WO2014089267A1 - Galectin-3 fusion proteins - Google Patents

Abstract

Isolated proteins comprising a galectin 3 carbohydrate binding domain and a therapeutic agent or diagnostic agent are disclosed. Also disclosed are compositions comprising the proteins and methods of using them, e.g., to provide increased residence time, e.g., in the eye, of a therapeutic agent or diagnostic agent.

Description

GALECTIN-3 FUSION PROTEINS

RELATED APPLICATIONS

This application claims priority to U.S. Application No. 61/733,642, which was filed on December 5, 2012, and to U.S. Application No. 61/819,047, which was filed on May 3, 2013. The entire content of each of the foregoing applications is hereby incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 4, 2013, is named D2046-7046WO_SL.txt and is 51,328 bytes in size.

FIELD OF THE INVENTION

The present disclosure relates to compositions and methods involving the use of a galectin-3 (Gal3) protein for extending the surface residence time and/or enhancing the potency of therapeutic and diagnostic agents.

BACKGROUND

One challenge for treating eye disease is achieving and maintaining a therapeutic amount of drug at the site of drug action for a suitable amount of time. One of the key limiting parameters in the penetration and effectiveness of topically applied ocular therapeutics is their mean residence time. In humans, most of an instilled solution clears through drainage, with first-order clearance about four times that of bulk tear flow (about 5 μΐ/minute). For protein drugs, the problem becomes more acute due to the additional burden of reduced penetrance through the cornea and conjunctiva due to their large size (e.g., compared to a small molecule drug). Various approaches for increasing the residence time of a drug on the surface of the eye have included increasing the viscosity of a formulation, receptor targeting (e.g., using transferrin conjugated liposomes), and mucoadhesives (e.g., polycarbophil, hydroxyethylcellulose, xanthan gum, and gellan gum). SUMMARY

Disclosed herein are compositions and methods related to the use of a galectin-3 protein to extend the residence time (e.g., the surface residence time), and/or increase the potency, of therapeutic agents (e.g., proteins) and diagnostic agents, e.g., in organs or tissues that express glycoprotein (e.g., MUC-1 and/or asialofetuin). For example, as described herein, a Gal3 protein (a protein that comprises at least one Gal3 carbohydrate binding domain (CBD) can extend the surface residence time of proteins around the ocular surface, e.g., when administered to the eye, e.g., when topically applied to the eye. Furthermore, as described herein, a Gal3 protein (a protein that comprises at least one Gal3 carbohydrate binding domain (CBD) can increase the potency of various agents, e.g., agents that modulate cytokine activity (e.g., cytokine agonists or antagonists).

Described herein is an isolated protein (e.g., an isolated fusion protein) that includes (i) a Gal3 protein (a protein that comprises at least one Gal3 carbohydrate binding domain (CBD)), and (ii) a therapeutic and/or diagnostic agent. In some embodiments, the Gal3 protein comprises multiple CBDs, e.g., two CBDs, three CBDs, four CBDs, or five CBDs. CBDs can be attached to each other and/or to the therapeutic and/or diagnostic agent by any means described herein or known in the art, e.g., via a covalent bond, disulfide bond, and/or linker sequence. In some cases, the Gal3 protein contains the amino acid sequence of a full length native Gal3 (e.g., a full-length human Gal3).

Also provided herein is a pharmaceutical formulation comprising the isolated protein.

Also provided herein is a method of increasing the residence time (as assessed, e.g., based on the half life or any other pharmacokinetic (PK) parameter) of a diagnostic or therapeutic agent, the method comprising associating (e.g., fusing) the diagnostic or therapeutic agent with a Gal3 protein.

Also provided herein is a method of improving the potency of a diagnostic or therapeutic agent, the method comprising associating (e.g., fusing) the diagnostic or therapeutic agent with a Gal3 protein. The diagnostic or therapeutic agent can be associated with the Gal3 protein by any means described herein or known in the art.

The Gal3 protein can be associated (e.g., connected or fused) with the therapeutic or diagnostic agent by any means known in the art, e.g., by a covalent bond such as a peptide bond or disulfide bond. In embodiments, the Gal3 protein is attached to the therapeutic or diagnostic agent via a linker, e.g., a linker as described herein or known in the art. In some embodiments, the Gal3 protein-linker-therapeutic or diagnostic agent are produced as a recombinant protein. The therapeutic agent can be any therapeutic agent known in the art. For example, the therapeutic agent can be a protein, a polypeptide, or a peptide. Alternatively, the therapeutic agent can be a small molecule. The therapeutic agent can be, e.g., an agent that is useful for treating a disease or disorder that affects the eye.

The therapeutic agent can modulate a target of interest, e.g., a cytokine. For example, the therapeutic agent can be a cytokine antagonist or a cytokine agonist. In some embodiments, the therapeutic agent is a modulator (e.g., an antagonist) of IL-1, IL-6, IL-17, or IL-23. In enbodiments, the therapeutic agent is a modulator (e.g., an antagonist) of IL-1, IL-2, IL-6, IL-17, IL-23, tumor necrosis factor (TNF), or VEGF. In some embodiments, the therapeutic agent is an IL-Ιβ, an IL-IRa, a heterologous molecule comprising fragments of an Il-lRa and an II- 1β, or an ST2. In embodiments, the therapeutic agent is a chimeric cytokine described in International Application No. PCT/US2011/045995. In embodiments, the therapeutic agent is an IL-6 antagonist disclosed in International Application No. PCT/US2013/069279.

In embodiments, the residence time (as assessed, e.g., based on the half life or any other pharmacokinetic parameter) of the diagnostic or therapeutic agent in a particular organ, tissue, or cell (e.g., in the eye or portion of the eye such as the ocular surface, vitreous, or retina) is increased. The organ, tissue, or cell can be any organ, tissue, or cell that expresses a glycoprotein to which the Gal3 protein (or a Gal3 CBD contained within the protein) binds.

In embodiments, the residence time of the diagnostic or therapeutic agent is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared with the corresponding diagnostic or therapeutic agent when it is not associated with the Gal3 protein. In embodiments, the residence time of the diagnostic or therapeutic agent is increased by at least 2-fold (e.g., by 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, or greater than 50-fold) compared with the corresponding diagnostic or therapeutic agent when it is not associated with the Gal3 protein.

In embodiments, the potency of the therapeutic agent is enhanced, relative to a therapeutic agent that is not associated with the Gal3 protein. In embodiments, the potency is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared with the corresponding diagnostic or therapeutic agent when it is not associated with the Gal3 protein. In embodiments, the potency of the diagnostic or therapeutic agent is increased by at least 2-fold (e.g., by 2-fold, 3-fold, 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, or greater than 50-fold) compared with the corresponding diagnostic or therapeutic agent when it is not associated with the Gal3 protein.

The improvement in potency can be an improvement in the modulatory (e.g., agonist or antagonist) activity of a therapeutic agent, e.g., a therapeutic agent described herein. In embodiments, the therapeutic agent inhibits cytokine activity. In embodiments, the therapeutic agent enhances cytokine activity. In embodiments, the therapeutic agent is 93:60, as described herein and in International Application No. PCT/US2011/045995. In embodiments, the therapeutic agent comprises or consists of amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of 93:60. In embodiments, the therapeutic agent is IL-lRa. In embodiments, the therapeutic agent comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of IL-lRa. In embodiments, the therapeutic agent is anakinra. In embodiments, the therapeutic agent is IL-Ιβ.Ιη embodiments, the therapeutic agent comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of IL-Ιβ.

In embodiments, the potency of the therapeutic agent is assessed based on its activity (e.g., its agonist or antagonist effect on a target, target pathway, or component of a target pathway). Activity may be assessed based on any test suitable for assessing the effect of a therapeutic agent on a target, target pathway, or component of a target pathway. In embodiments, the activity of an IL-1 modulator is assessed using the HEKBlue IL-Ιβ reporter cell-based assay, e.g., as described herein in the examples.

In embodiments, the potency (e.g., inhibitory activity) of the therapeutic agent is assessed based on its IC₅₀.

In embodiments, the potency of the therapeutic agent is assessed based on avidity for a target (e.g., a target cytokine receptor). In embodiments, the potency of the therapeutic agent is assessed based on affinity for a target (e.g., a target cytokine receptor).

In embodiments, the Gal3 carbohydrate binding domain comprises or consists of SEQ ID NO: 2 or SEQ ID NO: 4. In embodiments, the Gal3 carbohydrate binding domain is at least 85%, 86%, 87, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:2 or SEQ ID NO: 4.

In some embodiments, a diagnostic agent is included in the isolated protein. The diagnostic agent can be any diagnostic agent known in the art. In embodiments, the diagnostic agent is a fluorescent protein, for example, a green fluorescent protein, or a protein that can react to produce a fluorescent signal, e.g., a luciferase such as Gaussia luciferase. In an aspect provided herein is a pharmaceutical formulation for administration to the eye, the formulation comprising protein as described herein (e.g., an isolated protein comprising (i) a Gal3 protein (a protein that comprises at least one Gal3 carbohydrate binding domain (CBD)), and (ii) a therapeutic and/or diagnostic agent). In embodiments, the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier and/or an excipient.

In embodiments, the pharmaceutical formulation is suitable for topical administration to the eye (e.g., to the ocular surface or surrounding tissue). In embodiments, the pharmaceutical formulation is administered topically.

In embodiments, the pharmaceutical formulation is suitable for intravitreal

administration. In embodiments, the pharmaceutical formulation is administered intravitreally.

In embodiments, the pharmaceutical formulation is for use in the treatment of an eye disease or condition. In embodiments, the eye disease or condition is dry eye disease, Meibomian gland disorder, keratitis, episcleritis, conjunctivitis (e.g., allergic conjunctivitis,

keratoconjunctivitis sicca, or vernal keratoconjunctivitis), keratitis sicca, sicca syndrome, xerophthalmia, tear film disorder, decreased tear production, aqueous tear deficiency, dysfunctional tear syndrome, Meibomian gland dysfunction, dry eye associated with another condition (e.g., Sjogrens syndrome, graft versus host disease, or an autoimmune condition such as mucous membrane pemphigoid), blepharitis , a laser eye surgery or a corneal transplant.

Also provided herein is a method of treating a disease, disorder, or condition, the method comprising administering an isolated protein or pharmaceutical formulation described herein, thereby treating said disease, disorder, or condition. In embodiments, the disease, disorder or condition affects the eye. In embodiments, the method is a method of treating an eye disease or condition, e.g., dry eye disease, Meibomian gland disorder, keratitis, episcleritis, conjunctivitis (e.g., allergic conjunctivitis, keratoconjunctivitis sicca, or vernal keratoconjunctivitis), keratitis sicca, sicca syndrome, xerophthalmia, tear film disorder, decreased tear production, aqueous tear deficiency, dysfunctional tear syndrome, Meibomian gland dysfunction, dry eye associated with another condition (e.g., Sjogrens syndrome, graft versus host disease, or an autoimmune condition such as mucous membrane pemphigoid), blepharitis , a laser eye surgery or a corneal transplant.

Another aspect provided herein is a method of providing a therapeutic agent to a subject in need of treatment by administering a therapeutically effective amount of a protein described herein, e.g., to the surface of the eye.

Another aspect provided herein is a vector comprising a nucleic acid sequence encoding a protein described herein. Also provided herein is a cell comprising the vector. The present application discloses patents, scientific articles, and other publications. The contents of each such item is hereby incorporated by reference. In addition, the contents (as of the filing date of the application) of all websites referred to herein are incorporated by reference. DRAWINGS

Fig. 1 is a representation of the constructs comprising Gal3 CBDs. "(G₄S)₂" is disclosed as SEQ ID NO: 16 and "8 His" is disclosed as SEQ ID NO: 13.

Fig. 2 depicts data from experiments testing the in vivo retention of Gal3 fusions in murine eyes.

Fig. 3 is a graph depicting the results of an experiment in which the ability to inhibit IL-1 agonist activity was assayed for a known inhibitor (93:60) and a 2XGal3-His₈ composition ("His₈" disclosed as SEQ ID NO: 13).

Fig. 4 is a graph depicting the results of an experiment in which the ability to inhibit IL-1 agonist activity was assayed for 93:60 (squares), 93:60-2XGal3 (diamonds), and TAT -93:60 (triangles) at varying concentrations.

Fig. 5 is a graph depicting the results of an experiment in which plates were coated with micin-1, asialofetuin, IL-1R1 or an unglycosylated protein and tested for binding to a reporter protein containing Gal3 CBDs (Luc2XGal3).

Fig. 6 is a graph depicting the results of an experiment in which IL-IRa and IL-IRa linked to two copies of Gal3 CBD and His tagged (IL- lRa-2XGal3H8 ("H8" disclosed as SEQ ID NO: 13)) were tested for inhibitory activity in the HEK-Blue IL-Ιβ system using various concentrations ranging from lxlO^"15M to lxl0^"8M.

Fig. 7 is a graph depicting the results of an experiment in which IL-1 β and IL-1 β linked to two copies of Gal3 CBD were tested in the HEK-Blue IL-1 β system. "8H" is disclosed as SEQ ID NO: 13.

Fig. 8 is a graph depicting the results of an experiment testing retinal retention of intravitreally injected 93:60 and 93:60-2XGal3H8 ("H8" disclosed as SEQ ID NO: 13).

DETAILED DESCRIPTION

Short residence time limits the penetration and effectiveness of topical ocular therapeutics. Approaches for increasing pre-corneal residence time have included increasing formulation viscosity, receptor targeting, and use of mucoadhesives. Applicants have discovered that a galectin-3 (Gal3; also referred to as, for example, galactose-specific lectin 3, galactose- binding protein (GALBP), IgE binding protein, carbohydrate binding protein-35 (CBP-35), Mac- 2 antigen, 35 kDa lectin, L-31, laminin binding protein, and lectin L-29), a relatively small polypeptide that can be present as a pentameric ocular surface resident protein that cross-links O- type mucins, can be engineered to create fusion proteins with increased residence time. In particular, applicants have discovered that a protein containing at least one carbohydrate binding domain derived from a full-length galectin-3 is useful for creating fusion proteins with improved properties such as increased residence time at a delivery site or increased potency.

The surface of the eye includes a glycoprotein layer termed the glycocalyx, comprised of mucins and cross-linking proteins that are reported to increase the structural stability of the mucins and decrease the ability of large entities such as microorganisms or particles to reach the ocular surface. Full length native Gal3 is a protein that has been described to function as a pentamer to crosslink integral membrane proteins and has micromolar monovalent binding affinity for lactose. Human galectin-3 contains the following domains, signature sequences, or other structural features (for general information regarding PS and PF prefix identification numbers, refer to Sonnhammer et al., Protein 28:405, 1997): an N-terminal domain located at about amino acid residues 1 to 14 of SEQ ID NO: 1 ; a proline, glycine, and tyrosine -rich domain located at about amino acid residues 15 to 116 of SEQ ID NO: 1 ; a galactoside -binding domain located at about amino acid residues 117 to 247 of SEQ ID NO: 1 (also referred to as a carbohydrate binding domain or CBD); a galectin signature sequence (PROSITE No. PS51304; galactoside-binding lectin domain) located at about amino acids 181 to 200 of SEQ ID NO: l ; a potential N-glycosylation site (PROSITE No. PS00001) located at about amino acids 4 to 7 of SEQ ID NO: l ; two potential protein kinase C phosphorylation sites (PROSITE No. PS00005) located at about amino acids 137 to 139 and 194 to 196 of SEQ ID NO: 1 ; two potential casein kinase II phosphorylation sites (PROSITE No. PS00006) located at about amino acids 6 to 9 and 175 to 178 of SEQ ID NO: l ; and eight potential myristoylation sites (PROSITE No. PS00008) located at about amino acids 24 to 29, 27 to 32, 34 to 39, 43 to 48, 52 to 57, 61 to 66, 65 to 70, and 68 to 73 of SEQ ID NO: l. The N-terminal domain is involved in dimerization.

Featured herein are therapeutic and diagnostic compositions that bind to O-type mucins, e.g., on the surface of the eye. The compositions comprise a Gal3 CBD fused to a heterologous therapeutic moiety, e.g., a biologic moiety or other pharmaceutical moiety; or a Gal3 CBD fused to a diagnostic agent such as a fluorescent protein, e.g., a green fluorescent protein. Typically, the composition is a recombinant protein that includes a Gal3 protein sequence and the sequence of a non-Gal3 polypeptide biologic (termed herein a "heterologous polypeptide"). In some cases, the composition includes a Gal3 sequence covalently linked (e.g., via a disulfide bond) to the therapeutic moiety (e.g., heterologous polypeptide) or diagnostic agent. Linkages to a non-protein therapeutic agent can be made, e.g., using a free cysteine in a CBD amino acid sequence or by conjugation to the C-terminus of an amino acid sequence containing a CBD (for example, see protein conjugation technology, Almac, Souderton, PA). A composition can include more than one Gal3 CBD sequence, typically arranged in tandem, and optionally with one or more linker sequences.

Unless the context indicates otherwise, as used herein, a "galectin-3," a "Gal3", a "native Gal3", a "full length Gal3," a "full length native Gal3" and the like, refer to a native Gal3 protein, e.g., a human Gal3 protein or a native Gal3 protein from another species. A native Gal3 protein may include variations (e.g., natural variations such as polymorphisms, variations due to RNA editing, variations associated with post-translational modifications or lack thereof, etc.)

As used herein, a "Gal3 protein" comprises at least one Gal3 CBD as described herein. The Gal3 protein may further comprise one or more additional Gal3 CBDs and/or additional amino acids or sequences from a full length Gal3 protein. In some embodiments, the Gal3 CBDs are connected via a linker, e.g., a linker as described herein.

As used herein, an "isolated protein" (e.g., a "fusion protein") includes a Gal3 protein that is associated with a diagnostic or therapeutic agent by any means described herein or known in the art. For example, the Gal3 protein may be connected with the diagnostic or therapeutic agent by a covalent bond (e.g., a peptide bond or disulfide bond). In some instances, the Gal3 protein is connected with the diagnostic or therapeutic agent via a linker, e.g., a linker as described herein. The isolated protein may include non-protein components. For example, in some instances, the diagnostic or therapeutic agent is an entity that is not a protein.

A "Gal3 composition" is a composition comprising an isolated protein as described herein (a Gal3 protein that is associated with a diagnostic or therapeutic agent (e.g., a

heterologous biologic therapeutic agent)). A biologic therapeutic agent includes an agent that is a protein, polypeptide, or peptide. In embodiments, the therapeutic agent is useful for treatment of a disease or disorder, e.g., for treatment of an eye disorder, an eye disease, or other condition that affects the eye. A Gal3 formulation optionally further comprises a pharmaceutically acceptable carrier and/or an excipient.

In embodiments, a therapeutic agent that is associated with a Gal3 protein as described herein has an enhanced potency (e.g., compared with an appropriate control, e.g., compared with the potency of the therapeutic agent when it is not associated with a Gal3 protein). Potency can be assessed using assays or tests known in the art or described herein. The term "potency" includes any relevant activity, affinity, avidity and/or therapeutic effect of the therapeutic agent. The activity of a therapeutic agent includes its functional activity, e.g., its modulatory activity, e.g., agonist or antagonist activity. In general, the activity is assessed with respect to the effect of the therapeutic agent on a target (e.g., a target receptor, signaling pathway, or component of a signaling pathway).

Carbohydrate Binding Domains

A Gal3 composition described herein typically contains at least a Gal3 protein carbohydrate binding domain (a Gal3 CBD), e.g., a human Gal3 CBD, and a non-Gal3 therapeutic or a diagnostic agent. Typically, the composition is a recombinant protein. In some embodiments the Gal3 CBD is derived from a human galectin 3.

In some embodiments, the recombinant protein can include a polypeptide fusion that includes at least a segment (e.g., a functional segment) of a therapeutic agent (e.g., a therapeutic agent that is a peptide, polypeptide, or protein) and a Gal3 CBD. The segment of the therapeutic agent and the CBD can be optionally separated by a peptide linker. Non-limiting examples of such linkers include flexible linkers such as a (G₄S)_n (SEQ ID NO: 14) or a rigid linker such as (EAAAK)_n (SEQ ID NO: 15).

In some cases, the Gal3 composition is a recombinant protein and includes more than one Gal3 CBD. The recombinant composition typically can bind to a carbohydrate (e.g., a protein- linked glycosylation), e.g., to the glycocalyx. Such a protein can maintain a higher concentration on the eye and/or have a longer retention time on the eye than can the therapeutic agent alone. Typically, if the composition contains more than one Gal3 CBD sequence (e.g., two Gal3 CBD sequences), the Gal3 composition has greater binding to a carbohydrate than a composition that contains only one Gal3 CBD sequence. The multiple Gal3 CBDs can be separated by a linker, for example a flexible linker such as a (G₄S)_n (SEQ ID NO: 14) or a rigid linker such as (EAAAK)_n (SEQ ID NO: 15).

Galectins typically bind to β-galactoside sugars. Members of the galectin family have significant sequence similarity in the CBD. The structure of some exemplary CBDs have been determined (Lobsanov et al., J Biol Chem 267:27034, 1993 and Seetharaman et al., J Biol Chem 273: 13047, 1998). There are presently at least a dozen characterized eukaryotic members of the galectin family. Any Gal3 CBD that can bind to the targeted tissue or molecule with sufficient affinity to be useful can be used in embodiments described herein, e.g., a human gal3 CBD. In some embodiments, a Gal3 CBD can bind to asialofetuin with an affinity in the μΜ range or can bind to mucin-1 (MUC-1) with an affinity in the μΜ range. A useful Gal3 CBD can typically bind a carbohydrate moiety found on a MUC-1 or asialofetuin. In embodiments, a Gal3 CBD can bind a carbohydrate moiety found on a MUC-1. In embodiments, a Gal3 CBD can bind a carbohydrate moiety found on asialofetuin.

Human galectin-3 is about 250 amino acids long and has an approximate molecular weight of 26.1 kDa. The sequence of an exemplary Gal3 protein is as follows: MADNFSLHDA LSGSGNPNPQ GWPGAWGNQP AGAGGYPGAS YPGAYPGQAP PGAYPGQAPP GAYPGAPGAY PGAPAPGVYP GPPSGPGAYP SSGQPSATGA YPATGPYGAP AGPLIVPYNL PLPGGVVPRM LITILGTVKP NANRIALDFQ RGNDVAFHFN PRFNENNRRV IVCNTKLDNN WGREERQSVF PFESGKPFKI QVLVEPDHFK VAVNDAHLLQ YNHRVKKLNE ISKLGISGDI DLTSASYTMI (SEQ ID NO: 1).

In embodiments, the amino acid sequence of a human GAL3 CBD includes or consists of about residues 108-250 of the human protein (See UniProt identifier: P17931) as follows:

GAPAGPLIVP YNLPLPGGVV PRMLITILGT VKPNANRIAL DFQRGNDVAF HFNPRFNENN RRVIVCNTKL DNNWGREERQ SVFPFESGKP FKIQVLVEPD

HFKVAVNDAH LLQYNHRVKK LNEISKLGIS GDI DLTSAS YTM (SEQ ID NO: 2)

In embodiments, the amino acid sequence of a human GAL3 CBD includes or consists of SEQ ID NO: 4. SEQ ID NO: 4 is another useful amino acid sequence of a human GAL3 CBD, which differs from SEQ ID NO: 2 in that SEQ ID NO: 4 does not have the N terminal GAPA residues nor does it have the N C terminal I residue. The sequence of this human Gal3 CBD is provided below.

GPLIVPYNLP LPGGVVPRML ITILGTVKPN ANRIALDFQR GNDVAFHFNP RFNENNRRVI VCNTKLDNNW GREERQSVFP FESGKPFKIQ VLVEPDHFKV AVNDAHLLQY NHRVKKLNEI SKLGISGDID LTSASYTMI (SEQ ID NO: 4)

Certain Gal3 proteins useful in compositions and methods described herein include the amino acid sequence of a human Gal3 CBD (SEQ ID NO: 2 or SEQ ID NO: 4). Other useful Gal3 proteins include an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. The term "substantially identical" is used herein to refer to a first amino acid sequence that contains a sufficient or minimum number of amino acid residues that are identical to aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least 60%, or 65% identity, for example at least 75% identity, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2 or SEQ ID NO: 4, are termed substantially identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Proteins that contain alterations in SEQ ID NO: 2 or SEQ ID NO: 4, such as deletions, additions, substitutions or modifications of certain amino acid residues of SEQ ID NO: 2 or SEQ ID NO: 4 can be used in the compositions and methods described herein, provided that the sequences retain carbohydrate binding ability, e.g., when fused to a heterologous moiety (e.g., a therapeutic or diagnostic agent as described herein), and do not substantially interfere with the activity of the heterologous moiety in a fusion. A Gal3 protein can include regions represented by the amino acid sequence of a Gal3 derived from a non-human mammalian species including but not limited to bovine, canine, feline, caprine, ovine, porcine, murine, and equine species. In some embodiments, the free cysteine (e.g., the cysteine at residue 66 of SEQ ID NO: 2 or the cysteine at at residue 62 of SEQ ID NO: 4) is replaced to decrease the chance of interchain disulfide bond formation during production, concentration, or storage of the protein.

Calculations of sequence identity between sequences are performed as follows. To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid sequence for optimal alignment). The amino acid residues at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the proteins are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using an alignment software program using the default parameters. Suitable programs include, for example, CLUSTAL W by Thompson et al. (Nuc Acids Res 22:4673, 1994 (ebi.ac.uk/clustalw)), BL2SEQ by Tatusova and Madden (FEMS Microbiol Lett 174:247, 1999 (ncbi.nlm.nih.gov/blast/bl2seq/bl2.html)), SAGA by Notredame and Higgins (Nuc Acids Res 24: 1515, 1996 (igs-server.cnrs- mrs.fr/.about.cnotred)), and DIALIGN by Morgenstern et al. (Bioinformatics 14:290, 1998 (bibiserv.techfak.uni-bielefeld.de/dialign)). A Gal3 composition can be used for treatment of a subject, e.g., a mammal. The Gal3 protein contained in the composition can be derived from a native Gal3 protein of the species that is to be treated, or from a native Gal3 protein of another species. Sequences for other non-limiting examples of native galectin-3 proteins from which Gal3 CBDs can be derived for use in embodiments described herein include the following NCBI reference numbers NP_001095811.1 (Bos taurus); NP_001183972.1 (Cards lupus familiaris); NP_999756.1 (Gallus gallus);

NP_114020.1 (Rattus norvegicus); NP_001139425.1 (Mus musculus); NP_001090970.1 (Sus scrofa); NP_001075807 (Oryctolagus cuniculus); NP_001253292.1 (Macaca mulatto),

XP_005561371.1 (Macaca fascicularis) and BAA22164.1 (human). The Gal3 protein can also be derived from sequence variants of these or other native Gal3 proteins (e.g., sequences with one or more polymorphisms, variations due to RNA editing, or variations due to post-translational modifications or lack thereof).

In embodiments, the CBD is derived from the native galectin-3 protein of the species to be treated.

A Gal3 CBD can be a derivative of a native Gal3 CBD. The derivative typically comprises a sequence at least 90% identical to a native Gal3 CBD or to a Gal3 CBD described herein. In embodiments, the Gal3 CBD includes or consists of a sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a native Gal3 CBD sequence or to a Gal3 CBD sequence described herein. For example, the amino acid sequence can include one, two, three, four, five, six, between two and twenty, between two and fifteen, between two and ten, or between two and five substitutions, insertions, or deletions, e.g., substitutions, e.g., conservative substitutions. Methods of identifying and making conservative substitutions are known in the art. Suitable substitutions result in a Gal3 CBD derivative that retains the ability to bind carbohydrate, e.g., with an affinity in the μΜ to nM range, e.g., an affinity of at least 950 μΜ, at least 850 μΜ, at least 750 μΜ, at least 650 μΜ, at least 550 μΜ, at least 450 μΜ, at least 350 μΜ, at least 250 μΜ at least 150 μΜ, at least 100 μΜ, at least 50 μΜ, at least 5 μΜ, at least 1 μΜ, at least 950 nM, at least 850 nM, at least 750 nM, at least 650 nM, at least 550 nM, at least 450 nM, at least 350 nM, at least 250 nM at least 150 nM, at least 100 nM, at least 50 nM, at least 5 nM, or at least 1 nM.

In general, the Gal3 CBD of a composition includes a galectin signature sequence

(PROSITE No. PS51304; Hulo et al. Nucleic Acids Res. 34:D227-D230(2006).

A Gal3 CBD described herein can have a galactoside-binding domain that has a bit score for the alignment of the sequence to the consensus sequence PF00337 from PFAM of at least 100, 120, 150, 170, or greater. To calculate the bit score for the alignment of a particular sequence to the consensus sequence PF00337 from PFAM, the sequence of interest can be searched against the PFAM database of HMMs (e.g., the PFAM database, release 25.0) using the default parameters available at www.sanger.ac.uk/Software/Pfam. A description of the PFAM database can be found in Nucleic Acids Res 36 (Database issue): D281-8. In some embodiments, a CBD has the foregoing consensus sequence and is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to a CBD from a Gal3.

In some embodiments, a galactoside binding domain (e.g., of a galactoside binding lectin) has the following consensus sequence:

W-[GEK]-X-[EQ]-X-[KRE]-X(3,6)-[PCTF]-[LIVMF]-[NQEGSKV]-X-[GH]-X- (3)- [DENKHS]-[LIVMFC] (SEQ ID NO:3), where X can be any amino acid. In some embodiments, a CBD has the foregoing consensus sequence and is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to a CBD from a Gal3.

Therapeutic Agents

A Gal3 composition described herein can provide advantages for therapeutic treatments. In particular, a Gal3 composition can be useful for targeting a tissue of interest by virtue of binding to specific carbohydrate moieties, for example, for delivery to the ocular surface. Such compositions can also effectively provide enhanced pharmacokinetic properties. For example, a Gal3 composition can increase the residence time of the heterologous therapeutic agent at or near the surface of the eye, effectively increasing absorption and/or therapeutic effect of the therapeutic agent.

Non-limiting examples of therapeutic agents that can be associated with (e.g., fused to) a Gal3 protein include a cytokine (such as an interleukin or an interferon), a cytokine binding protein or soluble form of a cytokine receptor, an antibody or fragment thereof (such as an scFv or a Fab), an antibody mimetic, an alternative scaffold (e.g., an alternative scaffold from antibody technology), an Adnectin, a nanobody, a DARPIN, an anticalin, an Affibody, a knottin, a Kunitz domain, an avimer, and other biologic or pharmaceutical agents. In embodiments, the therapeutic agent is an inhibitor of cytokine signaling, e.g., an inhibitor of a TNF, a VEGF, an IL-1, an IL-2, an IL-6, or an IL-17.

For example, the agent can be an inhibitor of IL-1 signaling, e.g., a molecule described in PCT/US2012/022583, an ST2, an IL-IRa molecule (e.g., anakinra), an IL-1 agonist binding antibody such as a DVD Ig or an scFv that can specifically bind to an IL-Ιβ and/or an IL-1 a.

A Gal3 composition (e.g., a composition comprising a fusion protein described herein) can be used as a therapeutic, particularly, at a site of action at tissue that includes or is in the vicinity of glycocalyx, e.g., the ocular surface or a mucosa. For example, a Gal3 composition can be used to treat an ocular surface disease, such as a dry eye disease, Meibomian gland disorder, keratitis, episcleritis, or conjunctivitis (e.g., allergic conjunctivitis, keratoconjunctivitis sicca, and/or vernal keratoconjunctivitis). Examples of disorders affecting the surface of the eye and that can be treated using a Gal3 composition include conditions also referred to as dry eye disease, keratoconjunctivitis sicca, keratitis sicca, sicca syndrome, xerophthalmia, tear film disorder, decreased tear production, aqueous tear deficiency, dysfunctional tear syndrome, and Meibomian gland dysfunction, and other forms of dry eye associated with various etiologies, including Sjogrens syndrome, graft versus host disease, and other autoimmune conditions such as mucous membrane pemphigoid. Other conditions that can be treated with a Gal3 composition

(e.g., a composition comprising an appropriate heterologous therapeutic agent) include blepharitis and allergic conjunctivitis. Subjects who have undergone a medical procedure that affect the eye, e.g., eye surgery procedures such as, e.g., laser eye surgery (e.g., photorefractive keratectomy, laser-assisted sub-epithelial keratectomy (LASEK), laser epithelial keratomileusis, laser-assisted in situ keratomileusis (LASIK) surgery) or corneal transplant, can also be treated using a Gal3 composition described herein.

In some embodiments, the Gal3 protein (e.g., the protein comprising at least one Gal3 CBD) is associated with (e.g., fused to) an IL-1 antagonist (e.g., anakinra, or an IL-1 antagonist such as P05 identified in PCT/US 11/45995), or an IL-6 antagonist such as tocilizumab or a Fab or other fragment of an IL-6 antibody such as tocilizumab, a VEGf inhibitor such as ranibizumab or bevacizumab.

In some embodiments, the Gal3 protein (e.g., the protein comprising at least one Gal3

CBD) is associated with (e.g., fused to) an antagonist of IL-17 activity such as brodalumab, which targets the IL-17A receptor, or ixekizumab which binds to IL-17.

In embodiments, the Gal3 protein (e.g., the protein comprising at least one Gal3 CBD) is associated with (e.g., fused to) an IL-2 inhibitor such as aldesleukin.

In some embodiments, a Gal3 protein (e.g., the protein comprising at least one Gal3

CBD) is associated with (e.g., covalently linked to) a therapeutic such as an antibiotic (e.g., a polymyxin), steroid such as cyclosporine, or a non-steroidal anti-inflammatory agent (NSAID).

In some embodiments, a Gal3 protein (e.g., the protein comprising at least one Gal3

CBD) is associated with (e.g., covalently linked to) a therapeutic agent that is a small molecule.

In embodiments, the therapeutic agent is a small molecule linked through an engineered lysine or cysteine that works through extracellular interactions. In embodiments, the therapeutic agent is conjugated to the Gal3 protein using the technology of Ambrx, Inc. (La Jolla, CA). Gal3 compositions can also be used in methods for promoting corneal tissue healing. This includes treating corneal defects, e.g., corneal epithelial defects, caused by, for example, corneal ulcers, heat, radiation, phlyctenulosis, corneal abrasions or lacerations, medical procedures (e.g., photorefractive surgery for corrective myopia), foreign bodies and sterile corneal infiltrates; chemical burns caused by exposure to acids or alkali (e.g., hydrofluoric acid, formic acid, anhydrous ammonia, cement, and phenol) or other chemical agents such as white phosphorus, elemental metals, nitrates, hydrocarbons, and tar; keratopathies such as neurotrophic keratopathy, diabetic keratopathy and Thygeson's superificial punctate keratopathy; keratities such as viral keratitis (e.g., metaherpetic or herpetic keratitis) and bacterial keratitis; and corneal dystrophies such as lattice dystrophy, epithelial basement membrane dystrophy (EBMD) and Fuch's endothelial dystrophy.

In some embodiments, the Gal3 composition (e.g., the fusion protein) is administered locally, e.g., to the tissue and/or glycocalyx. For surface of the eye disorders, the fusion proteins are typically administered topically, e.g., in eye drops, a punctual plug, or contact lens.

Diagnostic Agents and Applications

Also provided herein is an isolated protein comprising a Gal3 protein and a diagnostic agent. The diagnostic agent can be any imaging agent (e.g., any contrast agent, label, or the like) known in the art. In embodiments, the diagnostic agent is used to evaluate properties of the eye surface. For example, the agent can be used to detect disruptions, tears, and/or breaks in the glycocalyx as well as other glycocalyx features.

The diagnostic agent, e.g., reporter, that can be fused to the Gal3 protein can be a fluorescent protein, e.g., a green fluorescent protein or variant thereof. Other non-limiting examples of reporters include lucif erase (e.g., firefly, Renilla, or Gaussia), beta-galactosidase, europium, fluorescent dyes (e.g., Alexa Fluor® dyes, Molecular Probes, Eugene, OR).

Testing Gal3 Compositions

Gal3 compositions can be tested for efficacy using methods known in the art, for example, animal models of a disorder to be treated. Additional methods are provided in the Examples (infra). Dry Eye

Gal3 compositions, for example, compositions comprising a Gal3 protein fused to an IL- 1 inhibitor, can be tested in murine models of dry eye. In one model, tear insufficiency is induced by applying scopolamine patches to the tail of wild type mice, according to the method reported in Dursun et al. (Invest Ophthamol & Vis Sci. 43:632-638, 2002) and Pflugf elder et al. (The

Ocular Surface, 1 :31-36, 2003). Another useful model uses MRL/lpr autoimmune mice (available from the Jackson Laboratories, Bar Harbor, ME), which develop lacrimal gland inflammatory lesions (Jabs et al., Invest Ophthamol Vis Sci. 32:371-380, 1991 ; Jabs et al., Curr Eye Res.

16:909-916, 1997). Another suitable model involves systemic administration of IL-la, producing an animal model of Sjogren's syndrome (Zoukhri, D. et al., Invest Ophthalmol Vis Sci 42: 925- 932, 2001). An animal model of diabetes can also be used to assay efficacy of a Gal3 dry eye composition, for example, induction of type 1 diabetes in rats using streptozotocin (Zagon et al., Diabetes. 2002; 51 : 3055-3062; Klocek et al., J Ocular Pharmacol Therapeutics. 2007; 23:89-102; Zagon et al., Arch Opthalmol. 2007; 125: 1082-1088).

Assays of Gal3 compositions useful for treating dry eye conditions can include in vivo assay of tear production, tear clearance, and corneal fluorescein staining in treated animals compared to control animals treated with vehicle. Biochemical assays and histopathology can also be conducted to compare treated and untreated animals. Effects specific to a Gal3 protein component of a composition can be determined by administering β-lactose, an inhibitor of Gal3.

An example of a method that can be used to evaluate the efficacy of a Gal3 dry eye composition is assay of tear secretion using the Schirmer Test. In this test, a Schirmer strip (Alcon Laboratories, Inc., Fort Worth Tex.) is inserted into the lower cul-de-sac of the eye for a selected period of time (e.g., 1 minute). The strip wetting length is measured to the nearest millimeter. Animals can be administered topical anesthetic such as Proparacaine hydrochloride Ophthalmic Solution 0.5% (Akorn, Inc., Buffalo Grove, IL) prior to testing. Testing is performed at specified intervals after treatment with the Gal3 composition or control (vehicle only).

Another assay that can be performed is testing corneal sensitivity. Corneal sensitivity can be determined using an aesthesiometer (Cochet and Bonnet-Aesthesiometer, Boca Raton, FL). The values (g/mm²) are determined directly from the protocol and conversion table supplied by the manufacturer.

In addition, slit-lamp observation can be used to examine general overall morphology and pathology (e.g., corneal edema, scarring, abrasions). Observations are made with a hand-held slit lamp (Zeiss HSO 10 Hand Slit Lamp, Dublin, Calif.). "Treating dry eye" includes complete or partial amelioration of all or some of symptoms of dry eye, and/or prevention or inhibition at least one symptom of dry eye.

Formulation

Gal3 compositions described herein can be prepared and administered using methods known in the art, including local administration and systemic administration routes. Local administration can include topical administration, for example, epidermal, transdermal, mucosal delivery (for example, intranasal, vaginal, or rectal), pulmonary (for example, by inhalation or insufflation), ophthalmic, pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal intramuscular or injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be included in such compositions and formulations.

In some embodiments, the Gal3 composition is administered as an ophthalmic formulation. The methods can comprise administration of the Gal3 composition and an ophthalmically acceptable carrier. In some embodiments, the ophthalmic formulation is a liquid, semi-solid, insert, film, microparticle, or nanoparticle.

In some embodiments, the Gal3 composition is formulated for topical administration, e.g., to the eye. The topical formulation can be a liquid formulation or semi-solid, for example, a topical formulation can include an aqueous solution, an aqueous suspension, an ointment or a gel. An ophthalmic Gal3 formulation can be topically applied to the front of the eye, under the upper eyelid, on the lower eyelid and in the cul-de-sac. Typically, the ophthalmic formulation is sterile. A Gal3 ophthalmic formulation can contain one or more pharmaceutical excipients suitable for the preparation of ophthalmic formulations. Examples of such excipients are preserving agents, buffering agents, chelating agents, antioxidant agents and salts for regulating the osmotic pressure. Ophthalmic formulations, including both ointments and suspensions, typically have a viscosity that is suited for the selected route of administration. In some embodiments, the ophthalmic formulation has a viscosity of from about 1,000 to about 30,000 centipoise (cp). In some embodiments, the ophthalmic formulation has a viscosity of less than 1,000 cp. In some embodiments the formulation has a viscocity of between 1 cp and 500 cp, e.g., between 1 and 400 cp, betweenl and 200 cp, or between 1 and 100 cp. In some embodiments, the ophthalmic formulation has a viscosity of less than 100 cp. In embodiments, the formulation has a viscosity of 1 to 20 cp, 1 to 30 cp, 1 to 40 cp, 1 to 50 cp, 1 to 60 cp, 1 to 70 cp, or 1 to 80 cp. In

embodiments, the ophthalmic formulation has a viscosity of less than 15 cp.

In some embodiments, treatment includes administering a pharmaceutical Gal3 composition to a patient, the pharmaceutical composition comprising the Gal3 composition and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is an oral dosage form. In some embodiments, the pharmaceutical compositions comprise, as the active ingredient, one or more of the agents above in combination with one or more pharmaceutically acceptable carriers (excipients). In making the compositions of the invention, the agent is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the formulations can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

In preparing a formulation, the Gal3 composition can be milled to provide the appropriate particle size prior to combining with the other ingredients. If the Gal3 composition is

substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the agent is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The

compositions can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.

A Gal3 composition can be formulated in a unit dosage form, each dosage containing from about 5 to about 1000 mg (1 g), more usually about 100 to about 500 mg, of the agent. The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

A Gal3 composition can be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It will be understood by those in the art that the amount of the agent actually administered is typically determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of

administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

For preparing solid a Gal3 formulation such as a tablet, the Gal3 composition is mixed with a pharmaceutical excipient to form a solid composition containing a homogeneous mixture of a Gal3 composition. Such formulations are typically provided in unit dosage forms, for example, about 0.1 to about 1000 mg of the Gal3 composition.

A tablet or pill comprising a Gal3 composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

A liquid formulation comprising a Gal3 composition can be prepared for oral delivery or for injection, for example, in an aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, or flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Formulations for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. In some embodiments, the formulations are administered by the oral or nasal respiratory route for local or systemic effect. Solution, suspension, or powder formulations can be administered orally or nasally from devices that deliver the formulation in an appropriate manner.

The amount and frequency of a Gal3composition administered to a patient will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration, and the like. In therapeutic applications, compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. Effective doses will depend on the disease condition being treated as well as by the judgment of the attending clinician depending upon factors such as the severity of the disease, the age, weight and general condition of the patient, and the like.

The therapeutic dosage of a Gal3 composition agents can vary according to, for example, the particular use for which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of an agent in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the agents can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral administration. In embodiments, an agent (e.g., an antibody or fragment thereof) is present at a concentration of up to 200 mg/ml, e.g., at a concentration of 1-200 mg/ml, 100-200 mg/ml, or 150-200 mg/ml. Some typical dose ranges are from about 1 μg/kg to about 1 g/kg of body weight per day. In some embodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kg of body weight per day. The dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

EQUIVALENTS

All technical features can be individually combined in all possible combinations of such features.

The invention may be embodied 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. All patents, patent applications, and references are incorporated herein for all purposes.

Further illustration is provided by the following non-limiting examples. EXAMPLES

Example 1: Construction of GAL3-G-lucif erase and control proteins.

Gal3 fusions with a Gaussia luciferase (Glue) were constructed in pTT5 vectors coding for Glue fused to the N-terminus of Gal3 CBD. The Glue sequence is provided below:

MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKE MEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPG FKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKI KGAGGD (SEQ ID NO: 5)

The human Gal3 CBD sequence used was:

GPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIV CNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEI SKLGISGDIDLTSASYTMI (SEQ ID NO: 4).

Three different constructs were prepared; a Glue with eight histidine residues at the C terminus (termed herein Gluc8 His or Glue) ("8 His" disclosed as SEQ ID NO: 13), a Gluc/Gal3 CBD fusion with eight histidine residues at the C terminus of the Gal3 moiety (termed herein Glue Gal3 8His or Gluc-Gal3) ("8 His" disclosed as SEQ ID NO: 13), and a Glue fusion with two tandem Gal3 sequences followed by a C terminal tag of eight histidines (termed herein Glue 2XGal3 8His or Gluc-Gal3-Gal3) ("8 His" disclosed as SEQ ID NO: 13). A (G₄S)₂ linker (SEQ ID NO: 16) was used between each moiety. Fig. 1 is a representation of the constructs. Using the vectors, protein was prepared and purified using immobilized metal ion affinity chromatography (IMAC). Briefly, 100 ml cultures of HEK 293 6E were transiently transfected with the different constructs at a final concentration of 0.89 x 10⁶/ml. The total amount of construct DNA used in the transfection was 50 μg. Cells were transfected using polyethylenimine (PEI) in a PEFDNA ratio of 2: 1. Twenty-four hours post-transfection, cells were fed with tryptone N-l (TN-1) to a final concentration of 0.5% w/v. Cells were harvested three days post-transfection with viability still at >95% .

Conditioned media (CM) samples were run undiluted, with or without lmM dithiothreitol (DTT) with IX SDS loading buffer (6X loading buffer was added to the CM to a concentration of IX loading buffer and 20 μΐ samples were loaded onto gels) and run using standard SDS running conditions on 4-12% Bis-Tris gel. Gels were run at 200V for 40 minutes with 20 μΐ samples loaded in each lane.

Proteins were then transferred onto Invitrolon™ (Invitrogen, Carlsbad, CA) membranes according to manufacturer's instructions. Transfer was carried out for 90 minutes at 30V. Blots were blocked for one hour with 5% non-fat milk in PBS containing 0.1% Tween® 20, and incubated overnight with primary antibody (rabbit anti-6X His tag® (SEQ ID NO: 17) (HRP) (Abeam, Cambridge, MA, Cat #Abl l87)). Blots were imaged with SuperSignal (Pierce Biotechnology, Rockford, IL) using a 20 second exposure. The proteins standard used was a different his-tagged protein diluted to relevant concentrations.

Estimated expression was about 50 mg/L of G-Luc-Gal 3, about lOmg/L G-Luc, 0.5 mg/L G-luc-Gal3-Gal3.

Protein purification

Expression products were purified using 1 mL HisTrap™ columns (GE Healthcare, Uppsala, Sweden) at a flow rate of 1 mL/minute. The first wash was in PBS followed by washes with PBS containing up to 40 mM imidazole. Protein products were eluted using PBS containing 250 mM imidazole.

Addition of Gal3 CBD moieties to luciierase did not affect either the specific or relative activities of luciierase. Luciierase was assayed using the BioLux® Gaussia Luciierase Flex Assay Kit from New England Biolabs following the manufacturer's instructions. This demonstrates that Gal3 fusion proteins can be constructed that do not adversely affect the function or activity of a heterologous fusion partner.

Example 2: Binding of Gal3 Fusion Proteins

To determine how creation of a Gal3 fusion protein affected the ability of the Gal3 moiety of the fusion to bind to a substrate, ELISA assays were performed testing the binding of the fusions described in Example 1 to asialofetuin. In these experiments, ELISA white plates (Sostar high binding white plates from Thermo Scientific) were coated overnight at 4°C with a solution of 4 mg/ml asialofetuin (Sigma catalog # A4781) or 10 μg/ml human mucin- 1 (MUC-1 ; Sino Biological, catalog #12123-H02H) in PBS, washed, and blocked for one hour with IX reagent diluent (R&D Systems Inc., Minneapolis, MN). Fusion proteins prepared as described supra and varying concentrations were added to wells (16 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, and 1 μΜ) and incubated for one hour at room temperature, then washed with PBS containing 0.05% Tween® 20, and bound protein activity was measured by detection of the reporter protein (Guassia luciierase (using the BioLux® Gaussia Luciierase Flex Assay Kit, New England Biolabs). Light output was integrated for 500 milliseconds.

In general, more Gluc-Gal3-Gal3 bound to plates than Gluc-Gal3, with Glue binding the least. For example, at ΙμΜ, Gluc-Gal3-Gal3 bound about 255 fold more than Glue, and Gluc- Gal3 about 21 fold more compared to Glue. Similarly, in an experiment in which ELISA plates were coated with 10 μg/ml recombinant MUC-1, at ΙμΜ, Gluc-Gal3-Gal3 bound about 310 fold more than Glue and Gluc- Gal3 about 10 fold more than Glue.

These data demonstrate that fusion of Gal3 to a heterologous sequence does not destroy the ability of Gal3 to bind to a β-galactoside and that increasing the number of Gal3 moieties in such a fusion can increase binding.

Example 3: In Vivo Retention of Gal3 Fusions in Mice

To test the ability of a Gal3 fusion protein to bind to and be retained on the eye surface in vivo, Gal3 fusion proteins prepared as described above were administered to mice. Briefly, 3μ1 of a ΙμΜ solution of a fusion protein was instilled into the eyes of mice. After 30 minutes, the animals were sacrificed and the eyes were enucleated. Eyes were kept in PBS containing 1% BSA on ice until lucif erase activity was measured (generally within about one hour).

The results are depicted in Fig. 2, and are the average of three readouts. Because the position of the eye influences the readout, the eyes were oriented in a consistent fashion to ensure consistency of the readout. In these experiments, about 95 times more Gluc-Gal3-Gal3 was retained on the eye compared to Glue and about 3 times more Gluc-Gal3 was retained on the eye compared to Glue.

These data provide evidence that Gal3 CBD containing fusions can be used to increase the retention time of a heterologous protein on the eye in vivo, and that multiple copies of a Gal3 CBD can further increase the retention time.

Example 4: Ex vivo retention of Gal3 Fusions in Rabbit Corneal Tissue

To further test the retention of Gal3 fusions on corneal tissue, binding to isolated rabbit corneal punches was examined. Rabbit corneas were purchased from Pel-Freez Biologicals. The corneas were washed in PBS and then using a 5 mm biopsy punch, two cornea punches were taken per cornea and transferred to a 24 well plate. The punches were divided into four groups and punches from the same cornea were separated to different groups. For a protein containing control (Gaussia luciferase alone), 200 μΐ of a 1 μΜ protein solution was added to the wells and for other samples, 200 μΐ of a ΙμΜ protein solution was added to the wells.

Corneal punches were incubated at room temperature for 30 minutes. The test solutions contained Gluc-Gal3, Gluc-Gal3-Gal3, Glue alone (protein control), or PBS (control). After incubation, the corneal tissue was transferred into 2 ml PBS and incubated at room temperature for 30 minutes. The tissue was then transferred to a 96 well white plate containing 200 μΐ of Gaussia lucif erase substrate and the light output was measured for 500 ms. Results were normalized for the specific activity of each protein.

Binding reflected as lucif erase activity was increased by 6.75- and 12.5-fold over the LUC -His incubated samples with Gluc-Gal3 or Gluc-Gal3-Gal3 compositions, respectively.

These data provide further evidence that Gal3 CBD containing fusions can drive corneal binding and be used to increase the retention time of a heterologous protein, and that multiple copies of a Gal3 CBD can further increase the retention time.

Example 5: Retention of Gal3 Fusions in Murine Eyes

Retention of Gal3 CBD containing compositions on the eye were assessed in a time- course study performed essentially as described in Example 3 except that samples were obtained at a series of time points; 1 minute, 15 minutes, 30 minutes, and 180 minutes. Animals were treated with 3 μΐ of a solution in PBS as follows; PBS only, Luc alone, Luc-Gal3, or Luc2XGal 3. Animals were euthanized after 1, 15, 30, or 180 minutes, then the eyes removed and stored in PBS for transport, which took about one hour. To immobilize the eyes and help with

standardization of the assay, the eyes were inserted into cut 10 μΐ pipette tips with the iris facing up and the tips placed in a 96 well white plate (with the iris oriented up). 200 μΐ of Gaussia luciferase substrate was added to each well and light output was measured for 500 ms.

Glue activity was at background levels in the PBS control. In the murine eyes by 15 minutes after administration, Gluc-Gal3 compositions had no detectable activity over background by about 30 minutes, while Gluc-Gal3-Gal3 required 180 minutes or more to reach background levels. The t_U2 of Gluc-Gal3-Gal3 was at least 6-fold higher than that of Glue alone. After one minute of incubation, there was 64 fold more Luc2XGal3 activity compared to Glue alone. Even after three hours, the Luc2XGal3 was detectable. The residence half-life of the Luc2XGal3 was about 32 minutes.

These data further demonstrate that the retention time of a protein composition can be increased using a heterologous fusion protein containing a Gal3 CBD and the protein, and that addition of a least one additional Gal3 CBD can further increase the retention time compared to a fusion that contains a single CBD.

Example 6: Gal3 Fusion Potentiation of Cytokine Activity

To further test whether fusion to Gal3 adversely affects activity of a fused moiety, an IL- 1 antagonist (termed 93:60) was fused to Gal3 using (G₄S)₃ (SEQ ID NO: 18) linkers in constructs described in Table 1 (infra). Because the inhibitor can be fused at either the amino or carboxy terminus, fusions were created containing one or two Gal3 binding domains on either terminus. The fusion proteins were produced in E coli and purified by affinity chromatography using a C-terminal poly His tag (H₈) ("H₈" disclosed as SEQ ID NO: 13). The sequence of 93:60 is as follows:

APVRSLNCRI WDVNQKTFYL RNNQLVAGYL QGPNVNLEEK FSMSFVQGEE SNDKIPVALG LKEKNLYLS CVLKDDKPTL QLESVDPKNY PKKKMEKRFV FNKIEINNKL EFESAQFPNW FLCTAMEADQ PVSLTNMPDE GVMVTKFYMQ FVSS

(SEQ ID NO:6)

The proteins were expressed and purified, then tested in a HEKBlue IL-Ιβ reporter cell- based assay (InvivoGen) as described in Hou, J. et al., Proc Natl Acad Sci U S A; 110(10): 3913- 3918 (5 March 2013). In these experiments which were performed substantially according to the manufacturer's instructions, cells were seeded at 50,000 cells/well in a 96 well plate and mixed with 0.1 ng/ml IL-Ιβ and dilutions of the 93:60 proteins. After 20 hours, activation of IL-1 activity was assayed using HEK -Blue IL-Ιβ cells that lack a TNF-a response and in which activity is assayed by monitoring the activation of the NF-κΒ using a secreted embryonic alkaline phosphatase reporter. The Quanti-Blue™ substrate for secreted alkaline phosphatase was used in these assays. The results are shown in Table 1.

Table 1

*A (G₄S)₂ linker (SEQ ID NO: 16) was included between 93:60 and Gal3 moieties for these constructs Surprisingly, the fusions of 93:60 with Gal3 CBD did not have a negative impact on the inhibitory activity of 93:60. In fact, the activity was dramatically increased. The maximum potency increase was 75 fold, observed for the fusion protein in which two adjacent Gal3 moieties were fused to the N terminus of 93:60.

Without committing to any particular theory, it may be that because Gal3 binds to cell surface receptors as well as mucins, the increase in potency of the IL-1 antagonist was due to a gain in avidity for cell surface binding

These data also demonstrate that the enhanced potency achieved by fusing a protein designed to modulate 11-1 activity to at least one Gal3 moiety (e.g., a Gal3 CBD).

Example 7: Inhibitory activity of a Gal3 moiety

To investigate whether the 2XGal3 moiety synergizes with the IL-1R1 inhibitor (93:60) to produce increased activity over the inhibitor alone, a mixture of 2XGal3-8H protein ("8H" disclosed as SEQ ID NO: 13) and 93:60 at equimolar concentration was used in HEK-Blue assays (described in Example 6) run essentially according to the manufacturer's instructions. The concentration ranges were from lxl0^"13M to lxl0^"8M. The results are shown in Fig. 3. At the concentrations used in this experiment, the mixture of the 2XGal3 moiety with 93:60 did not increase the inhibition of Il-lbeta activity compared with the inhibition provided by 93:60. This experiment indicates that 2xGal3 alone does not have IL1R1 inhibitory activity and that fusion of the 2xGal3 moiety to 93:60 is necessary for the observed increased potency.

Example 8: Specificity of Gal3 CBD effects

To determine whether coating the cell surface with an inhibitor is sufficient to increase the efficacy of the inhibitor, a TAT transduction peptide (GRKKRRQRRR (SEQ ID NO: 7)) was fused to the N-terminus of 93:60 (TAT-93:60). The TAT peptide has been shown to bind to heparin sulfate on the cell surface and would be expected to increase the local concentration of 93:60 on the cell surface without any direct binding to IL-1R1, which does heparin binding sites. The TAT-93:60 protein was produced in E. coli and purified on a heparin column. The assay was performed using the HEK-Blue IL-Ιβ kit as described in Example 6 above. The results are illustrated in Fig. 4.

The data demonstrated that the addition of TAT to 93:60 did not increase the inhibitory activity of 93:60. This indicates that the effect of the Gal3 CBD containing fusions is not merely an increase in the local concentration of the therapeutic agent at the cell surface. Without committing to any theory, it may be that the Gal3 CBD is effecting binding of the IL-1 inhibitor to IL-lRl glycans thereby effecting increased potency of the inhibitor.

To further elucidate the mechanism by which Gal3 CBD affects increased potency of a therapeutic agent, a reporter protein (Luc2XGal3; described above) was used in a luminescent ELISA assay. As described above, a white plate was coated with mucin-1, asialofetuin, IL-lRl, or an unglycosylated protein that was produced in E. coli, and each was incubated with the reporter protein before addition of the luciferase substrate. Light output was measured for 500 ms. The results are illustrated in Fig. 5.

The data from this experiment demonstrates that the 2XGal3 moiety can bind to IL-lRl but did not bind to unglycosylated protein. This indicates that a Gal3 CBD containing moiety can be used to increase potency of a therapeutic agent whose target has a β-galactoside, for example, a Gal3 CBD can be used to construct a fusion with any agent targeting an IL-lRl and increase the potency of the agent.

In another experiment, IL-IRa and IL-IRa linked to two copies of Gal3 CBD and His tagged (IL-lRa-2XGal3H8 ("H8" disclosed as SEQ ID NO: 13)) were tested for inhibitory activity in the HEK-Blue IL-Ιβ system using various concentrations ranging from lxlO^"15M to lxlO^"8M. The results of these experiments demonstrated that inhibition of IL-1 was effected at lower concentrations with the Gal3 CBD containing molecule compared to unmodified IL-IRa (Fig. 6).

In an additional experiment in which IL-1 β and IL-1 β linked to two copies of Gal3 CBD were tested in the HEK-Blue IL-1 β system, a more robust induction of activity was observed with the IL-i -2XGal3H8 ("H8" disclosed as SEQ ID NO: 13) than with unmodified 11 1 β. The results of this experiment are shown in Fig. 7. This experiment shows that Gal3 will potentiate the activity of the fused protein whether it is an agonist or an antagonist.

Example 9: In vivo retention of Gal3 CBD containing molecules

In vivo retention of Gal3 CBD containing molecules was tested using the 93:60- 2XGal3H8 construct ("H8" disclosed as SEQ ID NO: 13), which has the following sequence:

MAPVRS LNCRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKF SMSFVQGEE SNDK I PVA LGLKEKNLYL S CVLKDDKPTLQLE SVDPKNYPKKKMEKRFVFNK I E INNKLEFE SAQFP NWFLCTAMEADQPVS L TNMPDEGVMVTKFYMQFVS S GGGGS GGGGSRPL IVPYNLPLPG GVVPRML I T I LGTVKPNANRI ALDFQRGNDVAFHFNPRFNENNRRVI VCNTKLDNNWGR EERQ SVFPFE S GKPFK I QVLVEPDHFKVAVNDAHLLQYNHRVKKLNE I SKLG I S GD I DL T SASYTMI GGGGS GGGGS GGGGSPL IVPYNLPLPGGVVPRML I T I LGTVKPNANRIALD FQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQ SVFPFE S GKPFK I QVLVEPD HFKVAVNDAHLLQYNHRVKKLNE I SKLG I S GD I DL T SASYTMI S HHHHHHHH

(SEQ ID NO: 8). Rabbits were topically dosed with 30 μΐ/eye of either 4.8 mg/ml 93:60 or 14.2 mg/ml

2XGal3H8 ("H8" disclosed as SEQ ID NO: 13). Animals were euthanized and the eyes dissected at various times. The tissues (conjunctiva, cornea, sclera) were harvested, diluted 1 : 10 in Phosphate Buffered Saline (PBS) containing 125mM lactose, homogenized, and stored at -80°C until quantification by ELISA.

A sandwich ELISA assay was used for the detection and quantitation of 93:60 and 93:60 fusion proteins in tissue homogenates. Briefly, a mouse monoclonal antibody (mAb) to IL-Ιβ was immobilized on a 96-well microtiter plate overnight at 2 to 8°C then blocked for a minimum of 1 hour at room temperature (RT) and washed. Tissue homogenates containing known or unknown concentrations of 93:60 were added to the washed plates and incubated for one hour on a plate shaker at RT. Plates were washed and ready-to-use polyclonal anti-human IL-lra-horse radish peroxidase (HRP) conjugate was added to the wells and incubated for three hours without shaking at RT. After washing away any unbound conjugate, tetramethylbenzidine (TMB), a peroxidase substrate, was used to generate optical density at 450 nm that was proportional to drug concentration. Concentrations of 93:60 in tissue homogenates were determined by interpolation from a standard curve using a four-parameter logistic curve fit.

Eye tissues examined were conjunctiva, cornea, and sclera. Table 2 shows data from this experiment. Data are expressed as ng 93:60/ml of tissue lysate.

BLQ=beneath the level of quantification Tissue LLOD 0.25 hours 1 hour post 4 hours post 8 hours post 24 hours post dose dose dose dose post dose median median median median median

93:60-2XGalH8 ("H8" disclosed as SEQ ID NO: 13)

Conjunctiva 71.7+ 204.2 BLQ BLQ BLQ BLQ

8.6

Cornea 60.2 340.1 125.5 BLQ BLQ BLQ

Sclera 84.5 + BLQ BLQ BLQ BLQ BLQ

2.9

The ability to administer therapeutic agents to the vitreous can be limited by the frequency with which such treatments must be administered. To test the ability of a Gal3 CBD to increase residency of a molecule within the eye, rabbits were injected intravitreally with 50 μΐ of 6.6 mg/ml 93:60-2XGal3H8 ("H8" disclosed as SEQ ID NO: 13) or 50 μΐ 46 mg/ml 93:60.

Animals were euthanized and the eyes were harvested at 15 minutes, 24 hours, and 72 hours after injection and the retinas assayed for the amount of detectable 93:60. The retinas were harvested, diluted 1: 10 in Phosphate Buffered Saline (PBS) containing 125mM lactose, homogenized, and stored at -80°C until quantification by ELISA as previously described.

Fig. 8 illustrates the results of this experiment. By 24 hours a substantial rise in the amount of detectable 93:60 was observed in the animals injected with 93:60-2XGal3H8 ("H8" disclosed as SEQ ID NO: 13) and the amount was still elevated over 93:60 by 72 hours demonstrating the enhanced retinal retention of intravitreally injected molecules containing Gal3 CBD.

Example 10: Effect of Linker Length and Type on 93:60-Gal3 Fusion Proteins

To investigate the importance of the linker length between two Gal moieties we built four new 93:60-Gal3 fusion proteins (sequences below) with linkers of varying length (GS, G₄S (SEQ ID NO: 14), (G₄S)₂ (SEQ ID NO: 16) and (G₄S)₄ (SEQ ID NO: 19)) and tested their activity in the HEK blue ILip assay described in Example 6. All four proteins had similar activity as our original proteins (93:60-2xGal8H ((G₄S)₃ linker (SEQ ID NO: 18)) ("8H" disclosed as SEQ ID NO: 13)) indicating that the distance between the two Gals does not significantly affect the binding properties of 93:60.

We also investigated if the type of linker between the biologic and the Gal moieties could influence the activity. Since the original linker ((G₄S)₂ (SEQ ID NO: 16)) is considered a "flexible" linker we chose to replace it with a "rigid" linker. The "rigid" linker (EAAAK)₄ (SEQ ID NO: 20) was inserted between 93:60 and the 2xGal8H ("8H" disclosed as SEQ ID NO: 13). This protein (sequence below) also had the same activity as the original protein, indicating that in the case of 93:60, the linker did not have much impact. However, for other therapeutic agents, optimization of linker length and/or type may be necessary to retain their activity.

96:60-Gal-GS-Gal8H ("8H" disclosed as SEQ ID NO: 13)

MAPVRSLNCRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKFSMSFVQGEESNDKIPVA LGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFP NWFLCTAMEADQPVSLTNMPDEGVMVTKFYMQFVSSGGGGSGGGGSRPLIVPYNLPLPG GVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGR EERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEI SKLGI SGDIDL TSASYTMIGSPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNP RFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQ YNHRVKKLNEI SKLGI SGDIDLTSASYTMI SHHHHHHHH

(SEQ ID NO: 21).

96:60-Gal-G₄S-Gal8H ("G₄S" disclosed as SEQ ID NO: 14 and "8H" disclosed as SEQ ID NO: 13)

MAPVRSLNCRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKFSMSFVQGEESNDKIPVA LGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFP NWFLCTAMEADQPVSLTNMPDEGVMVTKFYMQFVSSGGGGSGGGGSRPLIVPYNLPLPG GVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGR EERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEI SKLGI SGDIDL TSASYTMIGGGGSPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFH FNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAH LLQYNHRVKKLNEI SKLGI SGDIDLTSASYTMI SHHHHHHHH

(SEQ ID NO: 22).

96:60-Gal-(G₄S)₂-Gal8H ("(G₄S)₂" disclosed as SEQ ID NO: 16 and "8H" disclosed as SEQ ID NO: 13)

MAPVRSLNCRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKFSMSFVQGEESNDKIPVA LGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFP NWFLCTAMEADQPVSLTNMPDEGVMVTKFYMQFVSSGGGGSGGGGSRPLIVPYNLPLPG GVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGR EERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEI SKLGI SGDIDL TSASYTMIGGGGSGGGGSPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGN DVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVA VNDAHLLQYNHRVKKLNEI SKLGI SGDIDLTSASYTMI SHHHHHHHH

(SEQ ID NO: 23).

96:60-Gal-(G₄S)₄-Gal8H ("(G₄S)₄" disclosed as SEQ ID NO: 19 and "8H" disclosed as SEQ ID NO: 13)

MAPVRSLNCRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKFSMSFVQGEESNDKIPVA LGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFP NWFLCTAMEADQPVSLTNMPDEGVMVTKFYMQFVSSGGGGSGGGGSRPLIVPYNLPLPG GVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGR EERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEI SKLGI SGDIDL TSASYTMIGGGGSGGGGSGGGGSGGGGSPLIVPYNLPLPGGVVPRMLITILGTVKPNAN RIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQV LVEPDHFKVAVNDAHLLQYNHRVKKLNEI SKLGI SGDIDLTSASYTMI SHHHHHHHH

(SEQ ID NO: 24).

93:60 (EAAAK)₄ -2XGal8H (rigid linker) ("(EAAAK)₄" disclosed as SEQ ID NO: 20 and "8H" disclosed as SEQ ID NO: 13)

MAPVRSLNCRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKFSMSFVQGEESNDKIPVA LGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFP NWFLCTAMEADQPVSLTNMPDEGVMVTKFYMQFVSSAEAAAKEAAAKEAAAKEAAAKAR PLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVI VCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNE I SKLGI SGDIDLTSASYTMIGGGGSGGGGSGGGGSPLIVPYNLPLPGGVVPRMLITILG TVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESG KPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEI SKLGI SGDIDLTSASYTMI SHHH HHHHH

(SEQ ID NO: 25).

Example 11: Safety of Gal3 CBD containing molecules

Galectin-3 has been implicated as a cardiovascular risk factor (deBoer et al., Curr Heart Fail Rep 7: 1-8, 2010) and an oncogenesis/metastasis risk factor (Zhao et al., Cancer Res

69:6799-806, 2009). These effects appear to derive from the ability of galectin-3 to oligomerize and cluster receptors at the cell surface thereby driving ligand-independent signaling. Fusions using only the C-terminal domain of Gal3 do not oligomerize and therefore are not predicted to demonstrate such adverse effects.

No adverse clinical effects were observed in the studies described herein: Luc 2xGal pharmacokinetic study (murine) in which there was short term dosing (one dose and assessment after three hours for acute toxicity), ST2 2xGal safety study (murine) (two doses/day for three days with evaluation on day four for toxicity and neovascularization), 93:602xGal - Formal Draize test (rabbits) (one dose then Draize test after 15 minutes, 1 hour, 4 hours, 8 hours and 24 hours; the data were negative at all time points).

In view of the accumulated safety-related data, Gal-3 fusions as described herein are predicted to be safe, e.g., for topical ophthalmic use.

Example 12: Gal-3 Anti-VEGF Fusion Protein

In one example, an anti-VEGF Fab-2xGal8H ("8H" disclosed as SEQ ID NO: 13) was constructed using the ranibizumab (LUCENTIS®) sequence, as disclosed in US Patent No. 6884879. This Fab had the following sequences:

Ranibizumab light chain sequence:

MVLQTQVFISLLLWISGAYGDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK VLIYFTSSLHSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE

(SEQ ID NO: 9)

Ranibizumab heavy chain 2xGal8H sequence ("8H" disclosed as SEQ ID NO: 13):

MDWTWRILFLVAAATGAHSEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQA PGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGT SHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKAAAGGGGSGGGGSGGGG SGPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNT KLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEI SKLGISGDI DLTSASYTMI SSGGDGGGGSGGGGSGPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQR GNDVAFHFNPRFNENNRRVI VCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDA HLLQYNHRVKKLNE I SKLGISGDI DLTSASYTMI SRHHHHHHHH

(SEQ ID NO: 10) Example 13: Additional exemplary Gal3 CBD-containing agents

In general, molecules that comprise a Gal 3 protein (that comprises at least one gal3-CBD containing sequence and optionally one or more additional CBD containing sequences and/or other amino acids or amino acid sequences from a Gal3 protein) and a therapeutic or diagnostic agent can be engineered as described herein and/or using methods known in the art. In one example, an anti-IL6 Fab 2xGal8H ("8H" disclosed as SEQ ID NO: 13) is constructed with the following sequences.

Anti-IL6 Fab light chain:

MVLQTQVFISLLLWISGAYGDI VMTQSPDSLAVSLGERATINCRASESVDNYGIPFMNW

YQQKPGQPPKLLIYAASNRGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSEEVPLTFGQ GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

(SEQ ID NO: 11)

Anti-IL6 Fab heavy chain-2xGal8H ("8H" disclosed as SEQ ID NO: 13):

MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGSSVKVSCKASGYALSNYLIEWVRQA PGQGLEWMGVITPGSGTINYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSRWDPLY YYALEYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCAAAGGGGSGG GGSGGGGSGPLI VPYNLPLPGGVVPRMLITILGTVKPNANRI ALDFQRGNDVAFHFNPRFNENNR RVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISK LGISGDIDLTSASYTMISSGGDGGGGSGGGGSGPLIVPYNLPLPGGVVPRMLITILGTVKPNANR IALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHF KVAVNDAHLLQYNHRVKKLNEI SKLGISGDIDLTSASYTMISRHHHHHHHH

(SEQ ID NO: 12)

In another example, a molecule containing the amino acid sequence of a Gal3 protein and an IL-6 modulator (e.g., IL-6 antagonist) as described in International Application No.

PCT/US2013/069279 is produced.

In further examples, a molecule containing the amino acid sequence of a Gal3 protein and another therapeutic agent is produced. The therapeutic agent can be, e.g., tocilizumab, sarilumab, bevacizumab, or ramucirumab (IMC-1121B).

Other embodiments are within the scope of the following claims.

Claims

What is claimed is:

1. An isolated protein comprising

a. a Gal3 carbohydrate binding domain (Gal3 CBD), and

b. a therapeutic or diagnostic agent.

2. The isolated protein of claim 1, wherein the protein comprises two Gal3 CBDs.

3. The isolated protein of claim 1, wherein the Gal3 CBD comprises SEQ ID NO:2 or SEQ ID NO: 4.

4. The isolated protein of claim 1 , further comprising a full length Gal3 amino acid sequence.

5. The isolated protein of any one of claims 1-4, wherein the therapeutic agent is a protein, a polypeptide or a peptide.

6. The isolated protein of any one of claims 1-5, wherein the therapeutic agent comprises a cytokine antagonist.

7. The isolated protein of any one of claims 1-5, wherein the therapeutic agent comprises a cytokine agonist.

8. The isolated protein of any one of claims 1-5, wherein therapeutic agent comprises an antagonist of IL-1, IL-6, IL-17, IL-23, TNF, or VEGF.

9. The isolated protein of any one of claims 1-4, wherein the therapeutic agent is 93:60 (SEQ ID NO: 6).

10. The isolated protein of any one of claims 1-4, wherein the therapeutic agent is an IL-Ιβ, an IL-IRa, a heterologous molecule comprising fragments of an Il-lRa and an II- 1β, or an ST2.

11. The isolated protein of any one of claims 1-10, wherein the diagnostic agent is a fluorescent molecule, enzyme, or heavy metal.

12. The isolated protein of claim 11, wherein the fluorescent protein comprises a green fluorescent protein.

13. A pharmaceutical formulation for application to the eye or surrounding tissue, the formulation comprising the isolated protein of any one of claims 1-12.

14. The pharmaceutical formulation of claim 13, wherein the formulation is suitable for delivery to the vitreous.

15. A method of providing a therapeutic agent to a subject in need thereof, the method comprising administering a therapeutically effective amount of the isolated protein of any one of claims 1-12, or the formulation of any one of claims 13-14, to the eye of the subject.

16. The method of claim 15, wherein the protein is administered intravitreally.

17. The method of claim 15, wherein the protein is administered topically.

18. A vector comprising a nucleic acid sequence encoding the protein of any one of claims 1-4.

19. A cell comprising the vector of claim 18.

20. The pharmaceutical formulation of claim 13, for use in the treatment of an eye disease or condition.

21. The pharmaceutical formulation of claim 20, wherein said eye disease or condition is dry eye disease, Meibomian gland disorder, keratitis, episcleritis, conjunctivitis (e.g., allergic

conjunctivitis, keratoconjunctivitis sicca, or vernal keratoconjunctivitis), keratitis sicca, sicca syndrome, xerophthalmia, tear film disorder, decreased tear production, aqueous tear deficiency, dysfunctional tear syndrome, Meibomian gland dysfunction, dry eye associated with another condition (e.g., Sjogrens syndrome, graft versus host disease, or an autoimmune condition such as mucous membrane pemphigoid), blepharitis , a laser eye surgery or a corneal transplant.