settings but may be subject to errors. Strips of filter paper, called Schirmer strips, are placed on the lower lid inside the tarsal conjunctiva. The patient is allowed to blink normally and the tear strip is scored according to the degree it wets in 5 minutes. There are two ways to perform this test: (a) without topical anesthesia (Schirmer test I) which evaluates the ability of the ocular surface to respond to surface stimulation; and (b) under topical anesthesia P T/IB2014/000866

(Schirmer test II) which evaluates basal tear secretion. Patients with tear soaking less than 10 mm are considered to have clinical dry eye and eyes with less than 5 mm wetting are diagnosed as severely dry. However, it is important to note that Schirmer tests are subject to environmental and physiologic changes with varying results over time. Javadi, et al., Dry Eye Syndrome, J. Ophthalmic Vis. Res. (201 1), 6(3): 192-198.

[00283] The lens for correcting vision and/or treating an ophthalmic disease or condition is a lens of copolymers prepared from hydrophilic monomers. Examples of useful lens-forming hydrophilic monomers include, for example, without being limiting: amides such as N,N-dimethylacrylamide and N,N-dimethylacrylaminde; cyclic lactams such as N- vinyl-2-pyrrolidone; meth(acrylated) alcohols, such as 2-hydroxyethyl methacrylate and 2- hydroethylacrylate; and meth(acrylated) poly(ethyleneglycol)s. In one embodiment, the lens forming material of the current invention is poly(methyl methacrylate) (PMMA).

[00284] The present disclosure provides methods of correcting vision and/or treating and/or preventing (or alleviating) an ophthalmic disease or condition with a device, e.g., a biomedical device, e.g., contact lens, coated with a fusion protein carrying carbohydrate moieties. The fusion protein comprises a mucin polypeptide or a fragment thereof and is conjugated to an immunoglobulin or a fragment thereof. The mucin polypeptide (or a fragment thereof) and the immunoglobulin (or a fragment thereof) is glycosylated with one or more sialyl glycans. The carbohydrate composition on mucins improves characteristics of the medical device such as the wetting, adsorption, surface forces and friction of the surface. In some embodiments, the fragment of a mucin polypeptide is the extracellular domain of the polypeptide, which is at least three amino acids in length.

[00285] The present disclosure provides methods of correcting vision and/or treating and/or preventing (or alleviating) an ophthalmic disease or condition with a device, e.g., a biomedical device, e.g. , contact lens, coated with a fusion protein comprising a mucin polypeptide carrying carbohydrate moieties. The mucin polypeptide used as a first polypeptide of a fusion polypeptide of the present disclosure to coat a device, e.g. , a medical device, e.g., a contact lens, includes full-length PSGL-1. In some embodiments, the first polypeptide of a fusion polypeptide used for coating a device, e.g., a medical device, e.g., a contact lens, comprises less than full-length PSGL-1 polypeptide, e.g. , a functional fragment of a PSGL-1 polypeptide. For example the first polypeptide of a fusion polypeptide used for coating a device, e.g., a medical device, e.g., a contact lens, is less than 400 contiguous amino acids in length of a PSGL-1 polypeptide, e.g., less than or equal to 300, 250, 150, 100, or 50, contiguous amino acids in length of a PSGL-1 polypeptide, and at least 25 contiguous amino acids in length (i.e. , 25-300 amino acids in length, 25-250 amino acids in length, 25-150 amino acids in length, 25-100 amino acids in length, or 25-50 amino acids in length) of a PSGL-1 polypeptide. The first polypeptide of a fusion polypeptide used for coating a device, e.g. , a medical device, e.g. , a contact lens, is, for example, the extracellular portion of PSGL- 1 , or includes a portion thereof.

[00286] The present disclosure provides methods of correcting vision and/or treating and/or preventing (or alleviating) an ophthalmic disease or condition with a device, e.g. , a biomedical device, e.g. , contact lens, coated with a fusion polypeptide comprising a first polypeptide, e.g. , a mucin polypeptide (or a fragment thereof) conjugated to a second polypeptide (or a fragment thereof). In some embodiments, the second polypeptide of a fusion polypeptide used for coating a device, e.g. , a medical device, e.g. , a contact lens, is a fragment of the immunoglobulin polypeptide. In some embodiments that second polypeptide of a fusion polypeptide used for coating a device, e.g. , a medical device, e.g., a contact lens, is the γ heavy chains or light chains of an IgG. In some embodiments, the immunoglobulin fragment is the Fc region of IgGl, IgG2, IgG3, or IgG4 for use in coating a medical device; and the medical device is coated as-such.

[00287] The present disclosure methods of correcting vision and/or treating and/or preventing (or alleviating) an ophthalmic disease or condition with a device, e.g., a biomedical device, e.g. , contact lens, coated with a fusion polypeptide comprising a mucin polypeptide or a fragment larger than 3 amino acids thereof, conjugated to IgA, IgD, IgE, IgM, or a domain fragment (at least 3 amino acids long) thereof, for use in coating a medical device; and the medical device is coated as-such. In some embodiments, the mucin polypeptide or a fragment at least 3 amino acids of a mucin polypeptide is conjugated to α, γ, δ, ε, or μ heavy chains of an immunoglobulin for use in coating a medical device; and the medical device is coated as-such. The present disclosure provides an extracellular domain of a mucin conjugated to α, γ, δ, ε, or μ heavy chains of an immunoglobulin for use in coating a medical device; and the medical device is coated as-such. In some embodiments, PSGL-1 , CD34, CD43, CD45, CD96, GlyCAM-1, MAdCAM-1 , or at least 3 amino acid long fragment thereof. The present disclosure provides a secreted mucin, a membrane associated mucin, or at least a 3 amino acids long fragment thereof conjugated to α, γ, δ, ε, or μ heavy chains of an immunoglobulin for use in coating a medical device; and the medical device is coated as- such. Secreted mucin is chosen from MUC2, MUC5AC, MUC5B, MUC6, MUC7, and MUC9, and the membrane associated mucin is chosen from MUC1 , MUC3A, MUC3B, MUC4, and MUC16, and is conjugated to α, γ, δ, ε, or μ heavy chains of an immunoglobulin for use in coating a medical device; and the medical device is coated as-such. In some embodiments, at least a 3 amino acid fragment of a secreted mucin chosen from MUC2, MUC5AC, MUC5B, MUC6, MUC7, and MUC9, or at least a 3 amino acid fragment of a membrane associated mucin chosen from MUC1, MUC3A, MUC3B, MUC4, and MUC16, is conjugated to α, γ, δ, ε, or μ heavy chains of an immunoglobulin for use in coating a medical device; and the medical device is coated as-such.

[00288] The present disclosure provides methods of correcting vision and/or treating and/or preventing (or alleviating) an ophthalmic disease or condition with a device, e.g., a biomedical device, e.g., contact lens, which is coated with a fusion polypeptide between a first polypeptide, e.g. , a mucin polypeptide such as the full-length PSGL-1 , and a second polypeptide. In some embodiments, the first polypeptide is not the full-length PSGL-1 polypeptide, e.g. , a functional fragment of a PSGL-1 polypeptide. For example, the first polypeptide is less than 400 contiguous amino acids in length of a PSGL-1 polypeptide, e.g., less than or equal to 300, 250, 150, 100, or 50, contiguous amino acids in length of a PSGL-1 polypeptide, and at least 25 contiguous amino acids in length of a PSGL-1 polypeptide. The first polypeptide for example, the extracellular portion of PSGL-1, or includes a portion thereof.

[00289] The present disclosure provides methods of correcting vision and/or treating and/or preventing (or alleviating) an ophthalmic disease or condition with a device, e.g. , a biomedical device, e.g., contact lens, which is coated with a fusion polypeptide between a first polypeptide and a second polypeptide, in which the first polypeptide is glycosylated by one or more glycosyltransferases. In some embodiments, the first polypeptide is glycosylated by 2, 3, 5 or more glycosyltransferases. In some embodiments, glycosylation is sequential or consecutive. In additional embodiments, glycosylation is concurrent or random, i. e., in no particular order. The first polypeptide is glycosylated by any enzyme capable of adding N- linked or O-linked sialic acid determinants to a protein backbone. For example the first polypeptide is glycosylated by one or more of the following: a core 2 p6-JV- acetylglucosaminyltransferase, a core 3 3-7V-acetylglucosaminyltransferase, a β4- galactosyltransferase, a 3-galactosyltransferase, an a3-sialyltransferase, an a6- sialyltransferase, and/or an a3-N-acetylgaIactosaminyltransferase. The first polypeptide is more heavily glycosylated than the native (i.e. wild-type) glycoprotein. For example, the first polypeptide may have about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, aboutlO fold, or more glycans than the native first polypeptide or a native glycoprotein. The P T/IB2014/000866 first polypeptide may contain equal to or greater that about 40%, equal to or greater that about 50%, equal to or greater that about 60%, equal to or greater that about 70%, equal to or greater that about 80%, equal to or greater that about 90% or equal to or greater that about 95% of its mass due to carbohydrate.

[00290] The present disclosure provides methods of correcting vision and/or treating and/or preventing (or alleviating) an ophthalmic disease or condition with a device, e.g. , a biomedical device, e.g. , contact lens, which is coated with a fusion polypeptide between a first polypeptide and a second polypeptide, in which the first polypeptide contains equal to or greater that about 40%, equal to or greater that about 50%, equal to or greater that about 60%, equal to or greater that about 70%, equal to or greater that about 80%, equal to or greater that about 90% or equal to or greater that about 95% of its mass due to carbohydrate.

[00291] For example, fusion polypeptide for use in coating a device, e.g. , a medical device, e.g., a contact lens, for use in correcting vision and/or treating and/or preventing (or alleviating) an ophthalmic disease or condition is a fusion of PSGL-1 with a second polypeptide, for example, the Fc region of IgG. In some embodiments, the fusion protein is PSGL-1 / gG4_.

[00292] Provided also are methods of increasing the tear film break-up time (TFBUT) of a subject's tear film, comprising administering to the eye surface of the subject in need thereof a formulation comprising a recombinant mucin, in a pharmaceutically acceptable carrier. Optionally, the ophthalmic formulation for increasing TFBUT may further comprise a tear substitute, or one or more components thereof.

[00293] Provided also are methods of increasing the ocular protection index (OP I) of a subject's eye. The method involves administering to the eye surface of the subject a formulation comprising a recombinant mucin, in a pharmaceutically acceptable carrier.

Optionally, the ophthalmic formulation for increasing OPI may further comprise a tear substitute, or one or more components thereof.

[00294] Provided also are methods for improving, treating, relieving, inhibiting, preventing, or otherwise decreasing ocular discomfort in a subject comprising administering to the eye surface of the subject in need thereof a formulation comprising a recombinant mucin in a pharmaceutically acceptable carrier. Optionally, the ophthalmic formulation for improving, treating, relieving, inhibiting, preventing, or otherwise decreasing ocular discomfort may further comprise a tear substitute, or one or more components thereof.

[00295] Provided also are method of improving overall ocular surface health of a subject's eye, comprising administering to the eye surface of the subject in need thereof a formulation comprising a low dose amount of at least one recombinant mucin in a

pharmaceutically acceptable carrier. Optionally, the ophthalmic formulation for increasing OPI may further comprise a tear substitute, or one or more components thereof.

[00296] The effective amount of the one or more recombinant mucins in the ophthalmic formulations of the invention will depend on absorption, inactivation, and excretion rates of the drug as well as the delivery rate of the compound from the formulation, and will be suitable for short or long term use for the treatment of acute or chronic conditions, respectively. It is to be noted that dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Typically, dosing will be determined using techniques known to one skilled in the art.

[00297] The dosage of the recombinant mucin of the present invention will vary depending on the symptoms, age and other physical characteristics of the patient, the nature and severity of the disorder to be treated or prevented, the degree of comfort desired, the route of administration, and the form of the supplement. Any of the subject formulations may be administered in a single dose or in divided doses. Dosages for the formulations of the present invention may be readily determined by techniques known to those of skill in the art or as taught herein.

[00298] An effective dose or amount, and any possible effects on the timing of administration of the formulation, may need to be identified for any particular formulation of the present invention. This may be accomplished by routine experiment. The effectiveness of any formulation and method of treatment or prevention may be assessed by administering the formulation and assessing the effect of the administration by measuring one or more indices associated with the efficacy of the composition and with the degree of comfort to the patient, as described herein, and comparing the post- treatment values of these indices to the values of the same indices prior to treatment or by comparing the post-treatment values of these indices to the values of the same indices using a different formulation.

[00299] The precise time of administration and amount of any particular formulation that will yield the most effective treatment in a given patient will depend upon the activity, pharmacokinetics, and bioavailability of a particular compound, physiological condition of the patient (including age, sex, disease type and stage, general physical condition,

responsiveness to a given dosage and type of medication), route of administration, and the IB2014/000866 like. The guidelines presented herein may be used to optimize the treatment, e.g. , determine the optimum time and/or amount of administration, which will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage and/or timing.

[00300] The combined use of several recombinant mucins formulated into the compositions of the present invention may reduce the required dosage for any individual component because the onset and duration of effect of the different components may be complimentary. In such combined therapy, the different recombinant mucins may be delivered together or separately, and simultaneously or at different times within the day.

[00301] Efficacy of the formulations and compositions of the invention in treating and preventing the signs and symptoms associated with dry eye disease and/or ocular irritation may be assessed by measuring changes in tear film break-up time (TFBUT), changes in ocular protection index (OPI), improved level of ocular comfort, decreased inflammation as measured by staining and/or redness, improved corneal sensitivity (e.g. , as measured by Cochet-Bonnet test), decreased blink rate, improved visual acuity (e.g. , as measured by the Inter-blink Interval Visual Acuity Decay (IV AD) test). An increase in TFBUT and/or OPI, and/or an improved level of ocular comfort, corneal sensitivity and/or visual acuity, and/or a decrease in the level of inflammation and/or blink rate in a subject following administration of the formulations and compositions of the invention to the ocular surface, as compared to the TFBUT, OPI, level of ocular discomfort, inflammation, corneal sensitivity, visual acuity, corneal staining and/or blink rate prior to administration to the ocular surface, indicates that the formulation is effective in treating and preventing signs and symptoms associated with dry eye disease and/or ocular irritation.

[00302] The ophthalmic formulations of the present invention effectively enhance tear film stability. One measure of tear film stability is an increase in tear film break up time (TFBUT) when measured post-instillation of the ophthalmic formulation into the eye as compared to TFBUT measured prior to instillation of the ophthalmic formulation into the eye (i.e., baseline TFBUT). For example, without limitation, TFBUT is increased by

approximately 0.5 to 10 seconds or more (or any specific value within said range) post- instillation as compared to baseline TFBUT. More particularly, TFBUT is increased by about 0.5 seconds, about 1 second, about 1.5 seconds, about 2 seconds, about 2.5 seconds, about 3 seconds, about 3.5 seconds, about 4 seconds, about 4.5 seconds, about 5 seconds, about 5.5 seconds, about 6 seconds, about 6.5 seconds, about 7 seconds, about 7.5 seconds, about 8 seconds, about 8.5 seconds, about 9 seconds, about 9.5 seconds, about 10 seconds, or more, when measured post instillation as compared to baseline TFBUT. 4 000866

[00303] One method of determining a clinically meaningful increase in TFBUT is an increase (i.e., improvement) in Ocular Protection Index (OPl) when measured post- instillation of the ophthalmic formulation into the eye as compared to OPl measured prior to instillation of the ophthalmic formulation into the eye (i. e. , baseline OPl). This approach to measuring clinically relevant alterations in TFBUT, known as the Ocular Protection Index (OPl) has proven useful in assessing factors that cause dry eye and evaluating its therapeutic agents.

[00304] When studying the relationship between TFBUT and the inter-blink interval (IBI=time between complete blinks), it may be suggested that their interaction assists in regulating the integrity of an ocular surface. A protected surface exists when the TFBUT is longer than the IBI. In contrast, an unprotected surface exists when the TFBUT is shorter than the IBI. Studies have shown that within one second of TFBUT, patients report ocular discomfort and shortly thereafter develop superficial punctate keratitis. To prevent these symptoms and signs, the TFBUT must match or exceed the inter-blink period, providing complete protection of the ocular surface. When quantifying an agent's effect on tear film stability, a binomial analysis may be performed. The index allows for two possible outcomes after treatment, 1) success=TFBUT either matches or exceeds the inter-blink period so that the ocular surface is protected and 2) failure=TFBUT remains shorter than the inter-blink period so that the ocular surface is unprotected. An OPl score≥ 1 is considered favorable since the patient has a tear protected ocular surface, resulting in fewer signs and symptoms associated with dry eye. An OPl score <1 is considered unfavorable since the patient has an exposed ocular surface, resulting in more signs and symptoms associated with dry eye.

[00305] The ophthalmic formulations of the invention effectively increase (/^'. e. , improve) OPL For example, without limitation, OPl is improved by about 0.1 to 10, or more (or any specific value within said range) when measured post-instillation of the ophthalmic formulation into the eye as compared to baseline OPl. More particularly, OPl is improved whereby the OPl is increased by about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.2, about 1.4, about 1.6, about 1.8, about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, about 3.0, about 3.2, about 3.4, about 3.6, about 3.8, about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.2, about 8.4, about 8.6, about 8.8, about 9.0, about 9.2, about 9.4, about 9.6, about 9.8, about 10.0, or more, when measured post instillation as compared to baseline OPl. Ocular irritation/discomfort is 4 000866 effectively decreased whereby patient assessment of ocular discomfort is less when measure post- instillation of the ophthalmic formulation into the eye as compared to ocular discomfort measured prior to instillation of the ophthalmic formulation into the eye.

[00306] TFBUT may be measured using various methods, including but not limited to illumination of the eye following instillation of sodium fluorescein in the eye, or equivalents thereof. OPI may be obtained by dividing the TFBUT by the time in seconds between blinks (the inter-blink interval, or "IBI")

[00307] An increase in ocular comfort or decrease in ocular discomfort in a subject following administration of the formulations and compositions of the invention as compared to ocular comfort level prior to administration, indicates that the formulation is effective in treating and preventing signs and symptoms associated with dry eye limited to subjective scales (for example but not limited to, standardized subjective scales that determine ocular discomfort as mild, moderate, sever, or 0, 1 , 2, 3, 4, etc., or other appropriate scale), reflexive response (e.g., flinch-reflex), and physiological response, including but not limited to changes in heart rate, blood pressure, and perspiration levels.

[00308] Efficacy of the formulations and compositions of the invention in improving overall ocular surface health may be assessed by measuring changes in corneal staining, conjunctival redness, corneal sensitivity, blink rate, and visual performance. Methods of assessing these parameters include: lissamine green or sodium fluorescein dyes, standardized assessment scales, Cochet Bonnet aesthesiometry or non-contact aesthesiometry, video recording and software analysis, and questionnaires or the Inter-blink Interval Visual Acuity Decay (IVAD) test, respectively.

PACKAGING

[00309] The device (e.g., contact lens) and/or formulations of the present invention may be packaged as either a single dose product or a multi-dose product. The single dose product is sterile prior to opening of the package and all of the composition in the package is intended to be consumed in one or several applications to one or both eyes of a patient. The use of an antimicrobial preservative to maintain the sterility of the composition after the package is opened is generally unnecessary. The formulations, if an ointment formulation, may be packaged as appropriate for an ointment, as is known to one of skill in the art.

[00310] Multi-dose products are also sterile prior to opening of the package. However, because the container for the composition may be opened many times before all of the composition in the container is consumed, the multi-dose products must have sufficient antimicrobial activity to ensure that the compositions will not become contaminated by PC17IB2014/000866 microbes as a result of the repeated opening and handling of the container. The level of antimicrobial activity required for this purpose is well known to those skilled in the art, and is specified in official publications, such as the United States Pharmacopoeia ("USP") and other publications by the Food and Drug Administration, and corresponding publications in other countries. Detailed descriptions of the specifications for preservation of ophthalmic pharmaceutical products against microbial contamination and the procedures for evaluating the preservative efficacy of specific formulations are provided in those publications. In the United States, preservative efficacy standards are generally referred to as the "USP PET" requirements. (The acronym "PET" stands for "preservative efficacy testing.")

[00311] The use of a single dose packaging arrangement eliminates the need for an anti-microbial preservative in the compositions, which is a significant advantage from a medical perspective, because conventional antimicrobial agents utilized to preserve ophthalmic compositions (e.g., benzalkonium chloride) may cause ocular irritation, particularly in patients suffering from dry eye conditions or pre-existing ocular irritation, or patients using multiple preserved products. However, the single dose packaging arrangements currently available, such as small volume plastic vials prepared by means of a process known as "form, fill and seal", have several disadvantages for manufacturers and consumers. The principal disadvantages of the single dose packaging systems are the much larger quantities of packaging materials required, which is both wasteful and costly, and the inconvenience for the consumer. Also, there is a risk that consumers will not discard the single dose containers following application of one or two drops to the eyes, as they are instructed to do, but instead will save the opened container and any composition remaining therein for later use. This improper use of single dose products creates a risk of microbial contamination of the single dose product and an associated risk of ocular infection if a contaminated composition is applied to the eyes.

[00312] While the formulations of this invention are preferably formulated as "ready for use" aqueous solutions, alternative formulations are contemplated within the scope of this invention. Thus, for example, the active ingredients, surfactants, salts, chelating agents, or other components of the ophthalmic solution, or mixtures thereof, can be lyophilized or otherwise provided as a dried powder or tablet ready for dissolution (e.g., in deionized, or distilled) water. Because of the self-preserving nature of the solution, sterile water is not required. KITS

[00313] In still another embodiment, this invention provides kits for the packaging and/or storage and/or use of the formulations and/or device coated with fusion proteins of the current invention is described herein, as well as kits for the practice of the methods described herein. Thus, for example, kits may comprise one or more containers containing one or more ophthalmic solutions, ointments, gels, sustained release formulations or devices, suspensions or formulations, tablets, or capsules of this invention. The kits can be designed to facilitate one or more aspects of shipping, use, and storage.

[00314] The kits may optionally include instructional materials containing directions (i.e. , protocols) disclosing means of use of the formulations provided therein. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g. CD ROM), and the like. Such media may include addresses to internet sites that provide such

instructional materials.

DEFINITIONS

[00315] As used herein, a "device" is any article that is designed to be used while either in or on mammalian tissues or fluid, and preferably in or on human tissue or fluids such as ophthalmic devices, for example, intraocular lenses and contact lenses. The devices are ophthalmic devices, for example, contact lenses, such as contact lenses made from silicone hydro gels.

[00316] As used herein, the terms "lens" and "ophthalmic device" refer to devices that reside in or on the eye. These devices can provide optical correction, wound care, drug delivery, diagnostic functionality, cosmetic enhancement or effect or a combination of these properties. The term lens includes but is not limited to soft contact lenses, hard contact lenses, intraocular lenses, overlay lenses, ocular inserts, and optical inserts.

[00317] As used herein, an "ophthalmic lens" refers to lenses which are placed in intimate contact with the eye or tear fluid, such as contact lenses for vision correction (e.g. , spherical, toric, bifocal), contact lenses for modification of eye color, ophthalmic drug delivery devices, ocular tissue protective devices (e.g., ophthalmic healing promoting lenses), and the like. Ophthalmic lens may be an extended- wear contact lens, especially extended- wear contact lenses for vision correction, with oxygen transmissibility or permeability, ion permeability, gas permeability, and other desirable transmissibility or permeability and 0866 features. As used herein, an "ocular environment" refers to ocular fluids (e.g. , tear fluid) and ocular tissue (e.g. , the cornea) which may come into intimate contact with a contact lens used for vision correction, drug delivery, wound healing, eye color modification, or other ophthalmic applications.

[00318] As used herein, an "outer surface" of an ophthalmic lens refers to the anterior surface of the lens which faces away from the eye during wear. The outer surface, which is typically substantially convex, may also be referred to as the front curve of the lens. The "inner surface" of a lens, as used herein, refers to the posterior surface of the lens which faces towards the eye during wear. The inner surface, which is typically substantially concave, may also be referred to as the base curve of the lens.

[00319] As used herein the term "monomer" is a compound containing at least one polymerizable group and an average molecular weight of about less than 2000 Daltons, as measure via gel permeation chromatography refractive index detection. Thus, monomers include dimers and in some cases oligomers, including oligomers made from more than one monomeric unit.

[00320] The term "acute" as used herein denotes a condition having a rapid onset, and symptoms that are severe but short in duration.

[00321] The term "analgesic" as used herein denotes a compound/formulation for the management of intermittent and/or chronic physical discomfort, suitable for long term use.

[00322] The term "anesthetic" or "anesthesia" as used herein denotes a

compound/formulation for the management of acute physical pain, suitable for short term, temporary use, which has an effect that produces numbing or decreased sensitivity in the body part/organ to which the compound/formulation is administered (e.g. , decreased corneal sensitivity of the eye).

[00323] The term "aqueous" typically denotes an aqueous composition wherein the carrier is to an extent of >50%, more preferably >75% and in particular 90% by weight water. The term "chronic" as defined herein is meant a persistent, lasting condition, or one marked by frequent recurrence, preferably a condition that persists/recurs for greater than 3 months, more preferably greater than 6 months, more preferably greater than 12 months, and even more preferably greater than 24 months.

[00324] The term "comfortable" as used herein refers to a sensation of physical well- being or relief, in contrast to the physical sensation of pain, burning, stinging, itching, irritation, or other symptoms associated with physical discomfort. 2014/000866

[00325] The term "comfortable ophthalmic formulation" as used herein refers to an ophthalmic formulation which provides physical relief from symptoms associated with dry eye disease and/or ocular discomfort, and only causes an acceptable level of pain, burning, stinging, itching, irritation, or other symptoms associated with ocular discomfort, when instilled in the eye, which are less than those seen with dosing with current concentrations on the market.

[00326] The term "dry eye" as used herein, refers to inadequate tear production and/or abnormal tear composition. Causes of dry eye disease as defined herein include but are not limited to the following: idiopathic, congenital alacrima, xerophthalmia, lacrimal gland ablation, and sensory denervation; collagen vascular diseases, including rheumatoid arthritis, Wegener's granulomatosis, and systemic lupus erythematosus; Sjogren's syndrome and autoimmune diseases associated with Sjogren's syndrome; abnormalities of the lipid tear layer caused by blepharitis or rosacea; abnormalities of the mucin tear layer caused by vitamin A deficiency; trachoma, diphtheric keratoconjunctivitis; mucocutaneous disorders; aging; menopause; and diabetes. Dry eye signs and/or symptoms as defined herein may also be provoked by other circumstances, including but not limited to the following: prolonged visual tasking; working on a computer; being in a dry environment; ocular irritation; contact lenses, LASIK and other refractive surgeries; fatigue; and medications such as isotretinoin, sedatives, diuretics, tricyclic antidepressants, antihypertensives, oral contraceptives, antihistamines, nasal decongestants, beta-blockers, phenothiazines, atropine, and pain relieving opiates such as morphine.

[00327] The term "wettability" as used herein, refers to the tendency for a liquid to spread over a solid surface, which is commonly characterized by measuring the contact angle at the liquid and solid interface. It is particularly of relevance to contact lenses because the lens surface needs to support a stable ocular tear film.

[00328] The phrase "effective amount" is an art-recognized term, and refers to an amount of an agent that, when incorporated into a pharmaceutical composition of the present invention, produces some desired effect at a reasonable benefit/risk ratio applicable to any medical treatment. In certain embodiments, the term refers to that amount necessary or sufficient to eliminate, reduce or maintain (e.g., prevent the spread of) a sign and/or symptom of dry eye and/or eye irritation, or prevent or treat dry eye and/or eye irritation. The effective amount may vary depending on such factors as the disease or condition being treated, the particular composition being administered, or the severity of the disease or condition. One of skill in the art may empirically determine the effective amount of a particular agent without necessitating undue experimentation.

[00329] A "patient," "subject," or "host" to be treated by the subject method refers to either a human or non-human animal, such as a primate, mammal, and vertebrate.

[00330] The phrase "pharmaceutically acceptable" is art-recognized and refers to compositions, polymers and other materials and/or salts thereof and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[00331] The phrase "pharmaceutically acceptable carrier" is art- recognized, and refers to, for example, pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition, or component thereof, from one organ, or portion of the body, to another organ, or portion of the body, or to deliver an agent to the surface of the eye. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient. In certain

embodiments, a pharmaceutically acceptable carrier is non-pyrogenic. Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, hydroxypropylmethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (1 1) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; (21) gums such as HP-guar; (22) polymers; and (23) other non-toxic compatible substances employed in pharmaceutical formulations.

[00332] The term "pharmaceutically acceptable salts" is art-recognized, and refers to relatively non-toxic, inorganic and organic acid addition salts of compositions of the present invention or any components thereof, including without limitation, therapeutic agents, excipients, other materials and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, ptoluenesulfonic acid, and the like. Examples of suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For purposes of illustration, the class of such organic bases may include mono-, di-, and trialkylamines, such as methylamine, dimethylamine, and triethylamine; mono-, di- or trihydroxyalkylamines such as mono-, di-, and triethanolamine; amino acids, such as arginine and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N- methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine;

(trihydroxymethyl)aminoethane; tromethamine, and the like. See, e.g. , J. Pharm. Sci., 66: 1- 19 (1977).

[00333] As used herein, the terms "tear substitute" and "artificial tear" may be used interchangeably, and each refers to one or more molecules or compositions, which lubricate, "wet," approximate the consistency of endogenous tears, aid in natural tear build up, or otherwise provide temporary relief of dry eye signs and/or symptoms and conditions upon ocular administration, including without limitation a polymer (e.g. , a cellulosic polymer), an ocular surface protectant, a demulcent, or other component found on the FDA monograph for tear substitutes. The term "tear substitute component" refers to one or more components thereof.

[00334] The term "treating" is an art-recognized term which refers to reducing or ameliorating at least one sign and/or symptom of any condition or disease. "Treating," includes any effect, e.g. , lessening, reducing, modulating, or eliminating, that results in the improvement of the condition, disease, disorder, etc. "Treating" or "treatment" of a disease state includes: (1) inhibiting the disease state, i.e. , arresting the development of the disease state or its clinical symptoms; (2) relieving the disease state, i.e. , causing temporary or permanent regression of the disease state or its clinical symptoms; or (3) reducing or lessening the symptoms of the disease state.

[00335] "Preventing" includes any effect in, e.g. , causing the clinical symptoms of the disease state not to develop in a subject that may be exposed to or predisposed to the disease state, but does not yet experience or display symptoms of the disease state. As used herein, "preventing" or "prevent" describes reducing or eliminating the onset of the symptoms or complications of the disease, condition or disorder. The term "preventing," when used in relation to a condition, such as dry eye and/or eye irritation, is art-recognized, and refers to formulation, composition and/or device (e.g. , contact lens) which reduces the frequency of, or delays the onset of, signs and/or symptoms of a medical condition in a subject relative to a subject which does not receive the composition.

[00336] As used herein, the term "alleviate" is meant to describe a process by which the severity of a sign or symptom of a disorder is decreased. Importantly, a sign or symptom can be alleviated without being eliminated.

[00337] As used herein the term "symptom" is defined as an indication of disease, illness, injury, or that something is not right in the body. Symptoms are felt or noticed by the individual experiencing the symptom, but may not easily be noticed by others. Others are defined as non-health-care professionals.

[00338] As used herein the term "sign" is also defined as an indication that something is not right in the body. But signs are defined as things that can be seen by a doctor, nurse, or other health care professional.

[00339] The term "more" as used in the present disclosure does not include infinite number of possibilities. The term "more" as used in the present disclosure is used as a skilled person in the art would understand in the context in which it is used. For example, 1 , 2, 3, or more glycosyltransferases implies, as a skilled artisan would understand, more than 3 glycosyltransferases that are known or will potentially be known in the art.

[00340] The term "about" is used herein to mean approximately, in the region of, roughly or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%.

[00341] As used in the present disclosure, whether in a transitional phrase or in the body of a claim, the terms "comprise(s)" and "comprising" are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases "having at least" or "including at least." When used in the context of a process the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a molecule, compound, or composition, the term "comprising" means that the compound or composition includes at least the recited features or components, but may also include additional features or components.

[00342] For the purposes of promoting an understanding of the embodiments described herein, reference made to preferred embodiments and specific language are used to describe the same. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. As used throughout this disclosure, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a

composition" includes a plurality of such compositions, as well as a single composition, and a reference to "a therapeutic agent" is a reference to one or more therapeutic and/or

pharmaceutical agents and equivalents thereof known to those skilled in the art, and so forth. All percentages and ratios used herein, unless otherwise indicated, are by weight.

[00343] The following examples are illustrative, but not limiting, of the methods and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in synthesis and use of the compounds of the present disclosure and that are obvious to those skilled in the art are within the spirit and scope of the present disclosure.

EXAMPLES

[00344] Examples of ophthalmic formulations and device coated with fusion protein of the present invention, illustrating the composition and the method of making such solutions, are noted below.

EXAMPLE 1

General Methods

[00345] Standard molecular biology protocols known in the art not specifically described herein are generally followed essentially as in Sambrook et al., Molecular cloning: A laboratory manual, Cold Springs Harbor Laboratory, New- York (1989, 1992), and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1988), and as in Ausubel et al , Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and as in Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988), and as in Watson et al., Recombinant DNA, Scientific American Books, New York and in Birren et al. (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York (1998) and methodology as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801 ,531 ; 5,192,659 and 5,272,057 and incorporated herein by reference. Polymerase chain reaction (PCR) was carried out as in standard PCR Protocols: A Guide To Methods and Applications, Academic Press, San Diego, Calif. (1990). In situ PCR in combination with Flow Cytometry (FACS) can be used for detection of cells containing specific DNA and mRNA sequences (Testoni et al., Blood 1996, 87:3822.) Methods of performing RT-PCR are well known in the art.

Cell Culture

[00346] HeLa cells (American Type Culture Collection) are cultured as described in Czauderna, et al (NAR, 2003. 31 :670-82). Human keratinocytes are cultured at 37°C in Dulbecco's modified Eagle medium (DMEM) containing 10% FCS. The mouse cell line, B16V (American Type Culture Collection), is cultured at 37°C in Dulbecco's modified Eagle medium (DMEM) containing 10% FCS. Culture conditions are as described in (Methods Find Exp Clin Pharmacol. 1997, 19(4):231-9).

Atomic Force Microscope (AFM) Colloidal Probe

[00347] An AFM (Veeco Instruments Inc.) was employed for force and friction measurements in a fused silica liquid cell (volume ~ 0.1 mL), using a Nanoscope Multimode III Pico Force system. The detection limit of the normal and friction force is in the range of piconewton. Rectangular tipless cantilevers (MikroMasch, CSC12/tipless/Cr-Au, F lever) with the approximate dimensions of 250 μιη in length, 35 μηι in width, and normal spring constants in the range 0.02 - 0.2 N/m were chosen for all the force and friction

measurements. The exact values of the normal and the torsional spring constants (k(p) were determined using the AFM Tune IT v2.5 software (Force IT, Sweden) according to a method based on thermal noise with hydrodynamic damping. See Green et al., Rev. Set lustrum. 2004, 75, 1988 and Sader et al., Rev. Sci. Instrum. 1999, 70, 3967. A spherical PMMA particle (Kisker, cat.#ppmma-10.0) with a diameter of approximately 10 μ^ηι was attached to the end of a cantilever with the aid of an Ependorf Micromanipulator 5171, a Nikon Optiphot 100S reflection microscope, and a small amount of epoxy glue (Araldite, 80806) after determining the spring constants of the cantilever. The lateral photodetector sensitivity (δ, V/rad) was calibrated using the method of tilting the AFM head as suggested by Pettersson et. al. See Pettersson et al., Rev. Sci. Instrum. 2007, (78), 93702.

[00348] The AFM experiments were started by measuring the normal forces between the PMMA surface and the PMMA probe across a 155 mM NaCl solution. Next, a 100 ppm solution of mucin (BSM or PSGL-l/mIgG_2b) in 155 mM NaCl was introduced to the fused silica liquid cell and the polymer was allowed to adsorb for 45 minutes. Then, the normal forces were measured at 25°C, followed by friction measurements. The normal forces were measured with a constant approach and retraction speed of 821 nm/second. The friction forces were determined by sliding the surfaces backwards and forwards 10 times at each normal load using a scanning angle perpendicular to the cantilever at a sliding speed of 2 μηι/sec.

[00349] In single asperity contacts the friction vs. load relationship can often be analyzed using the relation:

F_f = C + μΡ„

where the constant C is known as the critical shear stress and accounts for the adhesion between the surfaces, and μ is the friction coefficient. Since adhesion is only observed between the bare PMMA surfaces, the critical shear stress has a non-zero value in only this case. When discussing friction data it is sometimes better to report contact pressure than load, since the pressure values can be compared between experiments using different probe radius and materials with different Young's modulus (stiffness). The mean pressure that corresponds to a given load was calculated using JKR theory for bare PMMA surfaces where the adhesion contribution is significant, and the Hertz model for PMMA surfaces with mucin films where adhesion effects can be ignored. See Johnson et. al., Surface Energy and the Contact of Elastic Solids. Proceedings of the Royal Society of London. A. Mathematical and Physical Sciences 1971, 324 (1558), 301-313 and Hertz, Angew. Math. 1881 , 92, 156-171. For the calculation, the Young's modulus, 2.5 GPa, and Poisson's ratio, 0.38, for PMMA was used.

EXAMPLE 2

Protein Expression

[00350] PSGLl/mIgG2b was produced in CHO cells co-expressing the fusion protein and the genes coding for pi ,6-N-acetylglucosaminyltransferase I (C2 GnT-I) and

a l,3fucosyl transferase VII (FucT-VII). These cell line were produced to express the carbohydrate epitope sialyl Lewis x (Siaa2,3GaIpl ,4(Fucal,3)GlcNAc). The CHO cells co- expressing PSGL-l/mIgG2b, C2 GnT-1 , and FUT-VII were defined as C-PSLex. Fusion protein produced in this cell line mainly expressed mono- and/or disialylated core 1

(Siaa2,3Galpl,3(Sia 2,6)GalNAc), as well as sialylated type 2 on a core 2 chain

(Siaa2,3Galpl,4GlcNAcpl,6(Galpl ,3)GalNAc). Likewise, PSGL-l/hIgG4 is produced in non-glycoengineered CHO cells. The PSGL-l/hIgG4 expressed in these cells carries mainly mono- and disialylated core 1 and/or core 2 glycans.

[00351] PSGL-l/hIgG4 of the present disclosure is produced in non-glycoengineered CHO cells or CHO cells co-expressing the fusion protein and the gene coding for β1,6-Ν- acetylglucosaminyltransferase I (C2 GnT-I). This cell line is produced to express the carbohydrate epitope mono- or disialylated Core 2 (Siaa2,3Galpl,4GlcNAc). The CHO cells of the current invention co-expressing PSGL-l/hIgG4 and C2 GnT-1 are defined as C-PSC2 (sialylated Core 2). Fusion protein expresses mono- and/or disialylated core 1

(Siaa2,3Gaipi,3(Siaa2,6)GalNAc), as well as sialylated type 2 on a core 2 chain

(Siaa2,3Galpl ,4GlcNAcpl,6(Galpl ,3)GalNAc). In some embodiments, the CHO cells of the current invention also express an alpha2,3- or alpha2,6-sialyltransferase in order to increase the sialylation of the glycans

[00352] PSGL-l/hIgG4 and PSGLl/mIgG2b are expressed in Pichia pastoris. The fusion proteins are mannosylated.

EXAMPLE 3

Rabbit Model to Reverse the Onset of Dry Eye

[00353] Dry eye is created in rabbits by surgically closing the lacrimal gland excretory duct, and allowing the rabbits to remain untreated for at least four weeks. See Gilbard, J. P, 1996, "Dry Eye: phramcological approaches, effects, and progress" CLAO J. 22, 141-145. After confirming dry eye by Schirmer test, and ocular surface staining, formulation of the invention is instilled as a solution at concentrations of 0.01 , 0.1 , 1.0%, 5%, or 10% in neutral, isotonic buffered aqueous solution. The formulation is administered in one 50 microliter drop to the ocular surface up to 1-5 times a day, every day for 2-10 weeks. The symptoms of dry eye are monitored once a week for 2-10 weeks and an increase in Schirmer scores and/ or a decrease in the amount of ocular surface staining indicates the efficacy of the formulation of the current invention in the treatment of dry eye disease.

EXAMPLE 4

Wetting and adsorption properties of bovine submaxillary mucin (BSM) and PSGL- l/mIgG_2b produced in C-PSLex on poly(methyl methacrylate) (ΡΜΜΛ) surface

[00354] The effect of carbohydrate composition on mucins with regard to wetting, adsorption, surface forces and friction was investigated. PSGL-l/mIgG₂b produced in C- PSLex (CHO cells co-expressing PSGL-l/mIgG_2b, C2 GnT-I (core 2 β Ι,ό-N- acetylglycosyltransferase I) and FUT-VII (al ,3-fucosyltransferase VII)), produced a fusion protein carrying multiple negative charges. The wetting and adsorption properties of PSGL- l/mIgG_2b, produced in C-PSLex, were measured. PMMA was used, as an exemplary contact lens material, as the coated surface substrate for wetting and adsorption measurements. [00355] Materials used were: PMMA coated gold QCM crystal; BSM; and PSGL- l/mIgG_2b produced in C-PSLex.

[00356] Contact angle measurements (Data physics), to investigate the wetting properties of protein solutions. Contact angles on bare PMMA surface were measured using: Water, 50 ppm BSM solution, 50 ppm PSGL-l/mIgG_2b solution, water contact angles on BSM or PSGL-l/mIgG_2b coated PMMA surface were measured.

[00357] QCM-D: to measure amount of protein with coupled and trapped solvent adsorbed on PMMA surface. BSM and PSGL-l/mIgG_2b solution were found to have similar CA on PMMA surface to water, but the Re CA was much lower than for water, indicating superior wetting property due was to adsorption of the mucins. After coating by BSM and PSGL-l/mIgG_2b, the CA of the PMMA surface decreased by 20° and the Ad CA of PSGL- l/mIgG_2b was lowered even more, indicating that these mucins made the PMMA surface more hydrophilic. With prolonged time, PSGL- l/mIgG_2b solution gave smaller CA than BSM, showing superior wetting ability, and indicating water penetration into the layers.

Wetting and adsorption properties of PSGL-l/ItIgG4

[00358] The effect of carbohydrate composition on mucins with regard to wetting, adsorption, surface forces and friction are investigated. PSGL-l/hIgG4 are produced in C- PSLex (CHO cells co-expressing PSGL-l/mIgG_2b, C2 GnT-I (core 2 βΙ ,ό-N- acetylglycosyltransferase I) and FUT-VII (o l,3-fucosyltransferase VII)), which produces a fusion protein carrying multiple negative charges The wetting and adsorption properties of PSGL-l/hIgG4 produced in C-PSLex are measured. PMMA is used, as an exemplary contact lens material, as the coated surface substrate for wetting and adsorption measurements.

[00359] Similar experiments are performed with physiological tear fluid to compare with PSGL-l/hIgG4 or PSGLl/mIgG2b.

EXAMPLE 5

[00360] Adsorption of PSGL-l/mIgG_2b on PMMA surfaces was measured. BSM adsorption on PMMA surface was calculated as Af=-30 Hz, and PSGL-l/mIgG_2b adsorption on PMMA surface was calculated as Af=-67 Hz. The larger negative shift in Af, the larger mass of the layer (mucin+water) incorporated in the layer. The frequency change after adsorption of PSGL-l/mIgG_2b was larger than that of BSM, indicating more adsorption of purified PSGL-l/mIgG_2b on PMMA surface. The dissipation change of PSGL-l/mIgG_2b was also larger than that of BSM. Therefore, the adsorbed PSGL- l/mIgG_2b layer was less rigid than the BSM layer, possibly due to more extended polymer structure or a more viscous adsorbed layer. The study demonstrated that PSGL-l/mIgG_2b had a higher affinity for PMMA surface and a better wetting property than BSM.

[00361] The experiments of CA value calculation showed that water wets a PMMA surface coated with PSGL- l/mIgG_2b better than a PMMA surface coated with BSM.

[00362] Adsorption of PSGL-l/hIgG4 on PMMA surfaces is measured. The larger negative shift in Af, the larger mass of the layer (mucin+water) is incorporated in the layer. The frequency change after adsorption of PSGL-l/hIgG4 is larger than that of BSM, indicating more adsorption of purified PSGL-l/hIgG4 on PMMA surface. The dissipation change of PSGL-l/hIgG4 is also larger than that of BSM. Therefore, the adsorbed PSGL- l/hIgG4 layer is less rigid than the BSM layer, possibly due to more extended polymer structure or a more viscous adsorbed layer. The study demonstrates that PSGL-l/hIgG4 has a higher affinity for PMMA surface and a better wetting property than BSM.

[00363] The experiments of CA value calculation showed that water wets a PMMA surface coated with PSGL-l/mIgG_2b better than a PMMA surface coated with BSM. See Table 2. Similar experiments are performed with physiological tear fluid to compare with PSGL-l/hIgG4 or PSGLl/mIgG2b.

EXAMPLE 6

[00364] Stepwise adsorption of PSGL- l/mIgG_2b and BSM on PMMA surface was investigated. The stepwise adsorption was quantified as the sensed mass of PSGL-l/mIgG₂b or BSM as a function of concentration which was adsorbed on the PMMA surface. Sensed mass refers to adsorbed mass including coupled water derived from Voigt modeling. This model is based on the assumption that the adsorbed layer has a homogeneous thickness; and it takes into account the viscoelastic properties of the system. In this experiment mucin solutions of different concentrations (2, 25, 50, and 100 ppm in 155 mM NaCl) were introduced sequentially to the cell and allowed to adsorb for 45 minutes, with rinsing by 155 mM NaCl solution for 20 minutes in between each mucin solution as well as after the 100 ppm mucin solution.

[00365] The experiments showed that the sensed mass of BSM increased from 0.5 mg/m² to around 10 mg/m² when the concentration was increased from 2 ppm to 50 ppm before rinsing and remained at the same value as the concentration was increased further to 100 ppm. This suggests that the adsorption had reached saturation at 50 ppm. In all cases the T IB2014/000866 mass on the surface decreased by rinsing with 155 mM NaCl. This reveals that some of the adsorbed BSM molecules were rinsed away, and it also implies that the binding strength between BSM molecules and the PMMA surface is not strong enough to resist rinsing.

[00366] The experiments showed that the sensed mass of PSGL-l/mIgG _b increased constantly from 6 mg/m² (at 2 ppm) to around 16.4 mg/m² (at 100 ppm) before rinsing. Thus, the adsorbed mass of PSGL-l/mIgG₂b and water trapped in the layer is significantly larger, close to a factor of 2, than the mass of adsorbed BSM with associated water. Furthermore, the difference between the sensed mass before and after rinsing is negligible for PSGL- l/mIgG_2b, which contrasts to the significant desorption observed upon rinsing the PMMA surface coated with BSM. This means that the PSGL-l/mIgG_2b molecules had higher affinity towards the PMMA surface than BSM molecules and most PSGL-l/mIgG_2b molecules were permanently bound.

[00367] Stepwise adsorption of PSGL-l/hIgG4 on PMMA surface is investigated. The stepwise adsorption is quantified as the sensed mass of PSGL-l/hIgG4 as a function of concentration which was adsorbed on the PMMA surface. The PSGL-l/hIgG4 molecules have higher affinity towards the PMMA and most PSGL-l/hIgG4 molecules are permanently bound.

[00368] Table 3 shows the frequency and dissipation change for the 3^rd overtone when adsorbed from BSM and PSGL-l/mIgG_2b solution of 100 ppm, as well as the sensed mass and thickness extracted using the Voigt extended viscoelastic modeling of data obtained from the 3^rd and 7^th overtones. The frequency change after adsorption of PSGL- l/mIgG₂ was about twice the value of BSM, indicating more adsorption of PSGL-l/mIgG_2b on PMMA surface. Besides, the dissipation change of PSGL-l/mIgG_2b was also larger than that of BSM, which means that the adsorbed PSGL-l/mIgG_2b layer was more extended than the BSM layer. The modeling result further confirmed that PSGL-l/mIgG_2b molecules adsorbed more on the PMMA surface and formed a more extended layer, about 15 nm, than BSM molecules, about 8 nm. See Table 3. Similar experiments are performed with physiological tear fluid to compare with PSGL-l/hIgG4 or PSGLl/mIgG2b.

EXAMPLE 7

[00369] Contact angle measurements of water, BSM, or PSGL-l/mIgG_2b liquid phases on uncoated or BSM or PSGL-l/mIgG_2b coated PMMA surfaces were performed. Mucin solutions showed similar contact angles on PMMA surfaces. This means that neither of the two mucins spreads on the PMMA surface outside the droplet and they therefore do not promote water spreading on the uncoated PMMA. In contrast, after mucin adsorption on the PMMA surface, the contact angles were decreased to 52° for BSM coated surface and 5.0° for PSGL-l/mIgG_2b coated surface respectively, which means that adsorbed layers of both mucins increase the wettability of the PMMA surface, but the PSGL-l/mIgG_2b layer promotes wetting significantly more than the BSM layer.

[00370] Contact angle measurements of PSGL-l/hIgG4 liquid phases on uncoated or PSGL-l/hIgG4 coated PMMA surfaces are performed. Adsorbed layers of PSGL-l/hIgG4 increase the wettability of the PMMA surface. See Table 4. Similar experiments are performed with physiological tear fluid to compare with PSGL-l/hIgG4 or PSGLl/mIgG2b.

EXAMPLE 8

[00371] Contact angle measurements of water on BSM coated PMMA surface and PSGL-l/mIgG_2b coated PMMA surface as a function of time were performed. The contact angle for BSM coated surface almost remained constant, while the value for the PSGL- l/mIgG_2b coated surface decreased around 30° within 25 seconds. The data suggests that water was transported from the droplet to within the PSGL-l/mIgG_2b film, resulting in a force that spreads the water droplet over the surface to achieve close to complete wetting. Water penetration into the BSM film was less, and resulted in insignificant water spreading on the surface.

[00372] Contact angle measurements of water on PSGL-l/hIgG4 coated PMMA surface as a function of time is performed. The contact angle for the PSGL-l/mIgG_2b coated surface decreases around 30° within 25 seconds. Water is transported from the droplet to within the PSGL-l/hIgG4 film, resulting in a force that spreads the water droplet over the surface to achieve close to complete wetting.

[00373] Similar experiments are performed with physiological tear fluid to compare with PSGL-l/hIgG4 or PSGLl/mIgG2b.

EXAMPLE 9

[00374] Normal force-distance curves between PMMA surfaces across 155 mM NaCl solution, BSM layers adsorbed on PMMA surface across 100 ppm BSM solution, and PSGL- l/mIgG_2b layers adsorbed on PMMA surface across 100 ppm PSGL-l/mIgG_2b solution were obtained.

[00375] The normal force-distance curve between PMMA surfaces across 155 mM NaCl solution shows an attraction from around 40-50 nm. On separation, an adhesion force of 17IB2014/000866 around -3 mN/m is observed between the PMMA probe and PMMA surface. The large range of the attraction suggests that some PMMA chains extend from the surfaces and these bridges over to the opposing surface at small enough separations (< 50 nm), giving rise to a so-called bridging attraction.

[00376] The normal force-distance curve after adsorption of BSM on PMMA probe and PMMA surface demonstrated that forces experienced on approach were purely repulsive and of steric origin, i.e. due to compression of the BSM layer. On separation, a small adhesion force was observed, which may be due to the presence of small patches on the PMMA surface that are not covered by BSM molecules.

[00377] The normal force-distance curve between PSGL-l/mIgG₂b layers adsorbed on PMMA surfaces demonstrated a strong steric repulsion on approach and no or insignificant adhesion on separation. The (close to) lack of adhesion between the PSGL-l/mIgG₂b layers suggests a more complete coverage than was achieved for BSM. The hysteresis between trace and retrace force curves is small for PSGL-l/mIgG_2b layers, which suggests a rapid recovery of the initial conformation as the pressure is released. This, in turn, suggests that no new and long-lived PSGL-l/mIgG_2b -surface bonds are formed due to compression. This can be understood if one part of the PSGL-l/mIgG_2b molecule is strongly anchored to the surface, whereas the other part prefers contact with bulk solution.

[00378] Similar experiments are performed with physiological tear fluid to compare with PSGL-l/hIgG4 or PSGLl/mIgG2b.

EXAMPLE 10

[00379] Normal force-distance curves during compression and decompression were obtained. A comparison of normal forces during compression clearly demonstrated that a long-range attraction only exists between uncoated PMMA and PMMA surfaces. It also showed that the steric repulsion between PSGL-l/mIgG_2b layers is higher than that between BSM layers, which may be attributed to larger sensed mass and larger layer thickness of the PSGL-l/mIgG_2b layers.

[00380] A comparison of normal forces when the probe was withdrawn from the flat surface demonstrated a large pull-off force (adhesion, -3 mN/m) between PMMA and PMMA surfaces and it was reduced to around -0.2 mN/m by adsorption of BSM molecules, and no or insignificant attraction was observed between PSGL-l/mIgG₂b layers. Also, a significantly stronger steric repulsion between PSGL-l/mIgG_2b layers on retraction compared to that for BSM layers was observed. This is due to a more rapid recovery of the layer structure after compression of the PSGL-l/mIgG_2b layers.

[00381] Similar experiments are performed with physiological tear fluid to compare with PSGL- l/hIgG4 or PSGLl/mIgG2b.

EXAMPLE 11

[00382] Friction force Ff vs. load, F_n and F R between two bare PMMA surfaces, BSM coated PMMA surfaces, and PSGL-l/mIgG_2b coated PMMA surfaces was measured across 155 mM NaCl. It was observed that the friction is very high between PMMA coated surfaces across 155 mM NaCl, significantly smaller between BSM coated PMMA, and even smaller between PSGL- l/mIgG_2b coated PMMA surfaces.

[00383] The friction coefficient between PMMA surfaces in 155 mM NaCl is very high, μ=1.2 was obtained up to a load of 22.1 nN, corresponding to a pressure of 0.5 MPa. The friction coefficient was found to be around 0.7 for PMMA coated with BSM as measured up to the maximum load of 68.9 nN (pressure= 1.8 MPa). The friction versus load curve for PSGL- l/mIgG_2b nd PSGL- l/mIgG_2b is not linear with applied load, so the friction coefficients were calculated for different load regions. When the load was smaller than 1 1.6 nN (corresponding to a pressure of 1.0 MPa), a very small friction coefficient was obtained, about 0.06. The friction coefficient increased to 0.1 when the load was between 1 1.6 nN and 23.2 nN (1.3 MPa); and at even higher loads, up to 50.2 nN (1.6 MPa), another Amnotons' law like behavior was recovered and here μ= 0.37.

[00384] To compare with the load applied to a contact lens while blinking, the intraocular pressure (IOP) is applied to Hertz model. IOP is the tissue pressure of the intraocular contents and its normal range is 10-20 mmHg (1333.22 Pa to 2666.45 Pa) and it is maintained at this level throughout life. See Murgatroyd et al. , Intraocular pressure.

Continuing Education in Anaesthesia, Critical Care & Pain 2008, 8 (3), 100-103. The loads calculated from IOP are smaller than 1 nN using the contact area between mucin layers and PSGL-l/mIgG_2b layers at the maximum applied loads in experiments. Hence, the actual load on the contact lens should be smaller than 1 1.6 nN, which also means that the friction coefficient of 0.06 is relevant in a contact lens application.

[00385] At pressures below 1 MPa the friction coefficient for PMMA coated with PSGL- l/mIgG_2b is 0.06, for PMMA coated with BSM 0.7 and for bare PMMA surfaces 1.7. Thus, PSGL- l/mIgG_2b layers provide superior lubrication in aqueous environment compared to BSM. 14 000866

[00386] Similar experiments are performed with physiological tear fluid to compare with PSGL-l/hIgG4 or PSGLl/mIgG2b.

[00387] The foregoing detailed specification has been given for the purpose of explaining and illustrating the invention. It is to be understood that the invention is not limited to detailed information set forth, and that various modifications can be made. It is intended to cover such modifications and changes as would occur to one skilled in the art, as the following claims permit and consistent with the state of the prior art.

EXAMPLE 12

Recombinant mucin, PSGL-l/mIgG_2b

[00388] Substrates. Poly(methylmethacrylate), PMMA, coated gold sensors (AT cut quartz crystals) with a diameter of 14 mm, QSX 999 (Q-sense, Vastra Frolunda, Sweden), having a nominal resonance frequency of about 5 MHz, were used as the substrate for all experiments. The PMMA layer was spin-coated on the gold crystal surface and had a thickness of around 40 nm. The crystals were cleaned by rinsing with Milli-Q water and dried with a gentle flow of nitrogen gas before use. The water contact angle on the PMMA surface was determined to be 68°, as measured with a DataPhysics OCA40 micro (DataPhysics GmbH, Germany) instrument at 23 °C ± 0.5 °C and a humidity of 44%.

Quartz Crystal Microbalance with Dissipation (QCM-D)

[00389] A Q-sense E4 device (Q-sense, Sweden) was employed for studying adsorption of mucins on PMMA surfaces. This device has the capacity to continuously measure the change in frequency and dissipation at the fundamental frequency as well as at six overtone frequencies (15, 25, 35, 45, 55, 65 MHz). The frequency change observed during adsorption (Δ/) depended on the total mass added to the crystal, including solvent coupled to the adsorbed layer. Provided the adsorbed layer was thin, rigid and homogeneous, the sensed mass was directly proportional to the frequency change according to the Sauerbrey equation. However, in many cases the adsorbed layer was viscoelastic, and this required more elaborate analysis models. The QCM-D device also measured dissipation changes (ΔΖ⁾), which were energy losses in the adsorbed film. This allowed a more accurate estimation of the sensed mass by using a viscoelastic model to analyze changes in both frequency and dissipation for several overtones, e.g. using the Voigt representation, which treated the viscoelastic response of the layer as that of a spring and a dashpot coupled in parallel. The extended viscoelastic model was used in this study. PC17IB2014/000866

[00390] The extended viscoelastic model takes into account the frequency dependence of the viscoelastic properties of the adsorbed layer, and frequency and dissipation data from the 3^rd, 5^th, and 7^th overtones in the analysis were utilized.

Adsorption procedure

[00391] All QCM-D experiments were started by obtaining a stable baseline in 155 mM NaCl solution. In the sequential adsorption experiments, 100 ppm BSM or PSGL- l/mIgG₂b was injected into the cell. After the adsorption reached equilibrium, the system was rinsed by pumping 155 mM NaCl solution into the cell for 20 minutes. Next, the solution containing the other mucin type was injected into the cell and the response was followed until equilibrium was obtained. The surface was subsequently rinsed with 155 mM NaCl. In the stepwise adsorption experiments the surface was exposed to mucin solutions of increasing concentration (in 155 mM NaCl), and then the surface was rinsed with protein-free 155 mM NaCl. All experiments were performed at 25±0.2 °C.

Atomic Force Microscope (AFM) with Colloidal Probe

[003 2] N_anosc0p_e Multimode III Pico Force AFM (Veeco Instruments Inc.) was employed for force and friction measurements performed in a fused silica liquid cell (volume ~ 0.1 mL). Rectangular tipless cantilevers (MikroMasch, CSC12/tipless/Cr-Au) with the approximate dimensions of 250 μιη in length, 35 μπι in width, and normal spring constants in the range 0.02 - 0.2 N/m were chosen for all force and friction measurements. The exact values of the normal (k/S) and the torsional (kq>) spring constants were determined using the AFM Tune IT v2.5 software (Force IT, Sweden) adopting the method based on thermal noise with hydrodynamic damping. A spherical PMMA particle (Kisker,cat.#ppmma-10.0) with a diameter of approximately 10 μηι was attached to the end of the cantilever with the aid of an Ependorf Micromanipulator 5171 , a Nikon Optiphot 100S reflection microscope, and a small amount of epoxy glue (Araldite, 80806) after determining the spring constants of the cantilever. The lateral photodetector sensitivity (<5, V/rad) was calibrated using the method of tilting the AFM head proposed by Pettersson et al.

[00393] The normal force curves were measured with a constant approach and retraction speed of 1 μηι/s. In each case at least 10 curves were recorded. The force curves were then analyzed with the AFM Force IT software (Force IT). The deflection sensitivity obtained for the bare PMMA-PMMA system was used in all cases when defining the constant compliance region and generating force curves in the presence of adsorbed mucin layers. The reproducibility of the forces measured between mucin-coated surfaces was found to be good. The force curves presented in the result section were selected as one representative curve. Friction forces were measured by capturing "contact mode images" using a scanning angle perpendicular to the cantilever at different applied loads at a sliding speed of 4 μηι/s with a scan size of 2 μ^ηι. The frictional force values were recorded at the onset of the steric repulsion up to the hard wall region of the system and again on decreasing the load. The friction traces obtained were analyzed by employing the AFM Friction IT software (Friction IT).

Experimental procedure of normal and frictional forces measurements

[00394] Before each experiment, the fused silica cell and all other tools were cleaned by immersion in 2% Hellmanex (Hellma GmbH) solution for 1 hour and then rinsed excessively with Milli-Q water. They were then rinsed with ethanol before being dried with a stream of filtered nitrogen gas. The AFM experiments were started by measuring the normal forces between the PMMA surface and the PMMA colloidal probe across a 155 mM NaCl solution. This was followed by friction measurements. Next, a 100 ppm solution of mucin (BSM or PSGL-l/mIgG_2b) in 155 mM NaCl was introduced into the fused silica cell and the polymer was allowed to adsorb for 45 minutes before measuring the surface forces again. After the force curves were recorded, friction measurement was performed, and then followed again by force measurements. This was done to check the stability of the detector signal at zero load and to study if the friction measurements affected the mucin layers. After that, 155 mM NaCl solution was injected into the fused cell to replace the mucin solution. The same procedure (force - friction - force measurement) was repeated. All measurements were carried out at 25 °C.

Adsorption of mucins on PMMA

[00395] Stepwise adsorption experiments were performed by injecting mucin solutions of increasing concentrations into the QCM cell, allowing the adsorption to proceed for 45 minutes at each concentration. The Voigt mass, i.e. the mass of the adsorbed mucin and water associated with the layer, after each adsorption step is provided in Figure 22. This data set should not be regarded as reflecting the adsorption isotherm since adsorption of mucin from dilute solutions occurs very slowly, and within the time frame of 45 minutes only the data point obtained at 100 ppm mucin concentration can be regarded as reflecting the equilibrium situation (as judged from the absence of further changes in frequency and dissipation at the end of the adsorption process). The Voigt mass of the PSGL-l/mIgG_2b layer was significantly larger than that of the BSM layer. Sequential adsorption of BSM and PSGL-l/mIgG_2b

[00396] Two sequential adsorption experiments were performed to characterize the adsorption of BSM and PSGL-l/mIgG_2b to PMMA. Figure 23a shows the data obtained when first adsorbing BSM to PMMA and then allowing PSGL-l/mIgG_2b to interact with the BSM- coated surface.

[00397] The QCM-D response recorded during adsorption of BSM from 100 ppm solution was found to be quite complex. Initially a rapid decrease in frequency and increase in dissipation is observed as mass is added to the sensor surface during formation of a viscoelastic mucin layer. However, after about 10 minutes the response of the sensor reversed and now the frequency slowly increased and the dissipation decreased. This indicated structural rearrangements leading to a less extended layer structure. The adsorption reached equilibrium within 2 hours and only a limited amount of BSM was removed by rinsing. The frequency change resulting from the subsequent adsorption of PSGL-l/mIgG₂b was around - 25 Hz, and the dissipation of the composite BSM/PSGL-l/mIgG_2b layer was higher than that of the initial BSM layer. Clearly, PSGL- l/mIgG_2b can associate with the preadsorbed BSM layer, which increases the layer thickness (Table 5). It is also conceivable that some adsorbed BSM molecules are replaced by PSGL-l/mIgG_2b, even though this process requires penetration of the dimeric PSGL-l/mIgG_2b through the pre-adsorbed BSM layer.

[00398] The QCM-D response due to adsorption of PSGL-l/mIgG_2b on PMMA was less complex than that observed during BSM adsorption. In this case the frequency decreased and dissipation increased monotonically with time until equilibrium was reached as shown in Figure 23b. Exposure of this PSGL-l/mIgG_2b layer to a BSM solution only resulted in minor changes in frequency and dissipation. Clearly, BSM could not associate with the preadsorbed PSGL- l/mIgG_2b layer (Figure 23b) whereas PSGL-l/mIgG_2b did not interact with

preadsorbed layers of BSM (Figure 23a). This shows that the region of the PSGL- l/mIgG_2b molecule that associates with BSM is buried within the preadsorbed PSGL-l/mIgG_2b layer, and it is the IgG Fc part of PSGL-l/mIgG_2b that provides both the anchoring to PMMA and facilitates association with preadsorbed BSM, presumably via the non-glycosylated regions of BSM.

[00399] The Voigt mass of the two mucin layers formed from 100 ppm solutions is shown in Table 5. It was striking that the Voigt mass and Voigt thickness of the PSGL- l/mIgG_2b layer was more than 2 times higher than those of the BSM layer. The Voigt mass of the BSM layer decreased after rinsing, which could be explained by a limited desorption. In contrast, the Voigt mass increased slightly for the PSGL-l/mIgG₂b layer after rinsing, which 17IB2014/000866 was suggested to be due to the pH difference of the mucin solution (pH 4.8) and the buffer solution (pH 5.8) that resulted in higher charge of PSGL-l/mIgG_2b and increased the mass of water associated with the layer. The modeling result for the sequential adsorption

experiments are summarized in Error! Reference source not found.5. The standard deviation was calculated from three different experiments.

Adsorption of IgG-Fc to PMMA

[00400] To better understand the adsorption of PSGL- 1 /mIgG_2b to PMMA, experiments were also carried out with the IgG-Fc part of PSGL-l/mIgG₂b, and the results are shown in Figure 24.

[00401] IgG-Fc adsorption on PMMA resulted in a Voigt mass of about 6 mg/m² at a concentration of 25 ppm (almost 3 hours was required to reach equilibrium), increasing slightly to 6.5 mg/m² at 50 and 100 ppm IgG-Fc concentration. The corresponding Voigt layer thickness was around 5 nm. The weak dependence of the sensed mass on IgG-Fc bulk concentration was a sign of high surface affinity. Thus, the IgG-Fc fragment of PSGL- l/mIgG_2b mucin contributed significantly to the anchoring of this mucin to PMMA.

AD vs. A plots

[00402] The AD - Af plots for adsorption of the two mucins and IgG-Fc on PMMA are shown in Figure 25. This type of plot can shed light on structural transitions occurring as the adsorption proceeds. The AD - Af curve for IgG-Fc is rather featureless and display a linear relation between AD and Af. In contrast, the AD -Af curve for BSM consist of two regions, a first linear region up to 30 Hz and a second region with decreasing dissipation. This suggests that after the initial adsorption the adsorbed BSM molecules slowly change their

conformation to form a thinner layer to maximize the favorable interaction with the surface. The relatively small thickness of the BSM layer, around 7 nm, suggests that most molecules are oriented parallel to the surface.

[00403] The AD - Af curve for PSGL-l/mIgG_2b mucin was reminiscent of what has been observed for some synthetic bottle-brush polymers. The first linear region (up to 30 Hz) suggested that initially most PSGL-l/mIgG_2b molecules interacted with the PMMA surface in a similar way as BSM, i.e. with the chains mainly parallel to the surface. The decreasing slope in the second region (30 to 45 Hz) meant that the energy dissipated per unit sensed mass decreases, which signify that the layer became stiffer due to increased interactions between adsorbed polymer chains. The increasing slope in the last region (> 45 Hz) suggested a structural change towards a more extended layer conformation. 4 000866

Normal and friction forces - Normal forces between PMMA surfaces

[00404] The forces acting between two PMMA surfaces across 155 mM NaCl solution are shown in Figure 26. They were purely attractive except for the hard wall repulsion defining zero separation.

[00405] On approach, the attractive force was noted once the separation had decreased to 40-50 nm, and it gave rise of an adhesion with a magnitude of around 3 mN/m. On separation, the attraction persisted until the separation had increased to around 200 nm. The range of the attraction was larger than expected for a van der Waals force, suggesting that it originated from some PMMA polymer chains bridging between the surfaces, giving rise to a bridging attraction. The exact range of the attraction and the adhesion (2 to 6 mN/m) varied somewhat between measurements, which could be expected if bridging by a few polymer chains is the cause. The absence of any measurable electrostatic double-layer force did not mean that the surfaces must be uncharged, but it was a consequence of the high ionic strength (155 mM) that reduced the Debye-screening length to 0.8 nm and thus the range of any possible double-layer force accordingly.

Normal forces between mucin-coated PMMA

[00406] The reproducibility of the forces measured between BSM- and PSGL- l/mIgG₂b-coated surfaces was found to be good. The force curves were selected as one representative curve. The force curves measured after allowing BSM adsorption on the PMMA probe and PMMA surface for 45 minutes are shown in Figure 27a.

[00407] The forces experienced on approach are purely repulsive and of steric origin, i.e. due to compression of the BSM layer. On separation, a small but long range attractive force is observed, which was assigned to polymer bridging. The magnitude of the adhesion was reduced by about a factor of 10 compared to that observed before BSM adsorption. The presence of weak bridging attraction indicated that the surfaces were not fully covered by the adsorbed BSM molecules.

[00408] The normal force curves recorded between PSGL-l/mIgG₂b-coated PMMA are shown in Figure 27b. A strong steric repulsion was observed on approach and no or insignificant adhesion was noted on separation. The (close to) lack of adhesion between the PSGL-l/mIgG₂b layers suggested a more complete coverage than that achieved for BSM, which was consistent with the QCM-D results. The hysteresis between trace and retrace force curves was small for PSGL-l/mIgG _b layers, indicating rapid recovery of the initial conformation as the force is released. This, in turn, suggested that no new and long-lived PSGL-l/mIgG₂b - surface bonds were formed due to compression. This can be understood if 6 one part of the PSGL-l/mIgG_2b molecule (the IgG-Fc part) is strongly anchored to the surface, whereas the other part (the glycosylated bottle-brush part) prefers contact with the bulk solution. This demonstrated that the adsorbed mucin molecules were not removed by the combined action of shear and load.

[00409] The forces measured on approach between BSM-coated and PSGL- l/mIgG₂t,- coated PMMA surfaces are compared in Figure 28, using a logarithmic force scale.

[00410] An exponential decay of the steric repulsion was observed at large separations as expected in the dilute tail region, where the repulsion mainly originates from the increase in osmotic pressure caused by the higher segment density between the surfaces compared to in bulk solution. At further compression a more rapid increase in steric repulsion was expected and observed due to compression of the adsorbed polymer chains. A direct comparison of the magnitude of the steric repulsion in the two cases was not warranted since AFM colloidal probe force measurements, unlike measurements with the SFA, did not provide direct information on the adsorbed layer thickness. However, from the QCM-D measurements it was concluded that the PSGL-l/mIgG_2b layer was significantly thicker than the BSM layer.

Friction forces

[00411] The friction force measured between PMMA surfaces across 155 mM NaCl were high, significantly smaller between BSM-coated PMMA, and even smaller between PSGL-l/mIgG_2b-coated PMMA, as illustrated in Figure 21 a.

[00412 The friction vs. load relationship can often be analyzed using the relation:

where C = S_CA, with Sc being the critical shear stress and A the contact area. The quantity C/μ is sometimes referred to as the dynamic adhesion as it accounts for the adhesion between the surfaces during sliding motion, and μ is the friction coefficient. Since only a significant adhesion between the bare PMMA surfaces was observed, the critical shear stress had a non-zero value in only this case. The friction force and the friction coefficient between PMMA surfaces in 155 mM NaCl was very high. The friction coefficient, μ, equals 1.2 up to the highest load was investigated. Thus the friction force law (in nN) for the uncoated PMMA surfaces can be written as:

F_f = 50 ₊ l .2F_{n m}

[00413] For BSM-coated PMMA surface the critical shear stress, and thus the dynamic adhesion, was close to zero and the classical Amontons' rule Ff = μ-Ρ_η was recovered. The friction coefficient was found to be close to 0.7 up to the maximum load of 69 nN used in the experiment. Thus, in this case the friction force law is:

^Ff - ^{0 F}n _[3]

[00414] The friction versus load curve for PSGL-l/mIgG_2b layers on PMMA was not linear with applied load (see Figure 21 a), and in this case it is appropriate to define an effective friction coefficient, μ^, as: a - ^Ff

[4]

[00415] The effective friction coefficient as a function of load for PSGL-l/mIgG_2b- coated PMMA is shown in Figure 21b. The effective friction coefficient was low; increasing from 0.02 at low loads to 0.24 at the highest load explored, 50 nN. Thus, it is clear PSGL- l/mIgG_2b layers provide superior lubrication of PMMA surfaces in aqueous environment compared to BSM.

[00416] When discussing friction data it is sometimes better to report contact pressure than load, since the pressure values can be compared between experiments using probes with different radius and materials with different Young's moduli. The mean pressure that corresponds to a given load using JKR theory was calculated for bare PMMA surfaces where the adhesion contribution is significant, and the Hertz model for PMMA surfaces coated with mucin layers where adhesion effects can be ignored. The Young's modulus, 5 GPa, and Poisson's ratio, 0.38, for PMMA were used in these calculations, and the maximum force applied to the mucin-coated surfaces correspond to a pressure of 8-9 MPa.

INCORPORATION BY REFERENCE

[00417] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes. In the present disclosure the host document is identified with sufficient particularity and materials that are relevant to the disclosure is construed based on the context of the reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The invention having now been described by way of written description, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and the foregoing description and examples are for purposes of illustration and not limitation of the claims that follow. EQUIVALENTS

[00418] The invention can be embodies in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMS What is claimed is:

1. A device comprising a polymer surface coated with a fusion protein comprising a first polypeptide covalently conjugated to a second polypeptide, wherein the first polypeptide is a negatively charged polypeptide and the second polypeptide is an immunoglobulin

polypeptide or a fragment thereof, wherein the polymer surface is semi-rigid or soft, suitable for custom fitting with the curvature of an ocular surface.

2. The device of claim 1, wherein the first polypeptide is a mucin polypeptide or a fragment thereof.

3. The device of claim 2, wherein said mucin polypeptide comprises at least a region of a P-selectin glycoprotein ligand-1 (PSGL-1).

4. The device of claim 3, wherein said mucin polypeptide includes an extracellular portion of a P-selectin glycoprotein ligand-1.

5. The device of claim 2, wherein the mucin polypeptide is selected from the group consisting of PSGL-1, CD34, CD43, CD45, CD96, GlyCAM-1 , MAdCAM-1, and fragment thereof.

6. The device of claim 2, wherein the mucin polypeptide is a secreted mucin or a membrane associated mucin.

7. The device of claim 6, wherein the secreted mucin is MUC2, MUC5AC, MUC5B, MUC6, MUC7, or MUC9.

8. The device of claim 6, wherein the membrane associated mucin is MUC1 , MUC3A, MUC3B, MUC4, or MUC16.

9. The device of claim 2, wherein the mucin polypeptide is sialylated.

10. The device of claim 1 , wherein the immunoglobulin polypeptide comprises a region of a heavy chain immunoglobulin polypeptide.

1 1. The device of claim 10, wherein the immunoglobulin polypeptide comprises an Fc region of an immunoglobulin heavy chain.

12. The device of claim 1 , wherein the device is an ophthalmic device.

13. The device of claim 1 , wherein the fusion protein increases wettability of the polymer surface.

14. The device of claim 1, wherein the polymer is selected from the group consisting of unplasticized polyvinyl chloride (UPVC), polycarbonate (PC), polyethylene terephthalate (PET), polystyrene (PS), poly(methylmethacrylate) (PMMA), polytetrafluoroethylene (PTEE), silicone, silicone hydrogel, hybrid, gas permeable, hydrogel, and derivatives thereof.

15. The device of claim 1, wherein the polymer surface is coated with said fusion protein to form an inner layer, an outer layer, or both.

16. The device of claim 1, wherein the fusion protein is PSGL-l/mIgG2b.

17. The device of claim 1 , wherein the fusion protein is PSGL-l/hIgG4.

18. The device of claim 9, wherein the mucin polypeptide is mono or disialylated core 1 and/or core 2 polypeptide.

19. The device of claim 16, wherein the PSGL-l/mIgG2b is glycosylated with mono and disialylated core 1 and/or core 2 structures.

20. The device of claim 17, wherein the PSGL-l/hIgG4 is glycosylated with mono and disialylated core 1 and/or core 2 structures.