WNT replacement agents and methods for lacrimal gland regeneration

Abstract

Methods and compositions are provided for the treatment of dry eye disorders.In particular, the present disclosure provides methods and compositions for regulating WNT (Wingless and Int-1) signaling in a subject to regulate the regeneration of tear-producing acinar cells to treat aqueous tear-deficient dry eye disease.Furthermore, WNT signaling modulators used in these methods are disclosed.The present invention provides, for example, a method for regenerating lacrimal gland cells in a subject, comprising administering a WNT signaling modulator to the subject.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 291,243, filed December 17, 2021, each of which is incorporated by reference in its entirety herein.

[0002] (Sequence Listing Statement) A Sequence Listing XML associated with this application is provided in XML file format and is hereby incorporated by reference. The XML file containing the Sequence Listing XML is named SRZN_024_01WO_ST26.xml. The XML file is 61,424 bytes and was created on December 15, 2022. The XML file is submitted electronically via the USPTO Patent Center.

[0003] FIELD OF THEINVENTION The present disclosure relates to WNT signal modulators and methods of treatment for various ophthalmic related disorders. [Background technology]

[0004] BACKGROUND OF THEINVENTION The lacrimal gland is an exocrine gland located under the outer lateral portion of the upper eyelid in most vertebrates. The lacrimal gland produces a tear film that is secreted by the lacrimal duct and is associated with ocular health. (See Yao and Zhang, p. 939.) The tear film keeps the corneal surface and inner eyelid moist and protects the corneal and conjunctival epithelium from physical injury and immune responses. (See ibid.).

[0005] Lacrimal gland disorders and damage can lead to significant ocular disease pathology, particularly dry eye disease. (Dartt, Ocular Disease, p. 105). Dry eye disease is characterized by increased osmolality of the tear film and inflammation of the ocular surface. (See ibid.). Inadequate production of tears can result in irritation, pain, and potentially damage to the ocular surface.

[0006] There are two main types of dry eye disease: aqueous-deficient and evaporative. The aqueous-deficient type is in turn subdivided into Sjogren's syndrome (autoimmune) dry eye and non-Sjogren's syndrome (non-autoimmune) dry eye.

[0007] In aqueous-deficient dry eye, the lacrimal gland frequently becomes inflamed, leading to atrophy and cell death of the tear-producing acinar cells. After loss of acinar cells, the tear production problem worsens, resulting in a vicious cycle of increasing inflammation-atrophy. Thus, the lacrimal gland is a target in aqueous-deficient dry eye disease.

[0008] Currently, both aqueous-deficient and evaporative dry eye disease are treated by topical administration of artificial tears and / or anti-inflammatory drugs at the ocular surface. (See ibid., p. 106). Only a few treatments target the lacrimal gland itself by systemic administration of anti-inflammatory drugs, so-called secretagogues, immunosuppressants, and sex hormones. (See ibid.). None of these treatment approaches directly address the atrophy and / or damage of the lacrimal gland (including the tear-producing acinar cells). Consequently, there is a large unmet need for agents and methods to restore endogenous tear production and break the cycle of inflammation and atrophy through regeneration of acinar cells. Summary of the Invention [Means for solving the problem]

[0009] (overview) Provided herein are methods and compositions for treating dry eye disorders through activation and / or regeneration of the lacrimal gland and / or lacrimal acinar cells.

[0010] In one aspect, the present invention includes a method of regenerating lacrimal gland acinar cells, progenitor cells, ductal cells, myoepithelial cells, or immune cells in a subject, the method comprising administering a WNT signaling modulator to the subject. In one embodiment, the WNT signaling modulator can be an engineered WNT signaling modulator. In another embodiment, the WNT signaling modulator is an engineered WNT agonist or an engineered WNT superagonist, or an engineered WNT antagonist. In a particular embodiment, the cells are epithelial stem cells and / or epithelial progenitor cells, such as lacrimal gland epithelial stem cells and / or lacrimal gland epithelial progenitor cells.

[0011] In one embodiment of the method for regenerating lacrimal gland acinar cells, the WNT signaling modulator can comprise at least one engineered bispecific full-length IgG antibody that directly activates the classical WNT signaling pathway.In any embodiment, the engineered WNT agonist can be selected from (i) WNT3a; (ii) WNT mimic; or (iii) R-spondin mimic.The WNT mimic can be SWAP™ compound.The R-spondin mimic can be SWEETS™ compound.

[0012] In one embodiment of the method for regenerating lacrimal gland acinar cells, the WNT signaling modulator can affect the expression of any one or more of Fzd1, Fzd2, Fzd5, Fzd7, Fzd8, and Lrp6, and / or Lrp5. For example, the WNT signaling modulator can target any one or more of the group consisting of Fzd1, Fzd2, and Fzd7; or any one or more of the group consisting of Fzd5 and Fzd8, and also target Lrp6 and / or Lrp5. For another example, the WNT signaling modulator can affect the expression of any one or more of Fzd1, Fzd2, and Fzd7.

[0013] In one embodiment of the method of regenerating lacrimal gland acinar cells, the method further comprises administering at least one of the group consisting of RSPO2, a RSPO2 fragment, and an engineered RSPO2 mimetic.

[0014] In any embodiment, the WNT signaling modulator may be at a concentration of ≧1 nM. In any embodiment, the WNT signaling modulator may be administered in a therapeutically effective amount. In any embodiment, the subject may be a living mammal. In any embodiment, the subject may be a human patient.

[0015] In another aspect, the present invention comprises a method for treating lacrimal gland disorder in a subject, comprising administering a WNT signaling modulator to the subject.In one embodiment, the WNT signaling modulator can be an engineered WNT signaling modulator.In another embodiment, the WNT signaling modulator is an engineered WNT agonist or an engineered WNT antagonist.In a further embodiment, the WNT signaling modulator is an engineered WNT superagonist.

[0016] In one embodiment of the method for treating lacrimal gland disorder, the WNT signaling modulator can comprise at least one engineered bispecific full-length IgG antibody that directly activates the classical WNT signaling pathway.In any embodiment, the engineered WNT agonist can be selected from (i) WNT3a; (ii) WNT mimic; or (iii) R-spondin mimic.The WNT mimic can be SWAP™ compound.The R-spondin mimic can be SWEETS™ compound.

[0017] In one embodiment of the method for treating lacrimal gland disorder, the WNT signaling modulator can affect the expression of any one or more of Fzd1, Fzd2, Fzd5, Fzd7, Fzd8, and Lrp6, and / or Lrp5. For example, the WNT signaling modulator can target any one or more of the group consisting of Fzd1, Fzd2, and Fzd7; or any one or more of the group consisting of Fzd5 and Fzd8, and also target Lrp6 and / or Lrp5. For another example, the WNT signaling modulator can affect the expression of any one or more of Fzd1, Fzd2, and Fzd7.

[0018] In one embodiment of the method of treating a lacrimal gland disorder, the method further comprises administering at least one member of the group consisting of RSPO2, a RSPO2 fragment, and an engineered RSPO2 mimetic.

[0019] In any embodiment of the method for treating lacrimal gland disorder, the WNT signaling modulator can be at a concentration of ≧1 nM. In any embodiment, the WNT signaling modulator can be administered in a therapeutically effective amount. In any embodiment, the subject can be a living mammal. In any embodiment, the subject can be a human patient.

[0020] In yet another aspect, the present invention comprises a composition for treating a dry eye disorder in a subject, the composition comprising a WNT signaling modulator. In any embodiment of the composition, the dry eye disorder can be due to Sjogren's syndrome disorder, chronic graft-versus-host disease (cGHVD), rheumatoid arthritis (RA), Stephen's Johnson syndrome, ocular rosacea, chemotherapy, radiation oncology treatment, diabetes, lupus, etc.

[0021] In one embodiment of the composition, the WNT signaling modulator may comprise at least one engineered bispecific full-length IgG antibody that directly activates the canonical WNT signaling pathway. The at least one engineered bispecific full-length IgG antibody may be specific for any one or more of Fzd1, Fzd2, Fzd5, Fzd7, Fzd8, and Lrp6, and / or Lrp5.

[0022] In one embodiment, the composition may further comprise an anti-inflammatory agent or a lacrimal secretagogue.

[0023] In any embodiment of the composition, the composition may include a therapeutically effective amount of each of its components.

[0024] In any of the embodiments of the composition, the subject may be a living mammal. In any of the embodiments of the composition, the subject may be a human patient. [Brief description of the drawings]

[0025] [Figure 1]Figure 1 shows representative light micrograph images of organoid growth from primary mouse lacrimal gland tissue after 7 days using a standard 3D organoid protocol in Advanced DMEM supplemented with additional growth factors. Conditions included Base with R-spondin 1 (RSPO1) alone (control, top left), or Base with the addition of various WNT mimetic compounds, each at a concentration of 5 nM. "F" represents various FZD binding agents that bind to various FZD receptors (e.g., 18R5-FZD1,2,5,7,8 binding agent ("F12578"); R2H1-FZD1,2,7 binding agent ("F127"); 2919-FZD5,8 binding agent ("F58"); 5063-FZD4 binding agent ("F4"); and HB9L9.3-FZD10 binding agent (F10)); L represents an LRP binding agent (YW211.31.57) that binds to LRP5 and LRP6.

[0026] [Diagram 2] Figure 2 shows photomicrograph images illustrating lacrimal organoid morphology (left), fluorescent and synthetic (right) showing Mist1 acinar cell marker gene expression (center), scale bar 100 µm.

[0027] [Diagram 3] Figures 3A-C show bar graphs depicting relative gene expression of either proliferation markers or markers specific for lacrimal-derived and acinar cells in profiled organoids grown for 7 days in control medium or medium containing the L6-F12578 WNT mimetic compound. Genes measured include the WNT target Axin2 (Figure 3A), the lacrimal cell marker Krt7 (Figure 3B), and the lacrimal acinar cell marker Mist1 (Figure 3C).

[0028] [Figure 4]Figure 4 shows a graph quantifying organoid proliferation by luminescence in a 7-day WNT mimetic screening in acinar cell organoid cultures. The starting material was lacrimal gland acinar cell organoid cells. The medium contained either RSPO1 alone (control) or the addition of a 5 nM concentration of WNT mimetic.

[0029] [Diagram 5] Figure 5 shows optical micrographs showing the morphology of organoids at the end of WNT mimetic screening in control (left panel) or L6-F12578 (right panel). The solid budding morphology in WNT-rich cells is shared with outgrowths from primary tissues, indicating their identity as acinar cells. Scale bar is 200 μm.

[0030] [Figure 6] FIG. 6 shows a graph quantifying viable organoid cells by luminescence (in relative light units (RLU)) in organoid cells grown for 7 days with several doses of L6-F127 WNT mimetic (0.05 nM to 5 nM) with or without RSPO1 (500 ng / mL).

[0031] [Figure 7] FIG. 7 shows a schematic representation of the experimental design for measuring acute WNT target gene induction at 24 and 48 hours after exposure.

[0032] [Figure 8] Figure 8 shows representative light micrograph images of organoid cultures at the time of WNT stimulation at day 7 after plating. Scale bar is 200 μm.

[0033] [Figure 9] Figure 9 shows graphs quantifying WNT target Axin2 expression levels by qPCR at 24 hours (left) and 48 hours (right) after induction. Expression for the various conditions was normalized to actinB expression and compared to control (no WNT mimetic).

[0034] [Figure 10] FIG. 10 shows images of two non-dry eye human lacrimal glands that were used in the explant experiments.

[0035] [Figure 11] Figures 11A and 11B show optical micrographs of human lacrimal gland cell cultures after dissociation (Figure 11A) and after 24 hours in acinar cell medium (Figure 11B). Scale bars in both Figures 11A and 11B are 200 μm.

[0036] [Figure 12] 12 shows quantification of Axin2 expression levels by qPCR 24 hours after initiation of explant culture in RSPO1 alone (control) or RSPO1 + 5 nM WNT mimic. Expression for the various conditions was normalized to actinB expression and compared to control (no WNT mimic).

[0037] [Figure 13] 13 shows WNT receptor expression levels determined by in situ hybridization in naive mouse lacrimal gland tissue. Histology images of light micrographs containing pink probe signal, scale bar 100 μm.

[0038] [Figure 14] 14 shows WNT receptor expression levels determined by in situ hybridization in healthy human lacrimal gland tissue. Histology images of light micrographs containing pink probe signal, scale bar 100 μm.

[0039] [Figure 15]FIG. 15 shows camera-based fluorescence microscopy images of four naive mouse lacrimal cell samples stained with 4',6-diamidino-2-phenylindole (DAPI) and anti-Ki67, a marker for proliferative cells (green), after 14 days of exposure (10 mpk, IP, twice a week): a control (RSPO1 only) sample and three WNT mimetic samples.

[0040] [Figure 16] Figure 16 shows a graph quantifying the relative weight of the lacrimal gland (lacrimal gland weight / body weight) after 14 days of treatment with various WNT mimetics (WNT mimetics 3 mpk, RSPO2 0.1 mpk, IP, twice a week). Figure legend is from left to right on the graph.

[0041] [Figure 17] Figure 17 shows a schematic model of an in vivo mouse experiment in which dry eye disease was modeled by local injection of IL-1a. Red indicates the ipsilateral side where recombinant IL-1a and WNT mimic (or control anti-GFP) are injected. Black indicates the contralateral control side.

[0042] [Figure 18] Figures 18A and 18B show graphs quantifying the expression of WNT target genes Axin2 (Figure 18A) and Rnf43 (Figure 18B) after 8 and 24 hours of exposure to IL-1a + WNT mimic, and IL-1a + anti-GFP. Expression for the various conditions was normalized to actinB expression and compared to the control (8 hours exposure to GFP).

[0043] [Figure 19] FIG. 19 shows the average ipsilateral tear volume secretion over a 5-day time course for four experimental conditions: (1) local administration of WNT mimetic (10 μg, intralacrimal); (2) local administration of anti-GFP (10 μg, intralacrimal); (3) systemic administration of WNT mimetic (200 μg, IP); and (4) systemic administration of anti-GFP (200 μg, IP).

[0044] [Figure 20] Figure 20 shows bar graphs quantifying atrophy / degeneration pathology scoring and inflammation pathology scoring in mice 3 days after lacrimal gland injection with IL-1a and four different experimental treatment conditions: (1) local administration of WNT mimetic (10 μg, intralacrimal gland); (2) local administration of anti-GFP (10 μg, intralacrimal gland); (3) systemic administration of WNT mimetic (200 μg, IP); and (4) systemic administration of anti-GFP (200 μg, IP). Figure legends indicate left to right orientation for each category.

[0045] [Figure 21] Figures 21A and 21B show light micrograph images of lacrimal gland sections stained with hematoxylin and eosin (HE) dye from mice injected with IL-1a + WNT mimic, and IL-1a + anti-GFP. Scale bars in both images are 100 μm.

[0046] [Figure 22] Figure 22 shows salivary gland weights (in grams) after 14 days of twice weekly treatment with 3 mg / kg WNT mimetic. Figure legend indicates left to right orientation on the graph.

[0047] [Diagram 23] Figure 23 shows the histology of salivary glands after administration of various WNT mimetics at 3 mg / kg for 2 weeks. Panel A shows images of salivary gland histology stained with HE by treatment group on day 14. Panel B shows brown staining for Ki67 (proliferation marker) by treatment group on day 14. Scale bar 200 mm.

[0048] [Figure 24]Figures 24A and 24B show histology of salivary glands after 2 weeks of dosing at 10 mg / kg. Figure 24A shows representative images of salivary gland histology by HE staining by treatment group on day 14. Figure 24B shows quantification of mucinous acinar area (white) relative to serous acinar area by Image J by treatment group. Scale bar 200 μm.

[0049] [Diagram 25] 25 is a graph showing salivary gland weight (in grams) after two weeks of treatment with various concentrations of RSPO2-Fc. Figure legends indicate left to right orientation on the graph.

[0050] [Figure 26] Figure 26A and Figure 26B show mouse salivary gland organoid growth. Figure 26A shows the bright field image of the growth of salivary gland organoid from primary tissue on day 7 treated with RSPO1 or RSPO1+L-F12578. Scale bar 200 μm. Figure 26B shows the dose-dependent growth of mouse salivary gland organoid measured as cell viability on day 7 for WNT mimics with different FZD specificities.

[0051] [Figure 27] Figure 27 shows mouse salivary gland organoid WNT receptor profile. Gene expression levels of Fzd and Lrp genes in mouse submandibular gland organoids measured by quantitative PCR.

[0052] [Figure 28] Figures 28A and 28B show the effect of treatment on lupus mouse salivary glands. Figure 28A presents the salivary gland weights (in grams) for control (MRL / MpJ) and lupus (MRL-lpr) mice treated with anti-GFP or L-F12578 for 2 weeks. Figure 28B shows images of salivary gland histology by HE staining for lupus and control mice by treatment group at day 14. Scale bar 200 μm.

[0053] [Figure 29] Figure 29 shows the expression of the WNT target gene Axin2 24 hours after local injection in the IL-1α model. All treatment groups were injected at 10 μg. Data is normalized to the anti-GFP control group. Significant increase for the positive controls L-F12578 and 1SH1-03.

[0054] [Diagram 30] Figure 30 shows the expression of the WNT target gene Axin2 24 hours after local injection in the IL-1a model. All treatment groups except anti-GFP control at three different doses: 10 μg injection, 50 μg injection or 150 μg injection. Data is normalized to the anti-GFP control group. Significant increase for all 1SH1-03 groups and the high dose 1SH1-26 group.

[0055] [Diagram 31] Figure 31 shows tear volume measurements using phenol red thread in the IL-1a model. Data represents the average of 12 animals. Significant tear volume increases in several treatment groups compared to anti-GFP controls on days 2 and 3. On day 2, lines correspond from top to bottom to L-F12578, 1SH1-03, 1SH1-36, 1SH1-26, and anti-GFP.

[0056] [Diagram 32] Figure 32 shows the expression of WNT target gene Axin2 3 days after local injection in IL-1α model. 1SH1-03 treatment groups at two different doses: 10 μg injection or 150 μg injection. Data is normalized to anti-GFP control group. Significant increase of Axin2 in 1SH1-03 high dose on day 3.

[0057] [Diagram 33]Figure 33 shows tear volume measurements using phenol red in the IL-1α model. Data represents the average of 12 animals. Significant tear volume increases in several treatment groups compared to anti-GFP controls on days 2, 3, and 4. On day 3, the lines are, from top to bottom, L-F12578, 1SH1-36, 1SH1-03, 1SH1-26, and anti-GFP.

[0058] [Diagram 34] Figures 34A and 34B show the duct ligation model. Figure 34A is a schematic representation of lacrimal duct ligation on the ipsilateral side (blue). The other side (contralateral, red) was left as a control. Figure 34B is the study schedule of the duct ligation model; tear volume was measured after 3 days of occlusion and removal of the ligature and take down.

[0059] [Diagram 35] Figures 35A and 35B show the damage caused by duct ligation. Figure 35A shows tear volume measurements using phenol red thread after duct closure for 3 days. Ligation results in a strong decrease in tear volume, which recovers over 2-3 weeks. At day 0, the upper line is contralateral and the lower line is ipsilateral. Figure 35B shows a representative image of lacrimal gland histology after duct ligation. Some atrophy at day 7, which recovers slowly compared to controls at days 14 and 21.

[0060] [Diagram 36] Figure 36 shows the expression of WNT target gene Axin2 at 24 hours after 3 days of duct closure treatment in duct ligation model. Treatment groups with two different doses: 10 μg injection or 100 μg injection. Data is normalized to anti-GFP control group. Significant increase of Axin2 in 1SH1-03 and positive control L-F12578.

[0061] [Figure 37]Figure 37. One week tear volume measurement using phenol red thread after 3 days of duct occlusion. Significant increase in tear volume at day 7 in positive control L-F12578 and 100 μg of 1SH1-03. At day 7, the lines are, from top to bottom, L-F12578 (10 μg), 1SH1-03 (100 μg), 1SH1-03 (10 μg), and anti-GFP.

[0062] [Figure 38] Figure 38 shows quantification of proliferating acinar cells (Mist1+ Ki67+) in ipsilateral and contralateral glands on day 7 of the duct ligation study (from Figure 34). Increase in proliferative cells in the ipsilateral and contralateral sides of high dose 1SH1-03. For each set of bars, from left to right, the bars are GFP, L-12578 (10 μg), 1SH1-03 (10 μg), and 1SH1-03 (100 μg).

[0063] [Figure 39] Figure 39 shows 2-week tear volume measurements using phenol red thread after 3 days of duct occlusion. Significant increase in tear volume after 7 days in positive controls L-F12578 and 1SH1-03. At the last time point, the lines are, from top to bottom, L-F12578 (10 μg), 1SH1-03 (100 μg), 1SH1-03 (10 μg), and anti-GFP.

[0064] [Diagram 40] Figure 40 shows quantification of proliferating acinar cells (Mist1+ Ki67+) in ipsilateral and contralateral glands on day 14 of the duct ligation study (from Figure 39). Increase in proliferative cells in the ipsilateral and contralateral sides of high dose 1SH1-03. For each set of bars, from left to right, the bars correspond to GFP, G211-18R5, 1SH1-03 (10 μg), and 1SH-03 (100 μg).

[0065] [Diagram 41]Figure 41A and Figure 41B show the in vitro proliferation of acinar cells. Figure 41A shows representative images of lacrimal cells treated with control or 10 nM 1SH1-03. WNT activation using 1SH1-03 produces larger organoids at day 7. Figure 41B shows quantification of cell viability of organoids at day 7 grown in control medium or various doses of 1SH1-03. Dose-dependent effect with significantly more cells using 1SH1-03.

[0066] [Diagram 42] Figure 42 shows the quantification of salivary gland proliferation in animals during 2 weeks of systemic administration in animals. Administration of L-F12578 has more proliferative epithelial cells (Ki67+ / ECad+) on the 7th day compared with the control group and other time points. For each time point, vehicle is on the left and L-F12578 is on the right. For each time point, the left bar is vehicle and the right bar is L-F12578.

[0067] [Diagram 43] Figure 43 shows the quantification of salivary gland tissue weight in animals during 2 weeks of systemic administration in animals. Administration of L-F12578 significantly increases organ weight compared to control on days 7, 9, 11 and 14. For each time point, vehicle is on the left and L-F12578 is on the right. For each time point, the left bar is vehicle and the right bar is L-F12578. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] It will be readily apparent to one skilled in the art that the above diagrams are illustrative only and do not limit the scope of the present disclosure.

[0069] (Detailed Description) (I. Definition) As used in this application (including the appended claims), singular terms such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0070] All references cited herein are incorporated by reference to the same extent as if each individual publication, patent application, or patent was specifically and individually indicated to be incorporated by reference.

[0071] The "activity" of a molecule may describe or refer to the binding of the molecule to a ligand or receptor, catalytic activity, ability to stimulate gene expression, antigenic activity, modulation of the activity of other molecules, etc. The "activity" of a molecule may also refer to activity in modulating or maintaining cell-cell interactions (e.g., adhesion), or activity in maintaining the structure of a cell (e.g., cell membrane or cytoskeleton). "Activity" may also refer to specific activity, such as [catalytic activity] / [mg protein], or [immunological activity] / [mg protein], etc.

[0072] As used herein, the term "administering" or "introducing" or "providing" refers to the delivery of a composition to a cell, a subject's cell, a subject's tissue, and / or a subject's organ, or to a subject. Such administering or introducing can be performed in vivo, in vitro, or ex vivo.

[0073] As used herein, the term "antibody" refers to an isolated or recombinant binding agent that contains the necessary variable region sequence to specifically bind to an antigen epitope. Thus, an antibody is any form of antibody or fragment thereof that exhibits desired biological activity (e.g., binding to a specific target antigen). Thus, it is used in the broadest sense, specifically covering monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (including but not limited to scFv, Fab, and Fab2) as long as they exhibit desired biological activity.

[0074] An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments; diabodies; linear antibodies (e.g., Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments (called "Fab" fragments, each with a single antigen-binding site) and a residual "Fc" fragment (a designation reflecting the ability to crystallize readily). Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking to antigen.

[0075] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent (e.g., an antibody) and can be further used in an animal to generate antibodies capable of binding to an epitope of that antigen. In certain embodiments, a binding agent (e.g., a WNT surrogate molecule or binding region thereof, or a WNT antagonist) is said to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0076] The term "antigen-binding fragment" as used herein refers to a polypeptide fragment that contains at least one complementarity determining region (CDR) of an immunoglobulin heavy and / or light chain, or at least one complementarity determining region (CDR) of a VHH / sdAb (single domain antibody) or Nanobody® (Nab), that binds to an antigen of interest (particularly one or more FZD receptors, or LRP5 and / or LRP6). In this regard, the antigen-binding fragment of an antibody described herein may contain one, two, three, four, five, or all six CDRs of the VH and VL from an antibody that binds to one or more FZD receptors, or LRP5 and / or LRP6.

[0077] As used herein, the terms "biological activity" and "biologically active" refer to the activity resulting from a particular biological element in a cell. For example, the "biological activity" of a WNT agonist or its fragment or variant refers to its ability to mimic or enhance WNT signal. As another example, the biological activity of a polypeptide or its functional fragment or variant refers to the ability of the polypeptide or its functional fragment or variant to perform its native function, such as, for example, binding, enzymatic activity, etc. In some embodiments, a functional fragment or variant retains at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the activity of the corresponding native protein or nucleic acid. As a third example, the biological activity of a gene regulatory element (e.g., promoter, enhancer, Kozak sequence, etc.) refers to the ability of the regulatory element or its functional fragment or variant to regulate (i.e., promote, enhance, or activate the transcription of) the expression of the gene to which it is operably linked, respectively.

[0078] As used herein, the term "bifunctional antibody" refers to an antibody that contains a first arm that has specificity for one antigenic site and a second arm that has specificity for another antigenic site, i.e., the bifunctional antibody has dual specificities.

[0079] "Bispecific antibodies" are used herein to refer to full-length antibodies generated by quadroma technology (see Milstein et al., Nature, 305(5934):537-540 (1983)), by chemical conjugation of two different monoclonal antibodies (see Staerz et al., Nature, 314(6012):628-631 (1985)), or by a knob-into-hole approach or similar approach, which introduces mutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14):6444-6448 (1993)) to generate multiple different immunoglobulin species, only one of which is a functional bispecific antibody. A bispecific antibody binds one antigen (or epitope) in one of its two binding arms (one HC / LC pair) and binds another antigen (or epitope) in its second arm (another HC / LC pair). By this definition, a bispecific antibody has two antigen-binding arms that are distinct (in both specificity and CDR sequences) and is monovalent for each antigen to which it binds.

[0080] By "comprising," it is meant that the recited elements are required for, e.g., a composition, method, kit, etc., but that other elements may be included to form, e.g., a composition, method, kit, etc., that is within the scope of the claim. For example, an expression cassette that "comprises" a gene encoding a therapeutic polypeptide operably linked to a promoter is an expression cassette that may include other elements in addition to that gene and promoter (e.g., polyadenylation sequences, enhancer elements, other genes, linker domains, etc.).

[0081] By "consisting essentially of" is meant a limitation of the scope of the described, e.g., composition, method, kit, etc., to specific substances or steps that do not substantially affect the basic and novel characteristics of the described, e.g., composition, method, kit, etc. For example, an expression cassette "consisting essentially of" a gene encoding a therapeutic polypeptide operably linked to a promoter and polyadenylation sequence may include additional sequences (e.g., linker sequences) so long as they do not substantially affect the transcription or translation of the gene. As another example, a variant or mutant polypeptide fragment "consisting essentially of" a described sequence has the amino acid sequence of the described sequence plus or minus about 10 amino acid residues (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 less than the described boundary amino acid residues, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more than the described boundary amino acid residues) at the boundaries of the sequence, based on the full-length native polypeptide from which it is derived.

[0082] By "consisting of" it is meant the exclusion of any element, step, or ingredient from the composition, method, or kit not specified in the claim. For example, a polypeptide or polypeptide domain "consisting of" a recited sequence includes only the recited sequence.

[0083] A "control element" or "control sequence" is a nucleotide sequence involved in molecular interactions that contribute to the functional regulation of a polynucleotide, including the replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation can affect the frequency, rate, or specificity of the process, and can be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences, such as promoters and enhancers. A promoter is a DNA region that, under certain conditions, can bind RNA polymerase and initiate transcription of a coding region that is usually located downstream (3' direction) from the promoter.

[0084] An "expression vector" is a vector (e.g., a plasmid, a minicircle, a viral vector, a liposome, etc.) discussed herein or known in the art that contains a region encoding a gene product of interest and is used to effect expression of that gene product in an intended target cell. An expression vector also contains control elements (e.g., a promoter, an enhancer, an untranslated region (UTR), an miRNA target sequence, etc.) operably linked to the coding region to promote expression of the gene product in the target. The combination of a gene or genes operably linked to a control element for expression is sometimes called an "expression cassette", and many expression cassettes are known and available in the art or can be easily constructed from components available in the art.

[0085] As used herein, the term "FR set" refers to four adjacent amino acid sequences that form the CDRs of the CDR set of a heavy chain V region or a light chain V region. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen-binding site, and in particular, the FR residues are directly adjacent to the CDRs. Within FRs, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of about 90 amino acid residues. When the V regions are folded into the binding site, the CDRs are presented as protruding loop motifs that form an antigen-binding surface. It is generally recognized that there are conserved structural regions of FRs that affect the shape of the CDR loops folded into a certain "classical" structure, regardless of their exact CDR amino acid sequence. In addition, certain FR residues are known to participate in non-covalent interdomain contacts that stabilize the interaction between the heavy and light chains of the antibody.

[0086] The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans and non-human primates, including monkeys and humans; mammalian sport animals, such as horses; mammalian farm animals, such as sheep, goats, etc.; mammalian pets, such as dogs, cats, etc.; and rodents, such as mice, rats, etc.

[0087] A "humanized" antibody or fragment thereof refers to an antibody or fragment thereof derived from a non-human species whose protein sequence has been modified to increase its similarity to antibody variants naturally produced in humans. The process of "humanization" is typically applied to monoclonal antibodies developed for administration to humans.

[0088] "Monoclonal antibody" refers to a homogeneous antibody population, where the monoclonal antibody is composed of amino acids (naturally occurring and non-naturally occurring amino acids) involved in selective binding of an epitope. Monoclonal antibodies are highly specific and directed against a single epitope. The term "monoclonal antibody" includes not only intact and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain (scFv), single domain antibodies (sdAb (also known as nanobody)), variants thereof, fusion proteins containing antigen-binding fragments of monoclonal antibodies, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configurations of immunoglobulin molecules containing antigen-binding fragments (epitope recognition sites) of the required specificity and epitope binding ability, including the WNT surrogate molecules disclosed herein. It is not intended to be limited as regards to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as fragments such as those fragments listed above under the definition of "antibody."

[0089] The term "native" or "wild type" as used herein refers to a nucleotide sequence (e.g., a gene or gene product, e.g., RNA or protein) that exists in a wild-type cell, tissue, organ, or organism. The term "variant" as used herein refers to a variant of a reference polynucleotide sequence or a reference polypeptide sequence (e.g., a native polynucleotide sequence or a native polypeptide sequence), i.e., has less than 100% sequence identity with the reference polynucleotide sequence or the reference polypeptide sequence. In other words, a variant comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) compared to a reference polynucleotide sequence (e.g., a native polynucleotide sequence or a native polypeptide sequence). For example, a variant can be a polynucleotide that has 50% or more, 60% or more, or 70% or more sequence identity with the full-length native polynucleotide sequence (e.g., 75% or 80% or more, e.g., 85%, 90%, or 95% or more identity, e.g., 98% or 99% identity with the full-length native polynucleotide sequence). As another example, a variant can be a polypeptide having 70% or more sequence identity to a full-length native polypeptide sequence (e.g., 75% or 80% or more, e.g., 85%, 90%, or 95% or more identity, e.g., 98% or 99% identity, to the full-length native polypeptide sequence). A variant can also include a variant fragment of a reference (e.g., native) sequence that shares 70% or more sequence identity with a fragment of the reference (e.g., native) sequence (e.g., 75% or 80% or more, e.g., 85%, 90%, or 95% or more identity, e.g., 98% or 99% identity, to the native sequence).

[0090] "Operably linked" or "operably linked" refers to the juxtaposition of genetic elements in which they are in a relationship that allows them to function in an expected manner. For example, a promoter is operably linked to a coding region if the promoter helps initiate transcription of the coding sequence. There can be intervening residues between the promoter and the coding region so long as this functional relationship is maintained.

[0091] As used herein, the terms "polypeptide," "peptide," and "protein" refer to amino acid polymers of any length. The terms also include amino acid polymers that have been modified (e.g., to include disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component).

[0092] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be added before or after the assembly of the polymer. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide includes both its double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0093] A polynucleotide or polypeptide has a certain "sequence identity" percentage with another polynucleotide or polypeptide, which means that when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. The term "identical" or "identity" when used in the context of two or more nucleic acid sequences or polypeptide sequences refers to the number or percentage of residues that are the same in the sequence of interest and in the reference sequence. The percentage can be calculated by optimally aligning the sequence of interest with the reference sequence; comparing the two sequences over the entire length of the reference sequence; determining the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matched positions; dividing the number of matched positions by the total number of positions in the reference sequence adjusted by adding the number of gap positions introduced in the reference sequence when making the alignment; and multiplying the result by 100 to obtain the percentage of sequence identity. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Sequence "identity" may be determined by using a stand-alone executable BLAST engine program (bl2seq) for blast analysis of two sequences with its default parameters, which may be retrieved from the National Center for Biotechnology Information (NCBI) ftp site or from the World Wide Web at ncbi.nlm.nih.gov / BLAST / (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250, which is incorporated herein by reference in its entirety).

[0094] As used herein, a "promoter" includes a DNA sequence that induces binding to RNA polymerase, thereby facilitating RNA synthesis, i.e., a minimal sequence sufficient to induce transcription. The promoter and the expression of the corresponding protein or polypeptide can be ubiquitous, meaning that it is strongly active in a wide range of cells, tissues, and species, or cell type-specific, tissue-specific, or species-specific. The promoter can be "constitutive," meaning that it is continuously active, or "inducible," meaning that it can be activated or inactivated by the presence or absence of a biotic or abiotic factor. Enhancer sequences, which may or may not be contiguous with the promoter sequence, are also included in the nucleic acid construct or vector of the present invention. Enhancer sequences affect promoter-dependent gene expression, and can be located in the 5' or 3' region of the native gene.

[0095] "Recombinant" as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in constructs that differ from polynucleotides found in nature.

[0096] As used herein, "Sjoegren's syndrome" or "Sjogren's syndrome" is a chronic autoimmune disease affecting the lacrimal and salivary glands, defined according to conventional international diagnostic criteria as described in Vitali et al. (2002) Ann. Rheum. Dis. 61:554. Sjogren's syndrome is one of two major subclasses of aqueous-deficient dry eye disease, and is distinct from non-Sjogren's syndrome types.

[0097] As used herein, "SWAP™" (Surrozen WNT signal activating protein) refers to a WNT mimetic compound that includes an engineered bispecific full-length immunoglobulin G (IgG) antibody that directly activates the classical WNT signaling pathway in target tissues (e.g., lacrimal gland tissue) in a manner similar to WNT protein.

[0098] As used herein, "SWEETS™" (Surrozen WNT signal enhancer engineered for tissue specificity) refers to antibody-based R-spondin mimetic compounds as described in US20200048324.

[0099] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms (e.g., reducing the likelihood of the disease or its symptoms developing in a subject), and / or may be therapeutic, in terms of partially or completely curing a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, including: (a) inhibiting the disease, i.e., arresting or slowing its development; or (b) relieving the disease, i.e., causing a regression or reduction in the disease or reducing the severity of the disease. Therapeutic agents may be administered before, during, or after the onset of disease or injury. Treatment of ongoing disease is of particular interest if the treatment stabilizes or reduces undesirable clinical symptoms in the patient. Such treatment is desirably performed before complete loss of function in the affected tissue. The treatment is desirably administered during, and in some cases after, the symptomatic stage of the disease.

[0100] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology, microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are fully explained in such references as "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (edited by M. J. Gait, 1984); "Animal Cell Culture" (edited by R. I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (edited by D. M. Weir and C. C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (edited by J. M. Miller and M. P. Calos, 1987); "Current Protocols in Molecular Biology" (edited by F. M. Usubel et al., 1987); "PCR: The Polymerase Chain Reaction" (edited by Mullis et al., 1994); and "Current Protocols in Immunology" (edited by J. E. Coligan et al., 1991), each of which is expressly incorporated herein by reference.

[0101] Some aspects of the present invention are described below with reference to exemplary applications for illustration. It should be understood that numerous specific details, relationships, and methods are shown to provide a thorough understanding of the present invention. However, those skilled in the relevant art will readily recognize that the present invention can be implemented without one or more of those specific details, or with other methods. The present invention is not limited by the illustrated order of acts or events. This is because some acts may exist in another order and / or simultaneously with other acts or events. Moreover, not all illustrated acts or events are required to implement a method according to the present invention.

[0102] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including," "include," "having," "has," "with," or variations thereof are used in any of the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0103] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined (i.e., the limitations of the measurement system). For example, "about" can mean within one standard deviation or within more than one standard deviation, according to practice in the art. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within a 5-fold range, and more preferably within a 2-fold range of a value. When a particular value is described in this application and claims, unless specifically stated to the contrary, it should be assumed that the term "about" means within an acceptable error range for that particular value.

[0104] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited, and it is understood that the present disclosure supersedes any disclosure of the incorporated publications to the extent there is a conflict.

[0105] It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for use of exclusive terminology such as "solely," "only," and the like, or for use of a "negative" limitation in relation to the recitation of claim elements.

[0106] Unless otherwise indicated, all terms used herein have the same meaning as they would to a person skilled in the art, and the practice of the present invention employs conventional techniques of microbiology and recombinant DNA technology, which are within the knowledge of those skilled in the art.

[0107] (II. Overview) The present invention provides methods of modulating WNT signaling to treat lacrimal gland disorders resulting from, including but not limited to, Sjogren's syndrome disorders, chronic graft-versus-host disease (cGHVD), rheumatoid arthritis (RA), Stevens-Johnson syndrome, ocular rosacea, chemotherapy, radiation oncology treatment, diabetes, roasacea, lupus, and the like.

[0108] WNT ("Wingless-associated integration site" or "Wingless and Int-1" or "Wingless-Int") ligands and their signals play important roles in controlling the development, homeostasis and regeneration of many essential organs and tissues, including bone, liver, skin, stomach, intestine, kidney, central nervous system, mammary gland, taste buds, ovary, cochlea, lung, and many tissues (reviewed, for example, by Clevers, Loh, and Nusse, 2014;346:1248012). Modulation of the WNT signaling pathway has potential for the treatment of degenerative diseases and tissue damage.

[0109] One of the difficulties in modulating WNT signaling as a therapeutic approach is the existence of multiple WNT ligands and WNT receptors Frizzled 1–Frizzled 10 (Fzd1–Fzd10), with many tissues expressing multiple overlapping Fzds. In addition to Fzds, classical WNT signaling also involves low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) or low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) as co-receptors, which are widely expressed in various tissues. R-spondin1–R-spondin4 are a family of ligands that amplify WNT signaling. Each of these R-spondins functions through a receptor complex that contains Zinc and Ring finger 3 (ZNRF3) or Ring finger protein 43 (RNF43) at one end and Leucine-rich repeat-containing G protein-coupled receptor 4-6 (LGR4-LGR6) at the other end (e.g., reviewed by Knight and Hankenson, 2014, Matrix Biol. 37:157-161). R-spondins may also function through additional mechanisms of action. ZNRF3 and RNF43 are two membrane-bound E3 ligases that specifically target the WNT receptor (Fzd1-Fzd10 and LRP5 or LRP6) for degradation. Binding of R-spondin to ZNRF3 / RNF43 and LGR4-LGR6 causes removal or sequestration of the ternary complex, which removes the E3 ligase from the WNT receptor and stabilizes the WNT receptor, resulting in enhanced WNT signaling. Each R-spondin contains two Furin domains (1 and 2), Furin domain 1 binds ZNRF3 / RNF43, and Furin domain 2 binds LGR4-LGR6. An R-spondin fragment containing Furin domains 1 and 2 is sufficient to amplify WNT signaling. Although the effect of R-spondin depends on WNT signaling, both LGR4-LGR6 and ZNRF3 / RNF43 are widely expressed in various tissues, so the effect of R-spondin is not tissue specific.

[0110] Activating WNT signaling by WNT agonist can be used to treat various lacrimal gland diseases and disorders, including dry eye disease.Similarly, amplifying WNT signaling by RSPO or RSPO mimic can be used to treat various lacrimal gland diseases and disorders, including various dry eye disease and salivary gland disease.WNT agonist molecules can also be used to treat dry eye disease and salivary gland disorder.In particular, active WNT signaling can provide key stem cell maintenance signals, and plays an important role in regulating the regeneration of acinar cells in, for example, salivary gland.

[0111] III. Engineered WNT Agonists The present disclosure provides engineered WNT agonists, and the present disclosure contemplates the use of the engineered WNT agonists to stimulate, activate, or promote WNT signaling, for example, through the canonical WNT / β-catenin signaling pathway. Such engineered WNT agonists may also be referred to as WNT / β-catenin signaling agonists or Wnt mimetics.

[0112] The present disclosure provides engineered Wnt mimetics with drug-like properties, in particular bispecific antibodies that are recombinant forms that stimulate signaling with Fzd and Lrp together to mimic endogenous Wnt ligands.The Wnt mimetics of the present disclosure can freely diffuse and access damaged tissues, leading to tissue repair where Wnt signals are required.The present disclosure also provides Wnt mimetics that can repair damaged lacrimal or salivary gland tissues without being combined with RSPO.

[0113] In some embodiments, the WNT / β-catenin signaling antagonist or WNT / β-catenin signaling agonist may contain a binding agent or epitope binding domain that binds to one or more FZD receptors and inhibits or enhances WNT signaling. In certain embodiments, the agent or antibody specifically binds to the cysteine-rich domain (CRD) in the human frizzled receptor that it binds to. In addition, antagonistic binding agents that contain an epitope binding domain for LRP may also be used. In some embodiments, the WNT / β-catenin antagonist possesses a binding agent or epitope binding domain that binds to E3 ligase ZNRF3 / RNF43 and one or more FZD receptors or one or more LRP co-receptors and promotes the degradation of FZD receptors or LRP receptors, and the molecule may also contain a binding domain that binds to a cell type-specific epitope for targeting. The E3 ligase agonist antibody or fragment thereof may be a single molecule or may be combined with other WNT antagonists (eg, FZD receptor antagonists, LRP receptor antagonists, etc.).

[0114] As is well known in the art, an antibody is an immunoglobulin molecule capable of specifically binding to a target (e.g., carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one epitope-binding domain located in its variable region. As used herein, the term refers to intact polyclonal or monoclonal antibodies as well as fragments thereof comprising the epitope-binding domain (e.g., dAb, Fab, Fab', (F(ab')2, Fv, single chain (scFv), VHH (i.e., Nanobody®) or single domain antibodies (sdAb), DVD-Ig (also known as Fv-Ig), synthetic variants thereof, naturally occurring variants, fusion proteins comprising the epitope-binding domain, humanized antibodies, chimeric antibodies, and antigen-binding fragments thereof. The term "diabodies" includes any other modified configuration of an immunoglobulin molecule that contains a binding site or a fragment of the required specificity (epitope recognition site). "Diabodies" (WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA, 90, 6444-6448, 1993), which are multivalent or multispecific fragments constructed by gene fusion, are also specific forms of antibodies contemplated herein. Minibodies, which contain scFvs linked to a CH3 domain, are also included herein (S. Hu et al., Cancer Res., 56, 3055-3061, 1996). For example, Ward, E. S. et al., Nature, 341, 544-546 (1989); Bird et al., Science, 242, 423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883, 1988); PCT / US92 / 09965; WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA, 90, 6444-6448, 1993; Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996; S. Hu et al., Cancer Res., 56, 3055-3061, 1996; C. Bever et al., Anal See Bioanal Chem. 2016 Sep;408(22);5985-6002.

[0115] The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each contain a covalently linked heterodimer that contains an intact antigen binding site. The enzyme pepsin can cleave IgG molecules to provide several fragments, including the F(ab')2 fragment that contains both antigen binding sites. Fv fragments for use according to certain embodiments of the present disclosure can be generated by preferential proteolytic cleavage of IgM immunoglobulin molecules, and more rarely by proteolytic cleavage of IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly derived using recombinant techniques known in the art. The Fv fragments contain a non-covalent VH::VL heterodimer that contains an antigen binding site that retains much of the antigen recognition and binding capabilities of the native antibody molecule. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA, 69:2659-2662; Hochman et al. (1976) Biochem, 15:2706-2710; and Ehrlich et al. (1980) Biochem, 19:4091-4096.

[0116] In certain embodiments, single chain Fv or scFV antibodies are contemplated. For example, Kappa bodies (Ill et al., Prot. Eng., 10:949-57 (1997)); minibodies (Martin et al., EMBO J, 13:5305-9 (1994)); diabodies (Holliger et al., Proc. Nat. Acad. Sci. 90:6444-8 (1993)); or Janusins ​​(Traunecker et al., EMBO J, 10:3655-59 (1991) and Traunecker et al., Int. J. Cancer, Suppl. 7:51-52 (1992)) can be prepared using standard molecular biology techniques following the techniques of the present application for selecting antibodies with desired specificity. In yet other embodiments, bispecific or chimeric antibodies can be made that include the ligands of the present disclosure. For example, chimeric antibodies can contain CDRs and framework regions from different antibodies, but bispecific antibodies can be made that specifically bind to one or more FZD receptors through one binding domain and to a second molecule through a second binding domain. These antibodies can be made through recombinant molecular biology techniques or can be physically conjugated together.

[0117] Single-chain Fv (scFv) polypeptides are covalently linked VH:VL heterodimers, which are expressed from gene fusions containing VH- and VL-encoding genes linked by a peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci. USA, 85(16):5879-5883. Numerous methods have been described for identifying chemical structures for converting naturally aggregated (but chemically separated) polypeptide light and heavy chains derived from antibody V regions into scFv molecules, which fold into a three-dimensional structure substantially similar to the structure of the antigen-binding site. See, for example, U.S. Pat. Nos. 5,091,513 and 5,132,405 to Huston et al., and U.S. Pat. No. 4,946,778 to Ladner et al.

[0118] In certain embodiments, the antibody described herein is in the form of a diabody.A diabody is a multimer of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, and these two domains are linked (e.g., by a peptide linker) but cannot associate with each other to form an antigen-binding site: the antigen-binding site is formed by the association of the first domain of one polypeptide in the multimer with the second domain of another polypeptide in the multimer (WO94 / 13804).

[0119] A dAb fragment of an antibody consists of the VH domain (Ward, ES et al. (1989) Nature 341:544-546).

[0120] When bispecific antibodies are used, they can be conventional bispecific antibodies, which can be produced in a variety of ways (Holliger, P. and Winter G., Curr. Opin. Biotech., 4, 446-449 (1993)) (e.g., prepared chemically or from hybrid hybridomas), or can be any of the bispecific antibody fragments described above. Diabodies and scFvs can be constructed using only variable domains without the Fc region, potentially reducing the effects of anti-idiotypic reactions.

[0121] Bispecific diabodies are also particularly useful, as opposed to whole bispecific antibodies, because they can be easily constructed and expressed in E. coli. Diabodies (and many other polypeptides, such as antibody fragments) of appropriate binding specificity can be easily selected from libraries using phage display (WO94 / 13804). If one arm of the diabody is kept constant, for example with specificity for antigen X, a library can be made in which the other arm is varied, and antibodies of appropriate specificity can be selected. Whole bispecific antibodies can be made by knob-into-hole engineering (JBB Ridgeway et al., Protein Eng., 9, 616-621 (1996)).

[0122] In certain embodiments, the antibodies described herein may be provided in the form of a UniBody®. A UniBody® is an IgG4 antibody with the hinge region removed (see GenMab, Utrecht, The Netherlands; see also, e.g., US2009 / 0226421). This proprietary antibody technology creates a stable, smaller antibody format that is expected to have a longer therapeutic window than current small antibody formats. IgG4 antibodies are considered to be inert and therefore do not interact with the immune system. Fully human IgG4 antibodies can be modified by removing the antibody hinge region, resulting in half-molecule fragments with distinct stability characteristics compared to the corresponding intact IgG4 (GenMab, Utrecht). Halving the IgG4 molecule leaves only one region on the UniBody® that can bind to the cognate antigen (e.g., disease target), and therefore the UniBody® binds monovalently to only one site on the target cell.

[0123] In certain embodiments, the antibodies and antigen-binding fragments thereof described herein comprise a set of heavy chain CDRs and a set of light chain CDRs, which are inserted between a set of heavy chain framework regions (FRs) and a set of light chain framework regions (FRs), respectively, which provide support for the CDRs and define the spatial relationship of the CDRs to each other. As used herein, the term "set of CDRs" refers to the three hypervariable regions of the heavy chain V region or the light chain V region. Starting from the N-terminus of the heavy chain or the light chain, these regions are designated as "CDR1", "CDR2" and "CDR3", respectively. Thus, the antigen-binding site comprises six CDRs, which comprise a set of CDRs from each of the heavy chain V region and the light chain V region. A polypeptide comprising a single CDR (e.g., CDR1, CDR2 or CDR3) is referred to herein as a "molecular recognition unit". Crystallographic analyses of many antigen-antibody complexes have demonstrated that the amino acid residues in the CDRs make extensive contacts with the bound antigen, the most extensive of which are with the heavy chain CDR3, and thus these molecular recognition units are primarily responsible for the specificity of the antigen-binding site.

[0124] As used herein, the term "FR set" refers to four adjacent amino acid sequences that form the CDRs of the CDR set of a heavy chain V region or a light chain V region. Some FR residues may contact the antigen that binds; however, FRs are primarily responsible for folding the V region into the antigen-binding site, and in particular, the FR residues are directly adjacent to the CDRs. Within FRs, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide loop of about 90 amino acid residues. When the V regions are folded into the binding site, the CDRs are presented as protruding loop motifs that form an antigen-binding surface. It is generally recognized that there are conserved structural regions of FRs that affect the shape of the CDR loops folded into a certain "classical" structure, regardless of their exact CDR amino acid sequence. In addition, certain FR residues are known to participate in non-covalent interdomain contacts that stabilize the interaction between the heavy and light chains of the antibody.

[0125] "Monoclonal antibody" refers to a homogeneous antibody population, where the monoclonal antibody is composed of amino acids (naturally occurring and non-naturally occurring amino acids) involved in selective binding of an epitope. Monoclonal antibodies are highly specific and directed against a single epitope. The term "monoclonal antibody" includes not only intact and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain (scFv), Nanobody®, variants thereof, fusion proteins containing antigen-binding fragments of monoclonal antibodies, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configurations of immunoglobulin molecules containing antigen-binding fragments (epitope recognition sites) of the required specificity and epitope binding ability, including WNT surrogate molecules disclosed herein. It is not intended to be limited with respect to the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins as well as the fragments and the like referred to above under the definition of "antibody."

[0126] In certain embodiments, the antibodies of the present disclosure may take the form of single domain antibodies (sdAbs). sdAb technology was originally developed after the discovery and identification that camelids (e.g., camels, alpacas, and llamas) possess fully functional antibodies that consist only of heavy chains and thus lack light chains. These heavy chain-only antibodies contain a single variable domain (VHH) and two constant domains (CH2, CH3). The single variable domains, once cloned and isolated, have full antigen-binding capacity and are highly stable. These single variable domains, with their unique structural and functional properties, form the basis of sdAbs. The sdAb is encoded by a single gene and is efficiently produced in almost all prokaryotic and eukaryotic hosts, such as E. coli (see, e.g., U.S. Pat. No. 6,765,087), molds (e.g., Aspergillus or Trichoderma), and yeasts (e.g., Saccharomyces, Kluyvermyces, Hansenula, or Pichia (see, e.g., U.S. Pat. No. 6,838,254). The production process is scalable and can be used to produce multi-kilogram quantities of sdAb. b has been produced. sdAbs can be formulated as ready-to-use solutions with long shelf life. The Nanoclone® method (see, e.g., WO06 / 079372) is a proprietary method for generating sdAbs against desired targets, based on automated high-throughput selection of B cells. sdAbs are single-domain antigen-binding fragments of camelid-specific, heavy-chain-only antibodies. sdAbs (also called VHH antibodies) typically have a small size of about 15 kDa. See C. Bever et al., Anal Bioanal Chem., September 2016;408(22);5985-6002.

[0127] Another contemplated antibody fragment is the dual variable domain-immunoglobulin (DVD-Ig or Fv-Ig), an engineered protein that combines the functions and specificities of two monoclonal antibodies in one molecular entity. Fv-Ig is designed as an IgG-like molecule, except that each light and heavy chain contains two variable domains in tandem with a short peptide bond, instead of one variable domain in IgG. The orientation of the fusion of the two variable domains and the selection of the linker sequence are important for the functional activity and efficient expression of the molecule. Fv-Ig can be produced by conventional mammalian expression systems as a single species for manufacturing and purification. Fv-Ig has the specificity of its parent antibody, is stable in vivo, and exhibits IgG-like physicochemical and pharmacokinetic properties. Fv-Ig and methods for its production are described in Wu, C. et al., Nat Biotech, 25:1290-1297 (2007).

[0128] In certain embodiments, the antibody or antigen-binding fragment thereof disclosed herein is humanized. This refers to a chimeric molecule, generally prepared using recombinant techniques, having an antigen-binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise either a complete variable domain fused to a constant domain or just the CDRs grafted into appropriate framework regions in the variable domain. The epitope-binding site may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, although the possibility of an immune response against the foreign variable region remains (LoBuglio, AF et al. (1989) Proc Natl Acad Sci USA, 86:4220-4224; Queen et al., PNAS (1988) 86:10029-10033; Riechmann et al., Nature (1988) 332:323-327). Exemplary methods for humanization of the anti-Fzd or anti-LRP antibodies disclosed herein include those described in US Pat. No. 7,462,697.

[0129] Another approach focuses not only on providing constant regions of human origin, but also on modifying variable regions to reshape them as close as possible to human form. It is known that both heavy and light chain variable regions contain three complementarity determining regions (CDRs) that change in response to the epitope in question to determine binding ability, and these three complementarity determining regions (CDRs) are flanked by four framework regions (FRs) that are relatively conserved in a given species and are assumed to provide a scaffold for the CDRs. When a non-human antibody is prepared for a specific epitope, its variable region can be "redesigned" or "humanized" by grafting the CDRs from the non-human antibody into the FRs present in the human antibody to be modified. Application of this approach to a variety of antibodies has been reviewed in Sato, K., et al. (1993) Cancer Res 53:851-856; Riechmann, L., et al. (1988) Nature 332:323-327; Verhoeyen, M., et al. (1988) Science 239:1534-1536; Kettleborough, CA, et al. (1991) Protein Engineering 4:773-3783; Maeda, H., et al. (1991) Human Antibodies Hybridoma 2:124-134; Gorman, SD, et al. (1991) Proc Natl Acad Sci USA 88:4181-4185; Tempest, PR, et al. (1991) Bio / Technology 9:266-271; Co, MS, et al. (1991) Proc Natl Acad Sci USA, 88:2869-2873; Carter, P. et al. (1992) Proc Natl Acad Sci USA, 89:4285-4289; and Co, MS et al. (1992) J Immunol, 148:1149-1154. In some embodiments, the humanized antibody preserves all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs from the mouse antibodies).In other embodiments, a humanized antibody has one or more (one, two, three, four, five, six) CDRs that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0130] In certain embodiments, the antibody of the present disclosure may be a chimeric antibody. In this regard, a chimeric antibody is composed of an antigen-binding fragment of an antibody that is operably linked or otherwise fused to a heterologous Fc portion of a different antibody. In certain embodiments, the heterologous Fc domain is of human origin. In other embodiments, the heterologous Fc domain may be derived from a different Ig class from the parent antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In further embodiments, the heterologous Fc domain may be composed of a CH2 domain and a CH3 domain from one or more of the various Ig classes. As described above for humanized antibodies, an antigen-binding fragment of a chimeric antibody may contain only one or more of the CDRs of an antibody described herein (e.g., one, two, three, four, five, or six CDRs of an antibody described herein) or may contain the entire variable domain (VL, VH or both).

[0131] The structure and location of immunoglobulin CDRs and variable domains can be determined by reference to Kabat, EA et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 4th ed., US Department of Health and Human Services, 1987, and the latest edition thereof currently available on the Internet (immuno.bme.nwu.edu).

[0132] In certain embodiments, the antagonist or agonist binding agent has a dissociation constant (K D For example, in certain embodiments, FZD-binding agents or FZD antibodies described herein that bind to more than one FZD bind to those FZDs with a K of about 100 nM or less, about 20 nM or less, or about 10 nM or less. D In certain embodiments, the binding agent binds to its target antigen or antigens with an EC50 of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, or about 1 nM 20 or less.

[0133] The antibody or other agent of the present invention can be assayed for specific binding by any method known in the art. Immunoassays that can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as biolayer interference (BLI) analysis, FACS analysis, immunofluorescence, immunocytochemistry, Western blot, radioimmunoassay, ELISA, "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescent immunoassay, and protein A immunoassay. Such assays are conventional and well known in the art (see, for example, Ausubel et al., eds., 1994, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated herein by reference in its entirety).

[0134] For example, the specific binding of an antibody to a target antigen can be determined using ELISA. ELISA assays include the steps of preparing an antigen, coating the wells of a 96-well microtiter plate with the antigen, adding an antibody or other binding agent conjugated to a detectable compound (e.g., an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase)) to the wells, incubating for a period of time, and detecting the presence of the antigen. In some embodiments, the antibody or agent is not conjugated to a detectable compound, but instead, a second conjugated antibody that recognizes the first antibody or agent is added to the well. In some embodiments, instead of coating the wells with an antigen, the antibody or agent can be coated to the well, and a second antibody conjugated to a detectable compound can be added after the addition of the antigen to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art (see, e.g., Ausubel et al., eds., 1994, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Vol. 1, John Wiley & Sons, Inc., New York, 11.2.1).

[0135] The binding affinity of an antibody or other agent to a target antigen and the off-rate of the antibody-antigen interaction can be determined by competitive binding assay. One example of a competitive binding assay is a radioimmunoassay, which includes incubating a labeled antigen (e.g., Fzd, LRP), or a fragment or variant thereof, with an antibody of interest in the presence of increasing amounts of unlabeled antigen, and then detecting the antibody bound to the labeled antigen. The affinity and binding off-rate of the antibody can be determined from the data by scatter plot analysis. In some embodiments, BLI analysis is used to determine the on-rate and off-rate of an antibody or agent. BLI kinetic analysis includes analyzing the binding and dissociation of an antibody from a chip with an antigen immobilized on its surface.

[0136] IV. WNT AGONIST COMPOSITIONS AND METHODS OF USE Provided herein are methods and compositions for treating lacrimal gland diseases and disorders, including but not limited to, dry eye disorder, for example, by activation and / or regeneration of lacrimal gland acinar cells.

[0137] Those skilled in the art can easily understand that lacrimal glands are similar to meibomian glands and accessory lacrimal glands, and the methods and compositions described herein can be applied to treat meibomian glands and / or accessory lacrimal glands in aqueous deficient dry eye or evaporative dry eye.Meibomian glands are sebaceous glands that line the edge of the eyelid.These glands secrete oil into the eyelids that mix with natural tears to prevent excessive evaporation of the natural tears.Patients suffering from ocular rosacea often have meibomian gland dysfunction (MGD), in which the eyelids secrete less oil, resulting in dry eye.MGD is believed to be the main cause of dry eye disease.WNT agonist molecules can also be used to treat meibomian gland disorders. In particular, active WNT signaling may provide a maintenance signal to basal progenitor cells and plays an important role in regulating meibocyte regeneration (see, e.g., Parfitt et al. (2016) Stem Cell Rep., 7:399-410). In one aspect, the present invention includes a method of regenerating lacrimal acinar cells in a subject, the method comprising administering a WNT signaling modulator to the subject. In one embodiment, the WNT signaling modulator can be an engineered WNT signaling modulator (e.g., SWAP™ compound). In another embodiment, the WNT signaling modulator is an engineered WNT agonist or an engineered WNT antagonist. In yet a further embodiment, the WNT signaling modulator is a tissue-specific WNT signal enhancing molecule (e.g., SWEETS™ molecule). The WNT signaling modulator can also be a combination of a WNT agonist and a tissue-specific WNT signal enhancer.

[0138] In certain embodiments of any of the methods disclosed herein, the WNT agonist is selected from a WNT agonist disclosed in any of the following: PCT Application Publication No. WO2016 / 040895; U.S. Patent Application Publication No. 2017-0306029; U.S. Patent Application Publication No. 2017-0349659; PCT Application Publication No. WO2019 / 126398; PCT Application Publication No. WO2020 / 01030, PCT Application Publication No. WO2021 / 173726, or U.S. Patent Application Publication No. 17 / 806,624 (all of which are incorporated by reference in their entireties herein). In certain embodiments of any of the methods disclosed herein, the tissue-specific WNT signal enhancing molecule is selected from the tissue-specific WNT signal enhancing molecules disclosed in any of the following: PCT Application Publication No. WO2018 / 140821; U.S. Patent Application Publication No. 2020-0048324; or PCT Application Publication No. WO2020 / 14271, all of which are incorporated by reference in their entireties.

[0139] In one embodiment of the method of regenerating lacrimal gland acinar cells, the WNT signaling modulator may comprise at least one engineered bispecific full-length IgG antibody that directly activates the classical WNT signaling pathway. In any embodiment, the engineered WNT agonist may be selected from (i) WNT3a; (ii) a WNT mimetic; or (iii) an R-spondin mimetic. The WNT mimetic may be a SWAP™ compound. The R-spondin mimetic may be a SWEETS™ compound. The WNT mimetic may comprise one or more polypeptides comprising or having a polypeptide sequence as set forth in any of SEQ ID NOs: 1-14, or their isoforms and homologues, and a suitable expression vector therefor. The WNT mimetic may comprise one or more polypeptides having 80%-100% homology with any of the polypeptide sequences as set forth in SEQ ID NOs: 1-14. In a particular embodiment, the WNT mimetic comprises two polypeptide sequences having 80%-100% homology with any of the polypeptide sequences as set forth in SEQ ID NOs: 1-14. In certain embodiments, the WNT mimetic comprises two heavy chain polypeptide sequences and two light chain polypeptide sequences, each of which has 80%-100% homology to any of the polypeptide sequences set forth in SEQ ID NOs: 1-14. In certain embodiments, the heavy and light chain polypeptides present in the WNT mimetic have at least 80%, at least 90%, at least 95%, at least 98%, or 100% identity to any of the following combinations: SEQ ID NOs: 1 and 2; SEQ ID NOs: 3 and 4; SEQ ID NOs: 5 and 6; SEQ ID NOs: 7 and 8; or SEQ ID NOs: 9 and 10, or SEQ ID NOs: 11 and 14, or SEQ ID NOs: 12 and 14, or SEQ ID NOs: 13 and 14. In certain embodiments, the WNT mimetic has an IgG antibody structure comprising two heavy chains and two light chains, the two heavy chains being linked to each other and each of the light chains being linked to a separate one of the two heavy chains.

[0140] In one embodiment of the method for regenerating lacrimal gland acinar cells, the WNT signaling modulator can affect the expression of any one or more of Fzd1, Fzd2, Fzd5, Fzd7, Fzd8, and Lrp6, and / or Lrp5. For example, the WNT signaling modulator can target any one or more of the group consisting of Fzd1, Fzd2, and Fzd7; or any one or more of the group consisting of Fzd5 and Fzd8, and also target Lrp6 and / or Lrp5. For another example, the WNT signaling modulator can affect the expression of any one or more of Fzd1, Fzd2, and Fzd7.

[0141] In one embodiment of the method of regenerating lacrimal gland acinar cells, the method further comprises administering at least one of the group consisting of RSPO2, a RSPO2 fragment, and an engineered RSPO2 mimetic.

[0142] In some embodiments, the WNT signaling modulator can be a superagonist of WNT platform.This superagonist activity has been observed in the WNT molecule fused with LRP, FZD and RSPO, which acts as a strong activator of WNT signaling pathway.The WNT superagonist is selected from the WNT superagonists disclosed in PCT Application Publication No. WO2021 / 173726.

[0143] In any embodiment, the WNT signaling modulator can be at a concentration of ≧1 nM. For example, the WNT signaling modulator can be at a concentration of 1 nM, 1.1 nM, 1.5 nM, 2.0 nM, 2.5 nM, 3.0 nM, 3.5 nM, 4.0 nM, 4.5 nM, 5.0 nM. In certain embodiments, the WNT signaling modulator can be at a concentration of ≧5 nM. In any embodiment, the WNT signaling modulator can be administered in a therapeutically effective amount.

[0144] In any embodiment, the subject can be a living mammal. For example, the subject can be a mouse, rat, dog, cat, horse, or cow. In any embodiment, the subject can be a human patient.

[0145] In any embodiment, the WNT signaling modulator may be administered systemically or locally, for example, the WNT signaling modulator may be administered locally, by aqueous eye drops or by local intralacrimal injection.

[0146] In another aspect, the present invention comprises a method for treating lacrimal gland disorder in a subject, the method comprises administering a WNT signaling modulator to the subject.In one embodiment, the WNT signaling modulator can be an engineered WNT signaling modulator.In another embodiment, the WNT signaling modulator is an engineered WNT agonist or an engineered WNT antagonist.

[0147] In one embodiment of the method for treating lacrimal gland disorder, the WNT signaling modulator can comprise at least one engineered bispecific full-length IgG antibody that directly activates the classical WNT signaling pathway.In any embodiment, the engineered WNT agonist can be selected from (i) WNT3a; (ii) WNT mimic; or (iii) R-spondin mimic.The WNT mimic can be SWAP™ compound.The R-spondin mimic can be SWEETS™ compound.

[0148] In one embodiment of the method for treating lacrimal gland disorder, the WNT signaling modulator can affect the expression of any one or more of Fzd1, Fzd2, Fzd5, Fzd7, Fzd8, and Lrp6 and / or Lrp5.For example, the WNT signaling modulator can target any one or more of the group consisting of Fzd1, Fzd2, and Fzd7; or any one or more of the group consisting of Fzd5 and Fzd8, and also target Lrp6 and / or Lrp5.For another example, the WNT signaling modulator can affect the expression of any one or more of Fzd1, Fzd2, and Fzd7.

[0149] In one embodiment of the method of treating a lacrimal gland disorder, the method further comprises administering at least one member of the group consisting of RSPO2, a RSPO2 fragment, and an engineered RSPO2 mimetic.

[0150] In any embodiment of the method for treating lacrimal gland disorder, the WNT signaling modulator can be at a concentration of ≧1 nM. For example, the WNT signaling modulator can be at a concentration of 1 nM, 1.1 nM, 1.5 nM, 2.0 nM, 2.5 nM, 3.0 nM, 3.5 nM, 4.0 nM, 4.5 nM, 5.0 nM. In certain embodiments, the concentration of the WNT signaling modulator can be ≧5 nM. In any embodiment, the WNT signaling modulator can be administered in a therapeutically effective amount.

[0151] In any embodiment, the subject can be a living mammal. For example, the subject can be a mouse, rat, dog, cat, horse, or cow. In any embodiment, the subject can be a human patient.

[0152] In any embodiment, the WNT signaling modulator may be administered systemically or locally, for example, the WNT signaling modulator may be administered locally, by aqueous eye drops or by local intralacrimal injection.

[0153] In yet another aspect, the present invention comprises a composition for the treatment of a dry eye disorder in a subject, the composition comprising a WNT signaling modulator. In any embodiment of the composition, the dry eye disorder can be a Sjogren's syndrome disorder.

[0154] In one embodiment of the composition, the WNT signaling modulator may comprise at least one engineered bispecific full-length IgG antibody that directly activates the canonical WNT signaling pathway. The at least one engineered bispecific full-length IgG antibody may be specific for any one or more of Fzd1, Fzd,2, Fzd5, Fzd7, Fzd8, and Lrp6 or Lrp5.

[0155] In one embodiment, the composition may further include at least one additional drug, including an anti-inflammatory drug, an artificial tear agent, or a lacrimal gland secretagogue. In particular, the anti-inflammatory drug may be an antibiotic or a steroid drug, including cyclosporine A (e.g., Restasis®) and lifitegrast ophthalmic solution (e.g., Xiidra®). The artificial tear agent may include a non-prescription eye drop that stimulates tears, such as hydroxypropylcellulose (Lacrisert®) insert. The lacrimal gland secretagogue may include varenicline nasal spray, which selectively agonizes nicotinic acetylcholine receptors. Autologous serum eye drops are also contemplated.

[0156] In any embodiment of the composition, the composition may contain a therapeutically effective amount of each of its components.

[0157] In any of the embodiments of the composition, the subject can be a living mammal. For example, the subject can be a mouse, rat, dog, cat, horse, or cow. In any of the embodiments of the composition, the subject can be a human patient.

[0158] In a further embodiment, also disclosed is a pharmaceutical composition comprising an expression vector (e.g., a viral vector) comprising a polynucleotide comprising the nucleic acid sequence encoding the WNT antagonist molecule / WNT agonist molecule described herein, and one or more pharma- ceutically acceptable diluents, carriers, or excipients.In certain embodiments, the nucleic acid sequence encoding the WNT antagonist molecule and the nucleic acid sequence encoding the WNT agonist are in the same polynucleotide (e.g., an expression cassette).

[0159] The present disclosure further contemplates a pharmaceutical composition comprising a cell comprising an expression vector comprising a polynucleotide comprising a promoter operably linked to a nucleic acid encoding a WNT antagonist molecule / WNT agonist molecule, and one or more pharma- ceutically acceptable diluents, carriers, or excipients. In certain embodiments, the pharmaceutical composition further comprises a cell comprising an expression vector comprising a polynucleotide comprising a promoter operably linked to a nucleic acid sequence encoding a WNT antagonist and a WNT agonist. In certain embodiments, the nucleic acid sequence encoding the WNT antagonist molecule and the nucleic acid sequence encoding the WNT agonist molecule are in the same polynucleotide (e.g., expression cassette) and / or in the same cell. In certain embodiments, the cell is a heterologous cell or an autologous cell obtained from the subject to be treated.

[0160] In certain embodiments, the cell is a stem cell (e.g., an adipose-derived stem cell or a hematopoietic stem cell). The present disclosure contemplates a pharmaceutical composition comprising a first molecule for delivery of a WNT antagonist molecule as a first active agent, and a WNT agonist as a second molecule. The first molecule and the second molecule may be the same type of molecule, or may be different types of molecules. For example, in certain embodiments, the first molecule and the second molecule may each be independently selected from the following types of molecules: a polypeptide, an organic small molecule, a nucleic acid (optionally DNA or mRNA, optionally modified RNA) encoding the first or second active agent, a vector (optionally an expression vector or a viral vector) comprising a nucleic acid sequence encoding the first or second active agent, and a cell (optionally an expression cassette) comprising a nucleic acid sequence encoding the first or second active agent.

[0161] The molecules (alone or in combination) can be combined with pharma- ceutically acceptable carriers, diluents, excipients and agents that are generally safe, non-toxic and useful in preparing the desired formulations, which include excipients acceptable for use in mammals (e.g., humans or primates). Such excipients can be solid, liquid, semi-solid, or (in the case of aerosol compositions) gaseous. Examples of such carriers, diluents and excipients include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulations. The solutions or suspensions used for the formulation may include sterile diluents (e.g., water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents); antimicrobial compounds (e.g., benzyl alcohol or methylparabens); antioxidants (e.g., ascorbic acid or sodium bisulfite); chelating compounds (e.g., ethylenediaminetetraacetic acid (EDTA)); buffers (e.g., acetic acid, citric acid or phosphoric acid); surfactants (e.g., Tween® 20 to prevent aggregation); and compounds for adjusting tonicity (e.g., sodium chloride or dextrose). The pH may be adjusted with acids or bases (e.g., hydrochloric acid or sodium hydroxide). In certain embodiments, the pharmaceutical compositions are sterile.

[0162] The pharmaceutical compositions may further comprise sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In some cases, the composition is sterile, and the composition should be fluid so that it can be drawn into a syringe or delivered from a syringe to a subject. In certain embodiments, it is stable under the conditions of manufacture and storage, and is preserved against the contaminating action of microorganisms (e.g., bacteria and fungi). The carrier may be, for example, a solvent or dispersion medium, including, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. The proper fluidity may be maintained, for example, by the use of a coating (e.g., lecithin), by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonicity agents, such as sugars, polyalcohols (e.g., mannitol, sorbitol), sodium chloride in the composition. Prolonged absorption of the internal composition can be achieved by including agents that delay absorption in the composition (e.g., aluminum monostearate and gelatin).

[0163] Sterile solutions can be prepared by incorporating the WNT antagonist antibody / WNT agonist antibody or its antigen-binding fragment (or coding polynucleotide or cells containing it) in the required amount in a suitable solvent containing one or a combination of the above-listed ingredients, followed by sterile filtration if necessary. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required ingredients from the above-listed ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and lyophilization, which results in a powder of the active ingredient plus any additional ingredients desired from their previously sterile-filtered solutions.

[0164] In one embodiment, the pharmaceutical compositions are prepared with carriers that will protect the antibody or antigen-binding fragment thereof against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparation of such formulations will be apparent to those of skill in the art. These materials may also be obtained commercially. Liposomal suspensions may also be used as pharma- ceutically acceptable carriers. These may be prepared according to methods known to those of skill in the art.

[0165] It may be advantageous to formulate the pharmaceutical composition in dosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to a physically discrete unit suitable as a unitary dosage to a subject to be treated; each unit contains a predetermined amount of active antibody or antigen-binding fragment thereof calculated to produce a desired therapeutic effect, in combination with a required pharmaceutical carrier.The specifications for the dosage unit form are determined by and directly depend on the unique characteristics of the antibody or antigen-binding fragment thereof and the specific therapeutic effect to be achieved, as well as the limitations inherent in the field of formulating such active antibody or antigen-binding fragment thereof for the treatment of individuals.

[0166] The pharmaceutical compositions can be included in a container, pack, or dispenser (eg, an eye dropper, eg, a prefilled eye dropper) together with instructions for administration.

[0167] The pharmaceutical compositions of the present disclosure may be delivered to the subject in the form of a pill, capsule, cream, ointment, syrup, skin patch, suppository, intravenous drip, topical injection aqueous solution, non-aqueous solution, eye wash solution, or any combination thereof.

[0168] The pharmaceutical compositions of the present disclosure may be delivered to a subject by direct eye drops, by intramuscular injection, by intralacrimal injection, by subconjunctival injection, by meibomian gland injection, by intravenous injection, by intraperitoneal injection, intranasally, orally, rectally, or any combination thereof.

[0169] The pharmaceutical compositions of the present disclosure include any pharma- ceutically acceptable salts, esters, or salts of such esters, or any other compounds that are capable of providing (directly or indirectly) a biologically active antibody or antigen-binding fragment thereof upon administration to an animal, including a human.

[0170] The present disclosure includes the pharma- ceutically acceptable salts of the WNT antagonist molecules / WNT agonist molecules described herein.The term "pharma- ceutically acceptable salts" refers to physiologically and pharma- ceutical acceptable salts of the compounds of the present disclosure: that is, salts that retain the desired biological activity of their parent compounds and do not impart undesired toxicological effects to them.Various pharma- ceutical acceptable salts are known in the art and are described, for example, in "Remington's Pharmaceutical Sciences", 17th Edition, Alfonso R.Gennaro (ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and its more recent editions), "Encyclopedia of Pharmaceutical Technology", 3rd Edition, James Swarbrick (ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and J.Pharm.Sci.66:2 (1977). Also, for a review of suitable salts see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use", Stahl and Wermuth (Wiley-VCH, 2002). Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals, or organic amines.

[0171] Metals used as cations include sodium, potassium, magnesium, calcium, and the like. Amines include N-N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., "Pharmaceutical Salts," J. Pharma Sci., 1977, 66, 119). The base addition salts of the above acidic compounds are prepared in a conventional manner by contacting the free acid form with a sufficient amount of the desired base to produce the salt. The free acid forms may be produced in a conventional manner by contacting the salt form with an acid and isolating the free acid. The free acid forms differ somewhat from their respective salt forms in certain physical properties (e.g., solubility in polar solvents), but otherwise the salts are equivalent to their respective free acids for purposes of this disclosure.

[0172] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a WNT antagonist molecule / WNT agonist molecule or a pharma- ceutically acceptable salt thereof in admixture with a pharma- ceutically acceptable carrier, diluent and / or excipient, such as saline, phosphate buffered saline, phosphate salts, and amino acids, polymers, polyols, sugars, buffers, preservatives and other proteins. Exemplary amino acids, polymers and sugars are octylphenoxy polyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine serum albumin or human serum albumin, citrate, acetate, Ringer's solution and Hank's solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene and glycol. Preferably, the formulation is stable at 4° C. for at least 6 months.

[0173] In some embodiments, the pharmaceutical compositions provided herein comprise a buffer, such as phosphate buffered saline (PBS) or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water and other buffers known to those of skill in the art, such as those described by Good et al. (1966) Biochemistry, 5: 467. The pH of the buffer may range from 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

[0174] It will be appreciated from the above that, although specific embodiments of the present disclosure have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, the present disclosure is not to be limited except as by the appended claims.

[0175] The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the inventions to the specific embodiments. EXAMPLES

[0176] (Example) (I. Method) Standard methods of molecular biology were used, including those described, for example, in Maniatis et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif. Standard methods are also described in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc., New York, NY, which describe cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugate and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0177] Methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, are described, for example, in Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York. Chemical analysis, chemical alterations, post-translational modifications, generation of fusion proteins, and glycosylation of proteins are described, for example, in Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, Mo., pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, NJ, pp. 384-391. The production, purification and fragmentation of polyclonal and monoclonal antibodies are described, for example, in Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane (supra). Standard techniques for characterizing ligand / receptor interactions are available, for example, in Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York.

[0178] Methods for flow cytometry, including the Fluorescence Activated Cell Sorting Detection System (FACS®), are available, for example, in Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ. Fluorescent reagents suitable for modifying nucleic acids (including nucleic acid primers and probes), polypeptides, and antibodies, for example, for use as diagnostic reagents, are available, for example, in Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.

[0179] Standard methods for immune system histology are described, for example, in Muller-Harmelink (eds.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.; Louis et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY.

[0180] For example, software packages and databases for determining antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments are available from, e.g., GenBank, Vector NTI® Suite (Informax, Inc., Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp., Crystal Bay, Nev.); Menne et al. (2000) Bioinformatics, 16:741-742; Menne et al. (2000) Bioinformatics Applications Note, 16:741-742; Wren et al. (2002) Comput. Methods Programs Biomed., 68:177-181; von Heijne (1983) Eur. J. Biochem., 133:17-21; von Available in Heijne (1986) Nucleic Acids Res., 14:4683-4690.

[0181] II. Establishment and Expansion of Organoids Lacrimal and salivary glands from wild-type C57BL / 6 mice were dissected and stored in Advanced DMEM / F12 (GIBCO) on ice. Multiple lacrimal or salivary glands from mice were pooled in a Petri dish, and muscle, ducts, and connective tissue were removed and discarded as much as possible. The remaining glandular epithelium was minced into approximately 1 mm pieces using a scalpel. The tissue pieces were enzymatically digested in a collagenase (Sigma-Aldrich, C9407, 1 mg / mL) solution containing 10 μM ROCK inhibitor Y-27632 (Abmole, M1817) in Advanced DMEM / F12 (GIBCO) for approximately 15 minutes with shaking at 37°C. Before plating, the homogenous cell suspension was pelleted (1200 rpm for 5 minutes) and washed twice with Advanced DMEM / F12. The same protocol was applied to human post-mortem lacrimal gland material. Cells were plated in 20 μL droplets of Cultrex Pathclear Reduced Growth Factor Basement Membrane Extract (BME) (3533-001, Amsbio) or Matrigel™ GFR Membrane Matrix (Corning CB40230C). After 15 minutes of clotting, growth medium was added containing various mammalian growth factors and cytokines, RSPO1, and alternative WNTs (5 nM L-F12578; L-F127; L-F58; L-F4; or L-F10). As shown in Figure 1, cells treated with L-F12578, L-F127 or L-F58 proliferated rapidly with a solid budding morphology, whereas cells treated with control treatments and other alternative WNT treatments either had little proliferation or had a tubular morphology (see, e.g., Bannier-Helaouet et al. (2021) Cell Stem Cell, 28:1221-1232).

[0182] (III. Screening for Alternative WNTs in Acinar Cells) For proliferation activity screening of alternative WNTs (i.e., WNT mimetics), mouse primary lacrimal or salivary gland cells or organoid cells (<passage 5) were digested to near single cell suspensions using TrypLE (Thermo Fisher) for 10 minutes at 37°C. The basal medium for activity assays consisted of growth medium without RSPO1 or any alternative WNTs, supplemented with 1 μM porcupine inhibitor Wnt-C59 (#5148, Tocris) and 10 μM Y-27632 (#5092280001, MilliporeSigma). Experimental conditions consisted of one or a combination of 500 ng / mL recombinant RSPO1 and 5 nM alternative WNTs (L-F12578, L-F127, L-F58, L-F4, or L-F10) unless otherwise stated. All cells for all conditions were plated in 15 μL Matrigel droplets in 96-well plates and submerged in 120 μL of experimental medium in round-bottom 96-well plates. Cells were grown as organoids for 7 days before quantification. Growth efficiency was quantified using cell viability assay CellTiter-Glo® (G9683, Promega) and measured on a SpectraMax Paradigm microplate reader (Molecular Devices) according to the manufacturer's protocol. The results are shown in Figure 4 and Figure 5. For RSPO-dependent assays, organoids were cultured with a dose range of surrogate WNT (0.05 nM, 0.5 nM, 5 nM, 50 nM) with or without 500 ng / mL RSPO1. The results are shown in Figure 6.

[0183] For screening of WNT target gene expression upon alternative WNT stimulation, mouse acinar cell organoids were grown for 5 days at full growth time, followed by RSPO1, alternative WNT withdrawal, and addition of 1 μM porcupine inhibitor Wnt-C59 (#5148, Tocris) for 48 hours, as shown in Figure 7. After this WNT withdrawal period, RSPO1 and / or 5 nM alternative WNT (L-F12578, L-F127, L-F58, L-F4, or L-F10) were reintroduced for the indicated time periods to assess WNT target gene induction, as shown in Figure 8. RNA was extracted using a QIAprep miniprep kit (#27104, QIAGEN) according to the manufacturer's protocol. Axin2 expression was determined by qPCR using SYBR Green (#K0243, Thermo Scientific) according to the manufacturer's protocol. The results are shown in Figure 9.

[0184] Induction of human AXIN2 in an alternative WNT screen was performed in human lacrimal gland explant cultures derived from fresh human lacrimal gland tissue in complete growth medium (shown in FIG. 10). Cells were plated on Matrigel as described above (see FIG. 11A-B) and exposed to various alternative WNTs (L-F12578, L-F127, L-F58, L-F4, or L-F10) in combination with RSPO1 from the time of plating. After 24 hours of exposure, the RNA was extracted using a QIAGEN miniprep kit according to the manufacturer's protocol. Expression of AXIN2 was determined by qPCR using SYBR Green (Thermo Fisher) according to the manufacturer's protocol (primer sequences provided below). The results are shown in FIG. 12.

[0185] (IV. Immunofluorescence staining and in situ hybridization) Organoids were collected in cell collection solution (354253, Corning), fixed in 4% formaldehyde (R37814, Sigma-Aldrich) for at least 2 hours at room temperature, and permeabilized using 0.2% Triton® X-100 (#ICN19485450, Fisher Scientific) in PBS. Whole-mount staining was performed overnight in 2% donkey serum using rabbit anti-MIST1 (#14896, CST) and DAPI (#EN62248, Fisher Scientific), and the secondary antibody was Alexa Fluor 568 donkey anti-rabbit IgG (Life Technologies, A10042). Organoids were imaged with a Leica THUNDER imaging system (Figure 2). Immunofluorescence staining of mouse lacrimal glands was performed on sections from paraffin-embedded tissues. Briefly, sections were deparaffinized, heat antigen retrieved, permeabilized, blocked, and stained for Ki67 (#ab15580, Abcam) and DAPI (#EN62248, Fisher Scientific). Secondary staining was performed using the secondary antibody Alexa Fluor 568 donkey anti-rabbit IgG (Life Technologies, A10042) and imaging was performed on a Leica DMi8 system (Figure 15). In situ hybridization was performed using human or mouse specific probes for the indicated WNT receptors according to the manufacturer's protocol (ACDBio) and imaged on a Leica DMi8 system. The results are shown in Figures 13-14.

[0186] V. Animals and Treatments Female C57BL / 6 mice (8-10 weeks old) were intraperitoneally administered 10 mpk of various Wnt mimetics on days 0, 3, 7, and 10. On day 14, proliferating cells were detected in the L-F12578-, L-F127-, and L-F58-treated groups by Ki67 signaling ( FIG. 15 ). Similar timing of administration of WNT mimetics in combination with RSPO resulted in increased lacrimal gland weights in the L-F12578-, and L-F127-treated groups compared to controls and RSPO alone after 2 weeks ( FIG. 16 ).

[0187] Female C57BL / 6 mice (8-10 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME). All animal procedures were performed in accordance with IACUC committee regulations. Mice were anesthetized and their left extraorbital lacrimal glands were left untreated while their right lacrimal glands were injected with IL-1α or a cocktail of IL-1α and SWAP™ (i.e., a WNT mimetic) as shown in FIG. 17.

[0188] All animal studies were performed in accordance with national ethical guidelines as well as with guidelines and approval by the Institutional Animal Care and Use Committee (IACUC) of Surrozen, Inc. 12-week-old MRL-lpr (stock 000485) female mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed 4 per cage. Protein treatments at 3 mg per kg and 10 mg per kg were administered intraperitoneally to C57Bl / 6J and MRL-lpr as well as MRL / MpJ mice on days 0, 3, 7 and 10, respectively. RSPO2-nFcFc treatments at 0.1 mg, 0.3 mg, 1 mg and 3 mg per kg were administered intraperitoneally to C57Bl / 6J mice on days 0, 3, 7 and 10. Bone mineral density (BMD) and fat content of animals were measured on days 0, 7, and 13 by in vivo DEXA using a Faxitron UltraFocus (Faxitron Bioptics, Tucson, Arizona). Animals were anesthetized with isoflurane during imaging. Sample ROIs included the entire mouse skeleton, excluding material above the cervical spine due to increased radiographic intensity of the skull. BMD and fat content were calculated using accompanying Vision DXA software. Animals were sacrificed on day 14 and salivary glands were collected for histology.

[0189] (VI. Intralacrimal Injection) Intralacrimal injections were performed as previously described in Zoukhri et al., A Single Injection of Interleukin-1 Induces a Reversible Aqueous-tear Deficiency, Lacrimal Gland Inflammation, and Acinar and Ductal Cell Proliferation, Exp. Eye Res., 84, 894-904 (2007) with minor modifications. Briefly, a small incision was made in front of the ear of isoflurane-anesthetized animals to expose the extraorbital lacrimal gland. A volume of 1 μL was injected three times (total volume of 3 μL) into the exposed gland.

[0190] The following procedure was established for lacrimal gland duct ligation, adapted from Liu et al., 2017. The animal is anesthetized with isoflurane. After the mouse is no longer responsive to pinching of two limbs in two different quadrants of the body, the mouse is transferred to the stage of a dissecting scope and fitted with a nose cone to continue anesthesia with isoflurane. A small incision is made in front of the ear to expose the extraorbital lacrimal gland, which is elevated to expose its main drainage duct. The duct is then ligated using a size 4-0 silk suture. After ligation of the duct, the skin incision is closed and sutured, and antibiotic ointment is then applied to the skin. The contralateral lacrimal gland is not operated on and serves as a control. After 3 days, the skin suture is reopened to expose the lacrimal gland. The duct ligation is then released by cutting the silk suture with small scissors. In the same operation, 2mL of test substance is injected into the lacrimal gland ipsilateral to the ligation of the treatment group. After injection, the skin incision is closed and sutured, and then antibiotic ointment is applied. Then, 24 hours, 3 days, or 14 days after the release of the ligation, the animals are sacrificed and the ipsilateral and contralateral lacrimal glands are collected.

[0191] VII. Induction of Dry Eye and SWAP™ Treatment For induction of dry eye, 3 μL of 2 μg / μL recombinant human IL-1α (Peprotech) was injected into the lacrimal gland. For topical SWAP™ treatment, SWAP™ (3.5 μg / μL) was mixed with IL-1α (2 μg / μL) in a total volume of 3 μL and then injected into the lacrimal gland. For systemic SWAP™ treatment, SWAP™ was injected intraperitoneally at 10 mg / kg twice a week for 2 weeks, as shown in Figures 19-20.

[0192] (VIII. Measurement of Tear Secretion) Tear secretion was measured in restrained, non-anesthetized mice using phenol-impregnated cotton threads (Zone-Quick, Menicon). The threads were held with forceps and applied to the lateral canthus of both eyes for 30 seconds. The wetness of the threads, which turned from yellow to red on contact with tear fluid, was measured in millimeters. The results are shown in Figure 19.

[0193] (IX. WNT mimetic molecules induce salivary gland hypertrophy) The effect of systemic WNT mimetic administration in vivo on salivary gland weight and histology was examined. On day 14, a significant increase in salivary gland weight was observed in the L-F12578, L-F127, L-F4, and L-F10 groups administered at 3 mpk (101% (P<0.001), 114% (P<0.001), 29% (p<0.01), and 22% (P<0.05), respectively) compared to the vehicle group, with the effect of L-F12578 and the effect of L-F127 being the most prominent (Figure 22). None of these WNT mimetic molecules targeting various FZD receptors had any observable effect on the histology of submandibular gland in vivo by HE staining (Figure 23A). Ki67 staining for proliferating cells showed little difference between treatment groups, indicating that a possible earlier cell division peak drove the differences in organ weights (Figure 23B). Animals treated with the same molecule but at 10mpk (systemic treatment twice weekly for 14 days) had a distinct histopathological phenotype. L-F12578- and L-F127-treated animals showed differential staining in serous mucinous acini, and there was an increased amount of cytoplasmic basophilia as determined by an independent pathologist (Figure 24A). Automated quantification of the mucinous and serous portions of the glands by Image J shows a significant decrease in mucinous acinar area in L-F12578- and L-127-treated animals (Figure 24B). Systemic RSPO2 treatment also elicited a hypertrophic response in the glands. In a 2-week treatment with a range of RSPO2-nFc doses, the 1 mpk and 3 mpk doses have a significant increase in salivary gland weight at day 14 (FIG. 25).

[0194] To understand the effect of WNT activation on salivary gland cells, we utilized mouse salivary gland organoids (see, for example, Maimetz et al. (2016) Stem Cell, 6:150-162) as an experimental platform. Treatment with L-F12578 (5 nM) in combination with recombinant RSPO1 increased proliferation compared to RSPO1 alone, resulting in rapid growth of larger organoids (Figure 26A). The FZD specificity of this proliferation-promoting phenotype was investigated by screening WNT mimetics in a proliferation assay and comparing it to an anti-GFP antibody. In a dose-dependent manner, L-F12758 and L-F127 significantly increased proliferation efficiency measured as cell viability at day 10, with only a minor effect observed for high concentrations of L-F58 (Figure 26B). Fzd7, Fzd1 and Fzd2 were shown to be the most highly expressed Frizzled in mouse salivary gland organoids, consistent with their in vitro and in vivo effects on the salivary gland (Figure 27).

[0195] Sjögren's syndrome is a systemic autoimmune disease that specifically affects exocrine glands (e.g., salivary glands). Chronic inflammation in the glands, accompanied by immune cell infiltration, results in acinar cell atrophy leading to xerostomia (see, e.g., Jensen and Vissink (2014) Oral Maxillofac. Surg. Clin. North Am., 26:35-53). The MRL-lpr mouse strain (also known as lupus mice) exhibits systemic autoimmunity with salivary gland abnormalities (e.g., reduced saliva production and lymphocytic infiltration) similar to those in human Sjögren's syndrome (see, e.g., Ma et al. (2014) Diag. Pathol., 9:5). To test whether the L-F12578 WNT mimetic could increase and restore salivary gland weight in an ongoing damaging environment, it was administered for 2 weeks to aged lupus mice and MRL / MpJ control mice. Treatment with L-F12578 significantly increased salivary gland weight in both genetic strains without any observable adverse effects on histology (Figures 28A and 28B). Both the lupus mice and the control strain exhibited wet fur and mild color changes upon treatment with L-F12578.

[0196] X. Reagents and Materials WNT mimetics were constructed as described in WO2020 / 010308A1, which is incorporated herein in its entirety. All recombinant proteins were produced in Expi293F™ cells (Thermo Fisher Scientific) by transient transfection unless otherwise indicated. All IgG-based and Fc-containing constructs were first purified on Protein A resin and eluted with 0.1 M glycine (pH 3.5). All proteins were then purified by size-exclusion column in HBS buffer (10 mM HEPES pH 7.2, 150 mM NaCl). For long-term storage at -80°C, proteins were supplemented with glycerol to 10%.

[0197] Reagents used in lacrimal gland regeneration experiments include (but are not limited to): [Table 1]

[0198] Primers used in the experiments include: [Table 2-1] [Table 2-2]

[0199] The WNT mimetic construct used in the experiments contains the following amino acid sequence (bold = LRP-binding variable domain; underline = linker; italics = FZD-binding variable domain; no decoration (not bold, not underlined and not italic = Fc domain). [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0200] It will be readily apparent to one of skill in the art that the above constructs can be modified to be expressed as various homologs and isoforms, can be edited in non-binding domain sequences, and can be expressed using various synonymous nucleotide sequences using a variety of suitable expression vector systems. (References) The following references are incorporated herein in their entirety: [ka] [ka]

Claims

1. A composition for treating a lacrimal gland disorder in a subject, the composition comprising a WNT signaling modulator, and the subject being in need of lacrimal gland cell regeneration.

2. The composition of claim 1 , wherein the lacrimal gland cells are acinar cells, progenitor cells, ductal cells, myoepithelial cells, or immune cells.

3. 10. The composition of claim 1, wherein the WNT signaling modulator is an engineered WNT signaling modulator.

4. 4. The composition of claim 3, wherein the engineered WNT signaling modulator is a WNT agonist, the WNT agonist comprising at least one engineered bispecific antibody or antigen-binding fragment thereof that directly activates the canonical WNT signaling pathway.

5. 2. The composition of claim 1, wherein the WNT signaling modulator is an engineered WNT superagonist comprising an E3 ligase binding domain selected from the group consisting of a mutant R-spondin (RSPO) protein; and an E3 ligase-specific antibody or a functional fragment thereof.

6. 10. The composition of claim 1, wherein the WNT signaling modulator comprises at least one engineered bispecific IgG antibody or antigen-binding fragment thereof that directly activates the canonical WNT signaling pathway.

7. The composition of claim 6, wherein the bispecific IgG antibody or its antigen-binding fragment comprises a binding composition specific for at least one FZD receptor and a binding composition specific for at least one LRP receptor.

8. the WNT signaling modulator is selected from (i) WNT3a; (ii) a WNT mimetic comprising an FZD-binding composition and an LRP-binding composition; or (iii) an R-spondin mimetic; a) the FZD-binding composition binds to at least one of Fzdl, Fzd2, Fzd5, Fzd7, Fzd8, or a combination of the following Fzds: I. Fzd1, Fzd2, Fzd5, Fzd7, Fzd8; or II. Fzd1, Fzd2, and Fzd7; or III. Fzd5 and Fzd8 preferentially binds to one of the following: b) the LRP binding composition binds to at least one of Lrp6 and / or Lrp5; The composition of claim 1.

9. wherein the WNT signaling modulator comprises one or more polypeptides selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and any variants thereof of any of the foregoing; and optionally the WNT signaling modulator comprises one or more polypeptides selected from the group consisting of: a) two polypeptides of SEQ ID NO: 1 (or one or both variants thereof having at least 90% identity thereto) and two polypeptides of SEQ ID NO: 2 (or one or both variants thereof having at least 90% identity thereto); b) two polypeptides of SEQ ID NO: 3 (or one or both variants thereof having at least 90% identity thereto) and two polypeptides of SEQ ID NO: 4 (or one or both variants thereof having at least 90% identity thereto); c) two polypeptides of SEQ ID NO: 5 (or one or both variants thereof having at least 90% identity thereto) and two polypeptides of SEQ ID NO: 6 (or one or both variants thereof having at least 90% identity thereto); d) two polypeptides of SEQ ID NO: 7 (or one or both variants thereof having at least 90% identity thereto) and two polypeptides of SEQ ID NO: 8 (or one or both variants thereof having at least 90% identity thereto); or e) two polypeptides of SEQ ID NO: 9 (or one or both variants thereof having at least 90% identity thereto) and two polypeptides of SEQ ID NO: 10 (or one or both variants thereof having at least 90% identity thereto); f) two polypeptides of SEQ ID NO: 11 (or one or both variants thereof having at least 90% identity, at least 95% identity, or at least 98% identity thereto) and two polypeptides of SEQ ID NO: 14 (or one or both variants thereof having at least 90% identity thereto); g) two polypeptides of SEQ ID NO: 12 (or one or both variants thereof having at least 90% identity thereto) and two polypeptides of SEQ ID NO: 14 (or one or both variants thereof having at least 90% identity thereto); h) two polypeptides of SEQ ID NO: 13 (or one or both variants thereof having at least 90% identity thereto) and two polypeptides of SEQ ID NO: 14 (or one or both variants thereof having at least 90% identity thereto); or i) two polypeptides of SEQ ID NO: 15 (or one or both variants thereof having at least 90% identity thereto) and two polypeptides of SEQ ID NO: 16 (or one or both variants thereof having at least 90% identity thereto). The composition of claim 1 comprising:

10. The composition according to any one of claims 1 to 9, characterized in that at least one molecule from the group consisting of RSPO2, an RSPO2 fragment, and an engineered RSPO2 mimetic is co-administered.

11. The composition according to any one of claims 1 to 9, wherein the subject is a mammal.

12. The composition according to any one of claims 1 to 9, wherein the subject is a human.

13. The composition according to any one of claims 1 to 9, wherein the subject is in need of lacrimal gland cell regeneration due to lacrimal gland disorder, and the lacrimal gland disorder is dry eye disease.

14. 14. The composition of claim 13, wherein the dry eye disease is caused by Sjogren's syndrome, chronic graft-versus-host disease (cGHVD), rheumatoid arthritis (RA), Stevens-Johnson syndrome, ocular rosacea, chemotherapy, radiation oncology treatment, diabetes, lupus, or meibomian gland dysfunction (MGD).

15. The composition of claim 14, wherein the dry eye disease is Sjogren's syndrome.

16. 10. A pharmaceutical composition for the treatment of dry eye disorder in a subject, comprising the composition of any of claims 1 to 9 and a pharmaceutically acceptable excipient, diluent, or carrier.

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