EP3923960A1

EP3923960A1 - Stem cell therapy for lacrimal gland dysfunction

Info

Publication number: EP3923960A1
Application number: EP20704038.7A
Authority: EP
Inventors: Michael MØLLER-HANSEN; Steffen HEEGAARD; Ann-Cathrine LARSEN
Original assignee: Kobenhavns Universitet; Rigshospitalet Copenhagen University Hospital
Current assignee: Kobenhavns Universitet; Rigshospitalet Copenhagen University Hospital
Priority date: 2019-02-15
Filing date: 2020-02-14
Publication date: 2021-12-22
Also published as: WO2020165404A1

Abstract

Described herein are compositions comprising allogeneic adipose-derived stem cells and their use in treating or preventing lacrimal and/or meibomian gland dysfunction, e.g., in dry eye diseases, such as aqueous-deficient dry eye disease.

Description

STEM CELL THERAPY FOR LACRIMAL GLAND DYSFUNCTION

FIELD OF THE INVENTION

The present invention relates to compositions comprising human adipose tissue-derived mesenchymal stem cells (ASCs), preferably allogeneic cells, for use in treating or preventing the dysfunction of an exocrine gland, such as a lacrimal gland or meibomian glands.

BACKGROUND OF THE INVENTION

Dry eye disease (DED, keratokonjunctivitis sicca) is a common problem seen in patients all over the world with a reported prevalence of 5-50%, more often in women than in men and increasing with age (Stapleton et a/. , 2017) . Symptoms include ocular discomfort, visual disturbance, tear instability, damage to the ocular surface and inflammation of the ocular surface.

Particularly aqueous-deficient dry eye disease (ADDE) is common in elderly, as well as with use of systemic drugs such as anxiolytics, antipsychotics, and inhaled steroids. Possible causes of severe ADDE include trachoma, ocular chemical burns, and graft-versus-host disease (Gomes et a/. , 2017) . However, in the Western world the most common cause of severe ADDE is inflammatory infiltration of the lacrimal sac as seen in Sjogren syndrome (Craig et a/. , 2018) . Inflammation and the loss of tear volume in ADDE leads to stress of the ocular surface and frictional damage which causes epithelial injury and friction-related symptoms. With increasing severity, ADDE causes scarring and vascularization of the cornea leading to visual impairment. Current treatment is symptomatic, often in the form of topical ocular lubricants, but other options include tear conservation (e.g. punctal occlusion), topical and systemic secretagogues, autologous serum drops, and finally surgical interventions such as tarsorrhaphy. A curative treatment of severe ADDE does not, however, exist (Jones et al. , 2017) .

A recent undertaking by the Tear Film & Ocular Surface Society (TFOS) of an evidence-based review of current dry eye therapies and management options concluded that further evidence was required to support the introduction, and continued use, of many of the treatment options currently available to manage dry eye disease, as well as information of appropriate treatment starting points and an understanding of treatment specificity in relation to dry eye disease subtype (Jones et al. , 2017) . Mesenchymal stem cells (MSCs), including those derived from adipose tissue (ASCs), have been proposed as a new treatment option for a wide range of diseases and conditions (WO 2017/068140; WO 2014/203267; WO 2017/144552 Al), and have been studied in dogs suffering from dry eye disease as well as in animal models (Bittencourt et a/. , 2016; Villatoro et a/. , 2015; Wood et a/. , 2012; Beyazyildiz et a/. , 2014; WO 2012/166932 A2) .

Despite these and other advances in the art, however, there is still a need for safe, reproducible and clinically effective therapies for DED and other lacrimal gland dysfunctions in human subjects.

It is an object of embodiments of the invention to provide compositions and methods for such therapies, particularly based on ASCs.

SUMMARY OF THE INVENTION

It has been found by the present inventors that allogeneic ASCs are safe and effective for treating and/or preventing lacrimal gland dysfunction in human subjects.

So, in a first aspect the present invention relates to a composition comprising allogeneic adipose tissue-derived mesenchymal stem cells (ASCs) for use in treating or preventing dysfunction of a lacrimal gland in a human subject.

In a second aspect, the invention relates to a method of treating or preventing dysfunction of a lacrimal gland in a human subject, comprising administering a composition comprising allogeneic ASCs to the human subject, typically wherein the composition comprises a therapeutically effective amount of allogeneic ASCs.

In one embodiment, the composition is administered at a dosage of from about 1 x 10⁶ to about 5 x 10⁷ allogeneic ASCs. In a particular embodiment, the composition is administered at a dosage of from about 1 x 10⁶ to about 1 x 10⁷ allogeneic ASCs, such as about 2 x 10⁶, about 3 x 10⁶, about 4 x 10⁶, about 5 x 10⁶, about 7 x 10⁶, about 9 x 10⁶, or about 10 x 10⁶ allogeneic ASCs.

In one embodiment, the composition is administered at a volume of from about 0. 1 mL to about 0.5 mL, such as about 0.1 mL, about 0.2 mL or about 0.5 mL, directly into the lacrimal gland. In one embodiment, the composition is administered by transconjunctival injection into the palpebral lobe of the lacrimal gland by or transcutaneous injection into the orbital lobe of the lacrimal gland, optionally by injecting approximately equal volumes at two separate injection sites on the lobe.

In one embodiment, the administration is carried out under topical (eye drop) anesthesia.

In one embodiment, the composition is from a thawed, ready-to-use preparation of cryopreserved allogeneic human ASCs free of non-human animal proteins.

In one embodiment, the composition comprises a suspension of allogeneic adult ASCs in a protein-free cryoprotectant at a concentration of at least about 1.5 x 10⁷ allogeneic adult ASCs per mL, such as from about 2 x 10⁷ to about 5 x 10⁷ allogeneic adult ASCs per mL.

In one embodiment, the protein-free cryoprotectant comprises dimethylsulphoxide (DMSO) at a concentration of about 5% to about 15% (v/v), such as about 5% or about 10% (v/v). In a specific embodiment, the composition further comprises Trolox (6-hydroxy-2, 5,7,8- tetramethylchroman-2-carboxylic acid), Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl-, H₂P0^4-, HEPES,

lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine and glutathione.

In one embodiment, at least about 80% of the ASC population express CD90, CD73, CD13, CD105, CD29, CD166, CD10, CD140b, CD160, CD204, CD272, CD44, CD49a, CD54, CD9, Galectin 3, Galectin 9, HLA-G and LTPR and at most about 15% of the ASC population express CD45, CD19, CD14, CD106, CD31 and CD36.

In one embodiment, the composition is obtained or obtainable by a process comprising the steps of

(i) adding the stromal vascular fraction (SVF) of a lipoaspirate collected from a human donor to a bioreactor wherein at least one surface is pre-treated to promote adhesion of adult stem cells;

(ii) in the bioreactor, cultivating adherent cells to confluence in a serum-free culture

medium supplemented with human platelet lysate;

(iii) detaching the adherent cells;

(iv) freezing the detached cells in a cryoprotectant at a concentration of at least 1 x 10⁶ cells/mL;

(v) thawing the frozen cells and repeating steps (ii) and (iii) at least once,

(vi) freezing the detached cells in a cryoprotectant at a concentration of at least 1.5 x 10⁷ cells/mL; and (vii) thawing the frozen composition.

In one embodiment, the subject has, or is at risk for, dry eye disease (DED) . In one specific embodiment, the DED is aqueous-deficient dry eye disease (ADDE), evaporative dry eye disease, (EDE), or a combination thereof.

In one embodiment, the subject has, or is at risk for, severe ADDE.

In one embodiment, the human subject has primary or secondary Sjogren syndrome.

In a third aspect, the present invention relates to a composition comprising allogeneic adipose tissue-derived mesenchymal stem cells (ASCs) for use in treating or preventing dysfunction of meibomian glands in a human subject.

In a fourth aspect, the invention relates to a method of treating or preventing dysfunction of meibomian glands in a human subject, comprising administering a composition comprising allogeneic ASCs to the human subject, typically wherein the composition comprises a therapeutically effective amount of the allogeneic ASCs.

These and other aspects and embodiments are described in more detail below.

DETAILED DISCLOSURE OF THE INVENTION

Definitions

As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

As used herein, a "dysfunctional" lacrimal gland refers to a lacrimal gland with an abnormal tear production, resulting in insufficient tear production (hypolacrimation), surplus tear production (hyperlacrimation), and/or deficiencies in tear quality.

As used herein, "dry eye disease" (DED), "dry eye syndrome" (DES), "keratokonj unctivitis sicca" (KCS) or "xerophthalmia" refers to a condition characterized by hypolacrimation (aqueous-deficient dry eye disease, "ADDE") or excessive tear film evaporation (evaporative dry eye disease, "EDE"), or a combination of both. Several symptoms and signs define DED, such as one or more of ocular discomfort, visual disturbance, tear film instability, damage to the ocular surface, and inflammation of the ocular surface. To screen for DED, the Ocular Surface Disease Index (OSDI) is the most widely used and validated questionnaire. Based on 12 questions a score of 0- 100 is calculated : 0- 12 is normal, 13-22 mild, 23-32 moderate, and > 33 severe DED. However, to make the DED diagnosis, typically at least one of the following "homeostasis markers" indicate DED : a tear breakup time (TBUT) < 10 seconds, a tear osmolarity > 308 mOsm/L; Ocular Surface Staining > 5 corneal spots; > 9 conj unctival spots and/or lid margin ( >2 mm length & >25% width) . Non-limiting examples of methods to measure TBUT and tear osmolarity are provided in Example 1. If the DED diagnosis is established, the subtype of DED can range on a continuum from evaporative dry eye (EDE) to aqueous tear deficient dry eye disease (ADDE) .

ADDE is a condition in which the tear production in the lacrimal gland (LG) is impaired, resulting in lacrimal hyposecretion. In subjects with ADDE, the lacrimal gland does not produce sufficient tears to keep the entire conj unctiva and cornea covered by a complete layer. Inflammation and the loss of tear volume in ADDE leads to stress of the ocular surface and frictional damage which causes epithelial injury and friction-related symptoms, such as eye irritation, (e.g. , dryness, burning, sandy-gritty sensations, itching, stinging, fatigue, pain, redness, pulling sensations), and stingy discharge from the eyes. Typically, ADDE can be diagnosed using the Schirmer's I test without applying anaesthetic eye drops. In healthy subjects this test is > 10 mm in 5 minutes (Wolffsohn et a/. , 2017), which means that a Schirmer's I test of less than 10mm in 5 minutes is indicative of ADDE. A non-limiting example of the procedure for a Schirmer's I test is provided in Example 1.

Severe ADDE is usually caused by trachoma, ocular chemical burns, graft-versus-host disease and/or inflammatory infiltration of the lacrimal sac (as seen in primary or secondary Sjogren's syndrome) . With increasing severity ADDE causes scarring and vascularization of the cornea leading to visual impairment, and subjects with severe ADDE usually present with one or more of thickening of the corneal surface, corneal erosion, punctate keropathy, epithelial defects, corneal ulceration, corneal neovascularization, corneal scarring, corneal thinning, and/or even corneal perforation. Typically, severe ADDE can be diagnosed using one or two, preferably all, of the following criteria : an OSDI-score > 33; Schirmer's I test < 5 mm in 5 minutes and TBUT < 10 sec, using the respective methods described in Example 1.

Tear hyperosmolarity is believed to be the core mechanism of DED. It damages the ocular surface both directly and by initiating inflammation . The cycle of events is described as the Vicious Circle of DED. In ADDE, tear hyperosmolarity results when lacrimal secretion is reduced, in conditions of normal evaporation from the eye. In EDE, tear hyperosmolarity is caused by excessive evaporation from the exposed tear film in the presence of a normally functioning lacrimal gland. The homeostasis markers for ADDE and EDE are the same, however, in EDE the Schirmer's I test is usually normal while the degree of Meibomian gland dysfunction is usually more severe.

"Meibomian gland dysfunction" (MGD) refers to a chronic, diffuse abnormality of the meibomian glands, commonly characterized by terminal duct obstruction and/or

qualitative/quantitative changes in the glandular secretion. MGD may result in alteration of the tear film, symptoms of eye irritation, clinically apparent inflammation, and ocular surface disease. MGD is considered an "evaporative" dry eye condition, oftentimes due to a loss of amount and/or integrity of the lipid component of the precorneal tear film. MGD conditions include without limitation posterior blepharitis, meibomian gland disease, meibomitis, meibomianitis, and meibomian keratoconjunctivitis. The pathophysiological mechanisms can be broadly categorized into ( 1) low delivery of meibum (due to obstruction or

hyposecretion— either primary or secondary in nature) and (2) high delivery of meibum (either primary or secondary hypersecretion) . Typically, MGD is diagnosed by clinical examination, evaluating the appearance of the Meibomian gland ducts, lid margins, and lipid thickness.

Lacrimal gland hyperlacrimation occurs, e.g. , in crocodile tear syndrome (CTS) or gusto- lacrimal reflex, usually following a Bell's palsy or a traumatic facial paralysis.

As used herein, "adipose tissue-derived mesenchymal stem cells", "adipose tissue-derived stem cells," "adipose tissue-derived stromal cells" and the like, refer to multipotent stromal stem cells, also known as mesenchymal stem cells, multipotent stromal cells, multipotent stem cells, and mesenchymal stromal/stem cells, which are derived from adipose tissue, and are herein referred to as "ASCs". Certain criteria for identifying ASCs are known in the art and are described in, for example, Bourin et a/. (2013), which is incorporated by reference in its entirety. In some embodiments, ASCs are characterized by their ability to differentiate along adipocytic, chondroblastic and osteoblastic lineages under appropriate conditions. ASCs in culture may be characterized by expression of one or more of the following cell-surface markers: CD90, CD73, CD105 and lack of expression of CD45 and CD31. In some embodiments, they can be distinguished from bone-marrow-derived MSCs by their positivity for CD36 and negativity for CD 106.

The term "cryopreserve", "cryostore" or its various grammatical forms as used herein refers to preserving cells for storage in a cryoprotectant at sub-zero temperatures. For long-term storage, cryovials containing the cells and cryoprotectant are usually placed in liquid nitrogen. The term "cryoprotectant" as used herein refers to an agent that minimizes ice crystal formation in a cell or tissue, when the cell or tissue is cooled to sub-zero temperatures and results in substantially less damage to the cell or tissue after thawing in comparison to the effect of cooling without cryoprotectant.

A preparation of human ASCs "free of non-human animal proteins" means that the ASCs were produced by a process where they did not come into contact with proteins derived from non-human animals.

"Viability" as used herein refers to the feature of cells of not taking up membrane

impermeant dye (e.g. , Trypan Blue, FVS-780, SYTOX blue, propidium iodide), thereby demonstrating cell membrane integrity.

"Proliferative capacity" as used herein refers to the ability of cells to multiply in a suitable cultivation medium. Proliferative capacity can, for example, be represented by the relative number of cells after a 24h, 48h or 72h cultivation period as compared to the number of cells initially plated. This can also be expressed as "population doublings" during a certain period. For example, a population doubling of at least 1 during 48h in cell culture means that the number of cells seeded have doubled at least once during that period.

As used herein, the term "donor" refers to the human or mammal from which the adipose tissue is retrieved, typically by liposuction. Preferably, the human is an adult.

The terms "treatment," "therapy" and the like are used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e. , arresting its development; or (c) relieving the disease symptom, i.e. , causing regression of the disease or symptom.

As used herein, the term "pharmaceutical composition" refers to a composition intended for use in therapy of a human patient. A pharmaceutical composition according to the present disclosure typically comprises ACSs. Flowever, the pharmaceutical composition may additionally include other pharmaceutically acceptable, non-cellular components, such as pharmaceutically acceptable carriers. The phrase "pharmaceutically acceptable" is employed herein to refer to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical knowledge and j udgment by a person of skill in the art, suitable for use in contact with the tissues of human beings without excessive toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase

"pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, or excipient, which are known in the art.

As used herein, a "therapeutically effective amount" refers to the amount of an active agent (e.g. , ASCs) sufficient to induce a desired biological result (e.g. , prevention, delay, reduction or inhibition of one or more symptoms of a given lacrimal gland dysfunction) . A

"therapeutically effective amount" as used herein to denote any amount of the active agent (or agents) causing a measurable improvement in one or more symptoms; preferably an improvement which is significant at a predetermined level (e.g. , a two-sided p-value less than 0.05, using, e.g. , R Statistics software) . The amount may vary with the condition being treated, the stage of advancement of the condition, and the type and concentration of active agent applied . Appropriate amounts in any given instance will be readily apparent to those skilled in the art, such as a physician, or capable of determination by routine

experimentation.

In the context of therapeutic use of the disclosed pharmaceutical compositions, in

"allogeneic" therapy, the donor and the recipient are genetically different individuals of the same species, whereas in "autologous" therapy, the donor and the recipient is the same individual.

The terms "recipient", "subject" and "patient" are used interchangeably herein and refer to the subject for whom treatment or therapy is desired, particularly a human subject.

In the context of the present invention, unless contradicted by context, "about",

"approximately" or the like, typically refers to a variation (+/-) of at most 20%, such as at most 10%, such as at most 5%, from the reference value. So, for example, about 2.0 x 10^s cells may include or correspond to from 1.6 x 10⁸ to 2.4 x 10⁸ cells, such as from 1.8 x 10⁸ to 2.2 x 10⁸ cells, such as from 1.9 x 10⁸ to 2. 1 x 10⁸ cells. Specific embodiments of the invention

The present invention relates to a composition comprising allogeneic human ASCs for use in treating or preventing dysfunction of a lacrimal gland and/or meibomian glands in a human subject. The present invention also relates to a method of treating or preventing dysfunction of a lacrimal gland and/or meibomian glands in a human subject, the method comprising administering a composition comprising allogeneic ASCs to the human subject. The invention is based, at least in part, on the first-in-human trial on the safety and efficacy of allogeneic ASCs for treating patients with ADDE, described in Example 1.

The composition comprises ASCs isolated from healthy donors, preferably adult donors. Advantageously, the ASCs are isolated by two rounds of expanding the ASCs in a bioreactor separated by a cryopreservation step, resulting in a composition suitable for cryopreservation in a cell bank. The composition can be used as an off-the-shelf cryopreserved product, ready directly after thawing for administration to a huma n subject in which lacrimal gland dysfunction is to be treated or prevented .

In some embodiments, the ASC composition is administered locally, i.e. , in proximity to or directly into the lacrimal and/or meibomian glands. Without being limited to theory, once engrafted or transplanted, the ASCs stimulate and improve regeneration through paracrine and/or juxtacrine mechanisms, releasing factors and components promoting natural endogenous repair, including extracellular matrix remodelling, revascularization and anti inflammatory action. An additional property believed to be inherent in ASCs is also their active immunosuppression, a property which distinguishes them from other somatic cells and, e.g. , prevents rejection of the allogeneic ASC graft.

Dosages

The administered dosage of the ASCs may vary depending on the symptoms, age and body weight of the patients, the nature and severity of the lacrimal and/or meibomian gland dysfunction to be treated or prevented, and the route of administration. Suitable dosages for a patient or a group of patients can be determined by the skilled physician, particularly based on the present disclosure.

In some embodiments, the composition is administered at a dosage of from about 1 x 10⁶ to about 5 x 10⁷ allogeneic ASCs, such as about 1 x 10⁶ cells, about 2 x 10⁶ cells, about 3 x 10⁶ cells, about 4 x 10⁶ cells, about 5 x 10⁶ cells, about 7 x 10⁶ cells, about 9 x 10⁶ cells, about 10 x 10⁶ cells, about 11 x 10⁶ cells, about 13 x 10⁶ cells, about 15 x 10⁶ cells, about 17 x 10⁶ cells, about 20 x 10⁶ cells, about 22 x 10⁶ cells, about 25 x 10⁶ cells, about 30 x 10⁶ cells, about 35 x 10⁶ cells, about 40 x 10⁶ cells, about 45 x 10⁶ cells, or about 50 x 10⁶ cells.

In some embodiments, the composition is administered at a dosage of from about 4 x 10⁶ to about 3 x 10⁷ allogeneic ASCs.

In one preferred embodiment, the composition is administered at a dosage of from about 9 x 10⁶ to about 13 x 10⁶, such as between about 10 x 10⁶ to about 12 x 10⁶ allogeneic ASCs.

In one particular embodiment, the composition is administered at a dosage of about 10 x 10⁶ allogeneic ASCs. In one particular embodiment, the composition is administered at a dosage of about 11 x 10⁶ allogeneic ASCs.

In one preferred embodiment, the composition is administered at a dosage of from about 1 x 10⁶ to about 5 x 10⁶, such as between about 2 x 10⁶ to about 4 x 10⁶ allogeneic ASCs. In a particular embodiment, the composition is administered at a dosage of about 2.2 x 10⁶ allogeneic ASCs.

In one preferred embodiment, the composition is administered at a dosage of from about 2 x 10⁶ to about 7 x 10⁶, such as between about 3 x 10⁶ to about 6 x 10⁶ allogeneic ASCs. In a particular embodiment, the composition is administered at a dosage of about 4.4 x 10⁶ allogeneic ASCs.

In one preferred embodiment, the composition is administered at a dosage of from about 20 x 10⁶ to about 30 x 10⁶, such as between about 23 x 10⁶ to about 27 x 10⁶ allogeneic ASCs. In a particular embodiment, the composition is administered at a dosage of about 25 x 10⁶ allogeneic ASCs.

Administration

The composition comprising ASCs is typically administered parenterally, and preferably locally, i.e. , in proximity to the lacrimal and/or meibomian glands, so that the factors and components which are released from the engrafted or transplanted ASCs and which stimulate immunosuppression, vascularization and/or tissue regeneration can reach the gland and thereby prevent or treat the dysfunction.

In some embodiments, the composition is administered transconj unctivally {i.e. , through the conj unctiva and into e.g. the palpebral lobe of the lacrimal gland), subconj unctivally {i.e. , beneath the cunctiva), transcutaneously (/. e. , through the skin and into e.g. the orbital lobe of the lacrimal gland), perilacrirmally (/. e. , around the lacrimal gland), periocularly (/.e. , around the eye), or topically to the ocular surface. For example, in subjects who suffer from or is at risk for DED because of a dysfunctional lacrimal gland, the ASCs may be administered transconj unctivally or trancutaneously into the lacrimal gland . In subjects suffering from or at risk for DED because of meibomian gland dysfunction, the ASCs may, for example, be administered topically to the ocular surface or into the stroma of the eyelid, e.g. , at or near the lower lid margin. Preferably, the composition is administered within a distance of at most about 2 cm, such as at most about 1 cm, such as at most about 0.5 cm, such as at most 0.3 cm, such as at most 0.2 cm, from the perimeter of the gland . Most preferably, in subjects suffering from or at risk for DED because of a dysfunctional lacrimal gland, the composition is administered directly into the lacrimal gland, by transconj unctival or transcutaneous administration.

In some subjects, both lacrimal glands in a subject are dysfunctional. In such embodiments, both lacrimal glands can be treated according to the invention, optionally at the same occasion.

The administration of the composition may be carried out under general or local anaesthesia, typically in cases where the composition is to be administered via transconj unctival, subconj unctival or transcutaneous routes. In one embodiment, topical anaesthesia is applied, e.g. , using anaesthetic eye drops to provide local anaesthesia. Anaesthetic eye drops suitable for this purpose are well-known in the art. Suitable, non-limiting examples of anaesthetic agents suitable for administration via eye drops include oxybuprocain, lidocaine,

proparacaine, mepivacain, diclofenac and ***e, as well as pharmaceutically active salts thereof. In a particular embodiment, eye drops containing oxybuprocaine hydrochloride, e.g. , at a concentration of 0.4% w/v, is used for providing topical anaesthesia prior to

administration of the composition.

The volume of the composition to be administered is adj usted to the concentration of ASCs in the composition so as to administer a therapeutically effective amount of ASCs, /.e. , an amount of ASCs effective in preventing or treating the dysfunctional lacrimal and/or meibomian glands. In some embodiments, the composition is administered at a volume of from about 0.05 mL to about 1 mL, such as between about 0.05 mL to about 0.5 mL, such as about 0. 1 mL to about 1 mL, such as between about 0.1 mL to about 0.8 mL, such as between about 0.1 mL to about 0.5 mL, such as between about 0.2 mL to about 0.7 mL, such as about 0.1 mL, about 0.2 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL or about 1.0 mL. In one preferred embodiment, the total volume of the composition administered is between about 0.1 mL to about 0.3 mL, such as about 0. 1 mL or about 0.2 mL. In one preferred embodiment, the total volume of the composition administered is between about 0.4 mL to about 0.6 mL, such as about 0.5 mL.

At a given administration event, the total volume of composition may be administered as a single dose, or may be divided into several doses, such as 2, 3, 4 or 5 doses, e.g. , 2 doses, typically of approximately equal volume.

In some embodiments, the injection volume is adapted to less than 50% of the estimated volume (mL) of the lacrimal gland. The volume of the lacrimal gland can be estimated using, e.g. , magnetic resonance imaging (MRI) . So, in one aspect, the composition is administered using a method comprising the following steps: (a) estimating the volume of the lacrimal gland, and (b) administering a predetermined volume of the composition directly into the lacrimal gland, wherein the predetermined volume is no more than about 50% of the estimated volume of the lacrimal gland as determined in step (a) .

In one embodiment, the predetermined volume is selected from about 0. 1 mL, about 0.2 mL, about 0.3 mL, about 0.4 mL and about 0.5 mL. Preferably, the predetermined volume selected form this list is the volume closest to 50% of the estimated volume of the lacrimal gland.

In some embodiments, the composition is administered at a volume of from about 0. 1 mL to about 1 mL, such as from about 0. 1 mL to about 0.5 mL, such as about 0. 1 mL, about 0.2 mL or about 0.5 mL, directly into the lacrimal gland. In one embodiment, the composition is administered to the lacrimal gland by transconjunctival injection. This may, for example, be carried out according to the method described by Nava-Castaneda et a/. (2006), using a single eversion of the upper eyelid . In one specific embodiment, the composition is administered by transconjunctival injection into the palpebral lobe of the lacrimal gland, optionally by injecting approximately equal volumes at two separate injection sites into the palpebral lobe.

In one particular embodiment, suitable for subjects suffering from or at risk for DED because of a dysfunctional lacrimal gland, topical anaesthesia is given, the upper eyelid is everted, the lacrimal gland identified, and a total volume of about 0.5 mL of the composition injected transconj unctivally directly into the palpebral lobe of the lacrimal gland at two separate injection sites; at approximately equal volumes at each injection site, e.g. , between about 0.2 and about 0.3 mL, such as about 0.25 mL, at each site. In one preferred embodiment, the about 0.5 mL composition administered comprises from about 5 x 10⁶ to about 20 x 10⁶, such as from about 7 x 10⁶ to about 15 x 10⁶, such as about 9 x 10⁶ to about 13 x 10⁶, such as from about 10 x 10⁶ to about 12 x 10⁶ allogeneic ASCs, such as about 11 x 10⁶ ASCs. In another preferred embodiment, the about 0.5 mL composition administered comprises from about 20 x 10⁶ to about 25 x 10⁶, such as from about 23 x 10⁶ to about 25 x 10⁶ allogeneic ASCs, such as about 25 x 10⁶ allogeneic ASCs.

In one particular embodiment, suitable for subjects suffering from or at risk for DED because of a dysfunctional lacrimal gland, topical anaesthesia is given, the upper eyelid is everted, the lacrimal gland identified, and a total volume of about 0.2 mL of the composition injected transconj unctivally directly into the palpebral lobe of the lacrimal gland ; optionally at two separate injection sites and approximately equal volumes at each injection site, e.g. , about 0.1 mL at each site. In one preferred embodiment, the about 0.2 mL composition

administered comprises from about 2 x 10⁶ to about 10 x 10⁶, such as from about 3 x 10⁶ to about 6 x 10⁶, such as about 4 x 10⁶ to about 5 x 10⁶, such as about 4.4 x 10⁶ ASCs. In another preferred embodiment, the about 0.2 mL composition administered comprises from about 8 x 10⁶ to about 10 x 10⁶, such as from about 9 x 10⁶ to about 10 x 10⁶, such as about 10 x 10⁶ allogeneic ASCs.

In one particular embodiment, suitable for subjects suffering from or at risk for DED because of a dysfunctional lacrimal gland, topical anaesthesia is given, the upper eyelid is everted, the lacrimal gland identified, and a total volume of about 0.1 mL of the composition injected transconj unctivally directly into the palpebral lobe of the lacrimal gland ; optionally at two separate injection sites and approximately equal volumes at each injection site, e.g. , about 0.05 mL at each site. In one preferred embodiment, the about 0. 1 mL composition administered comprises from about 1 x 10⁶ to about 3 x 10⁶, such as from about 1.5 x 10⁶ to about 2.7 x 10⁶, such as about 2 x 10⁶ to about 2.5 x 10⁶, such as about 2.2 x 10⁶ ASCs. In another preferred embodiment, the about 0. 1 mL composition administered comprises from about 2 x 10⁶ to about 10 x 10⁶, such as from about 3 x 10⁶ to about 6 x 10⁶, such as about 4 x 10⁶ to about 6 x 10⁶, such as about 5 x 10⁶ ASCs.

In one particular embodiment, suitable for subjects suffering from or at risk for DED because of dysfunctional meibomian glands, a total volume of about 0.05 to about 0. 1 mL of the composition is administered topically to the ocular surface, e.g. , in the form of eye drops, such as 1 or 2 eye drops. In one preferred embodiment, an about 0.05 mL composition administered comprises from about 1 x 10⁶ to about 2.5 x 10⁶ ASCs, such as about 1 x 10⁶, about 1.5 x 10⁶, about 2 x 10⁶, or about 2.5 x 10⁶ ASCs. In another preferred embodiment, an about 0. 1 mL composition administered comprises from about 2 x 10⁶ to about 5 x 10⁶ ASCs, such as about 2 x 10⁶, about 3 x 10⁶, about 4 x 10⁶, or about 5 x 10⁶ ASCs.

The composition may be administered using a delivery device suitable for the present purpose, i. e. , the delivery of cells in proximity to the lacrimal and/or meibomian glands. In embodiments where the composition is to be administered via transconj unctivital, subconj unctival or transcutaneous routes, the cell delivery device is usually a syringe, such as a sterile, disposable syringe with a volume of 1 mL, luer lock, markings for every 0.01 mL and a 28G needle with a length of 6 mm. Suitable cell delivery devices for the present compositions and uses are described in WO 2012/ 166932, hereby incorporated by reference in its entirety.

Compositions

The compositions for use according to the aspects and embodiments described herein comprise human allogeneic ASCs, i.e. , ASCs that are obtained or isolated from a healthy donor. Several different allogeneic ASC compositions proposed for pharmaceutical use together with methods for preparing them have been described (see, e.g. , WO 2017/068140; WO 2014/203267; WO 2017/144552 Al), all of which are contemplated for use in the treatments described herein.

Typically, the concentration of the ASCs in the composition is at least about 1.5 x 10⁷, such as at least about 2 x 10⁷, such as at least about 3 x 10⁷, such as at least about 5 x 10⁷ of allogeneic ASCs per mL. In one embodiment, the concentration is from about 1.8 x 10⁷ to about 2.6 x 10⁷ ASCs per mL, such as from about 2.0 x 10⁷ to about 2.4 x 10⁷ ASCs per mL, such as about 2.2 x 10⁷ ASCs per mL. In one embodiment, the concentration is from about 4 x 10⁷ to about 5 x 10⁷ ASCs per mL, such as from about 45 x 10⁶ to about 5 x 10⁷ ASCs per mL, such as about 50 x 10⁶ ASCs per mL.

The ASCs suitable for the composition for the uses as described herein can be characterized by their multipotent capacity, marker profile, and/or and by functional characteristics, such as proliferation capacity, viability, recovery and immunosuppressive capability, even after cryopreservation. Such characteristics are described in more detailed below. Suitable methods for determining these and other characteristics are described in WO 2017/068140 Al, which is hereby incorporated by reference in its entirety.

The ASCs are, in particular, characterized by their ability to differentiate along adipocytic, chondroblastic and osteoblastic lineages under appropriate conditions, e.g. , when cultured in differentiation medium according to the method described in Example 4 of WO 2017/068140 Al .

The ASCs can also or alternatively be characterized according to their phenotype, i. e. , marker profile, regarding their expression of markers in common with other mesenchymal stromal/stem cells, including CD90, CD73, CD105, and CD44, and maintaining low or negligible expression levels of CD45 and CD31 (Bourin et al. , 2013). Marker profiles can, for example, be conveniently determined by flow cytometry using fluorescence-labelled antibodies against each marker.

The compositions for use in the therapeutic methods described herein comprise an ASC population which is substantially homogenous, meaning that the majority of the cells comply with ASC standards.

Accordingly, in some embodiments, at least about 80% of the ASC population express CD90, CD73, CD13, CD105, CD29, CD166, CD10, CD140b, CD160, CD204, CD272, CD44, CD49a, CD54, CD9, Galectin 3, Galectin 9, HLA-G and LTPR and at most about 15% of the ASC population express CD45, CD19, CD14, CD106, CD31 and CD36.

In some embodiments, of the ASC population, at least 90% express CD90, CD73, CD13, CD29 and CD166; at most 5% express CD45, CD19, CD14 and CD31; at most 10% express CD106; between 2 and 15% express CD36; at least 10% express CD146; at least 80% express CD105 and at most 40% express CD34; and/or

- at least 90% express CD10, CD140b, CD160, CD204, CD272, CD44, CD54, CD9, Galectin 3, Galectin 9, HLA-G and LTPR; at least 80% express CD49a; at least 60% express CD258 and CD270 and at least 5% express CD200; at most 15% express CD15, CD152, CD163, CD18, CD274, CD39, CD40, CD62L, CD80 and CD86; and at most 30% express CXCR4.

The ASCs may further be characterized by their immunosuppressive properties. For example, the ASCs may be characterized by one or more or all of the following : suppressing activation of dendritic cells (DCs), suppressing proliferation of peripheral blood mononuclear cells (PBMCs), cell surface markers indicative of immunomodulation, especially

immunosuppression, or by a change in one or more cell surface markers in response to a cytokine such as interferon-gamma.

In one embodiment, the ASCs of the invention suppress activation of DCs, e.g. , reducing the expression of CD40, CD80, CD86 and HLA-DR by DCs mixed with ASCs as compared to DCs not mixed with ASCs (/.e., a positive control). In a specific embodiment, the assay of Example 9 of WO 2017/068140 A1 is used, wherein ASCs and DCs are seeded to result in approximately a 1 : 1 ratio; the DCs being stimulated with 1 pg/mL lipopolysaccharide (LPS) and 20 ng/mL interferon-gamma and incubated for 24 h; and the respective expression level of CD40, CD80, CD86 and HLA-DR is reduced, in average, to at most 80%, 65%, 70% and 80%, respectively, of the positive control.

In one embodiment, the ASCs of the invention suppress the proliferation of PBMCs, e.g. , as determined in a Mixed Lymphocyte Reaction (MLR). This type of assay is well-known in the art, and may comprise mixing ASCs with stimulated PBMCs from an allogeneic donor in different ratios, e.g. , in the range 1 : 20 to 1 : 1, using PBMCs without ASCs as positive controls, and measuring after a 4-day co-culture period, the PBMC incorporation of 3H- thymidine (25 pSi/ml) during an 18-20 h incubation period. Using this type of assay, as compared to the positive control, a 1 : 20, 1 : 10, 1 : 5 and 1 : 1 ratio of ASCs to PBMCs may result in an average 3H-thymidine incorporation of at most about 80%, 75%, 55%, and 25%, respectively, of the positive control.

In some embodiments, the ASCs are also or alternatively characterized by specific markers indicative of immunomodulation, especially immunosuppression, such as CD10, CD140a, CD160, CD204, CD258, CD270, CD272, CD44, CD49a, CD54, CD9, Galectin 3, Galectin 9, HLA-G, LTPR and combinations thereof. Without being limited to theory, these markers are associated with immune signalling, cell-cell and cell-ECM adhesion, homing, pattern recognition, T cell inhibition, up-regulation of growth factor receptors and inactivation of pro- inflammatory proteins.

In a further embodiment, the ASCs of the invention are also or alternatively characterized by a change in one or more cell surface markers in response to a pro-inflammatory cytokine such as interferon-gamma. This may advantageously be tested according to the assay of Example 11 of WO 2017/068140 Al, measuring a change in one or more ASC markers in Table 16 and 17 of that Example, showing a positive or negative change in the percentage of the ASC population expressing the marker in at least 5% of the ASC population, or

a positive- or negative change in the expression level of the marker on the portion of cells expressing the marker of at least 0.5-fold, when cultivated for 3 days in the presence of 50 ng/ml IFN-gamma, as compared to a control, such as cells from the same ASCs which have not been stimulated with IFN-gamma.

For example, in some embodiments, upon INF-gamma stimulation, the percentages of the ASC population expressing CD200, CD270, CD9, CXCR4 are reduced; the percentages of the ASC population expressing CD274 and CD49a are increased, and the expression level of CD54 on CD54-positive cells is increased. In one specific embodiment, at most about 30%, such as at most about 20%, such as at most about 15%, such as at most about 10% of the ASC population expresses CD274 whereas upon interferon-gamma stimulation, at least 70%, such as at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 95% of the ASC population expresses CD274, e.g. , when cultivating the ASCs for 3 days in the absence and presence of 50 ng/ml IFN-gamma, respectively.

In one specific embodiment, at least 95% of the ASC population expresses CD54 and upon interferon-gamma stimulation, the expression level of CD54 on CD54-expressing cells is increased by at least 20-fold, such as at least 30-fold . The marker CD54 (ICAM- 1) illustrates the mobilisation of an intercellular adhesion molecule necessary for the stabilisation of ASC- leukocyte interactions and signal transduction. ICAM- 1 is a ligand for LFA- 1 (integrin), a receptor found on leukocytes.

In a particular embodiment, upon interferon-gamma stimulation according to Example 11 of WO 2017/068140 Al, the percentage of the ASC population expressing CD274 is increased to at least 80% and the expression level of CD54 on CD54-positive cells is increased at least 25-fold.

In some embodiments, the composition is from a thawed, ready-to-use preparation of cryopreserved allogeneic adult human ASCs free of non-human animal proteins. In a preferred embodiment, the composition comprises a suspension of allogeneic adult human ASCs in a protein-free cryo protectant at a concentration of at least about 1.5 x 10⁷ allogeneic adult human ASCs per mL. In some embodiments, the concentration is from about 2 x 10⁷ to about 5 x 10⁷ allogeneic adult human ASCs per mL in a protein-free cryoprotectant. For example, in one embodiment, the concentration is from about 1.8 x 10⁷ to about 2.6 x 10⁷, such as from about 2.0 x 10⁷ to about 2.4 x 10⁷, such as about 2.2 x 10⁷ ASCs per mL in a protein-free cryoprotectant. In one embodiment, the concentration is from about 4 x 10⁷ to about 5 x 10⁷, such as from about 45 x 10⁶ to about 5 x 10⁷, such as about 50 x 10⁶ ASCs per mL in a protein-free cryoprotectant.

In some embodiments, the ready-to-use preparation has a total volume of up to 5 mL, such as from about 0.5 to about 2 mL, such as from about 1.0 mL to about 1.5 mL, such as about 1.1 mL or 1.3 mL. In one specific embodiment, the ready-to-use preparation has a total volume of about 1.3 mL and comprises about 29 million allogeneic adult human ASCs suspended in added protein-free cryoprotectant. In one specific embodiment, the ready-to- use preparation has a total volume of about 1. 1 mL and comprises about 55 million allogeneic adult human ASCs suspended in added protein-free cryoprotectant. The cryoprotectant used for preparing the compositions is typically protein-free, endotoxin- free and sterile.

In the first-in-human clinical trial of Example 1, it is found that a cryopreserved ASC product based on high-concentration human allogeneic ASCs and a protein-free cryoprotectant comprising 10% DMSO is safe and effective when administered directly into lacrimal glands. So, in one embodiment, the cryoprotectant comprises DMSO, preferably at a concentration of about 5% to about 15% (v/v). In one embodiment, the cryoprotectant comprises about 5%, about 6%, about 8%, about 10%, about 12% or about 15% DMSO. Preferably, the cryoprotectant comprises about 5% DMSO (v/v) or about 10% DMSO (v/v). Alternatively, the DMSO can be replaced by a glucan such as, for examples dextran, having an average molecular weight in the range of 35000 to 45000 Da, such as, e.g. , Dextran-40.

In one embodiment, the cryoprotectant comprises a 1 : 10 to about 1 : 20 mixture of DMSO and an aqueous solution comprising

(a) one or more electrolytes selected from the group consisting of potassium ions at a concentration ranging from about 35-45 mM, sodium ions ranging from about 80-120 mM, magnesium ions ranging from about 2-10 mM, and calcium ions ranging from about 0.01-0.1 mM ;

(b) a macromolecular oncotic agent having a size sufficiently large to limit escape from the circulation system and effective to maintain oncotic pressure equivalent to that of blood plasma and selected from the group consisting of human serum albumin, polysaccharide and colloidal starch;

(c) a biological pH buffer effective under physiological and hypothermic conditions;

(d) a nutritive effective amount of at least one simple sugar;

(e) an impermeant and hydroxyl radical scavenging effective amount of mannitol;

(f) an impermeant anion impermeable to cell membranes and effective to counteract cell swelling during cold exposure, said impermeant ion being at least one member selected from the group consisting of lactobionate, gluconate, citrate and

glycerophosphate;

(g) a substrate effective for the regeneration of ATP, said substrate being at least one member selected from the group consisting of adenosine, fructose, ribose and adenine; and

(h) glutathione.

In one embodiment, the cryoprotectant comprises a 1 : 10 to about 1 : 20 mixture of DMSO and an aqueous solution comprising a), one or more electrolytes selected from the group consisting of potassium ions at a concentration ranging from 35-45 mM, sodium ions ranging from 80-120 mM, magnesium ions ranging from 2-10 mM, and calcium ions ranging from 0.01-0.1 mM; b). a macromolecular oncotic agent having a size sufficiently large to limit escape from the circulation system and effective to maintain oncotic pressure equivalent to that of blood plasma and selected from the group consisting of human serum albumin, polysaccharide and colloidal starch;

c). a biological pH buffer effective under physiological and hypothermic conditions;

d). a nutritive effective amount of at least one simple sugar;

e). an impermeant and hydroxyl radical scavenging effective amount of mannitol;

f). an impermeant anion impermeable to cell membranes and effective to counteract cell swelling during cold exposure, said impermeant ion being at least one member selected from the group consisting of lactobionate, gluconate, citrate and

glycerophosphate;

g). a substrate effective for the regeneration of ATP, said substrate being at least one member selected from the group consisting of adenosine, fructose, ribose and adenine, and

h). at least one agent which regulates apoptotic induced cell death.

While several suitable cryoprotectants are commercially available or otherwise known in the art, non-limiting examples of cryoprotectants contemplated for the ASC compositions of the present invention are CryoStor® (BioLife Solutions), including CryoStor CS2, CryoStor CS5 and CryoStor CS10; and ProFreeze (Lonza). CryoStor freeze media are sterile serum-free and protein-free, having a pH 7.5 - 7.7, and an endotoxin level under 1 EU/mL. In one embodiment, the cryoprotectant is Hypothermosol® (CMS, Rockville, Md.) plus 10% DMSO (WO 2000/002572 Al). Hypothermosol® comprises Trolox (6-hydroxy-2, 5,7,8- tetramethylchroman-2-carboxylic acid), Na⁺, K⁺, Ca²⁺, Mg²⁺¹Q-, H₂P0₄-, HEPES,

lactobionate, sucrose, mannitol, glucose, Dextran-40 (/.e. , dextran with an average MW of 40,000 Da), adenosine and glutathione (WO 2010/064054 Al). According to the

manufacturer, ProFreeze should be supplemented with 10% DMSO at time of use. WO 2000/002572 Al and WO 2010/064054 Al are hereby incorporated by reference in their entireties.

In one specific embodiment, the cryoprotectant in which the ASCs are suspended is protein- free and comprises DMSO at a concentration of about 5% to about 15% (v/v), such as about 5% (v/v) or 10% (v/v), and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl-, H₂P0₄-, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine and glutathione. In one specific embodiment, the composition comprises a suspension of allogeneic adult human ASCs at a concentration of about 2.2 x 10⁷ per mL, the composition prepared by suspending allogeneic adult human ASCs in a protein-free cryoprotectant comprising about 10% (v/v) DMSO and, optionally, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid), Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl-, H₂P0₄-, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine and glutathione.

In one specific embodiment, the composition comprises a suspension of allogeneic adult human ASCs at a concentration of about 5 x 10⁷ per mL, the composition prepared by suspending allogeneic adult human ASCs in a protein-free cryoprotectant comprising about 10% (v/v) DMSO and, optionally, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid), Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl-, H₂P0₄-, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine and glutathione.

In one specific embodiment, the composition comprises a suspension of allogeneic adult human ASCs at a concentration of about 2.2 x 10⁷ per mL, the composition prepared by suspending allogeneic adult human ASCs in a protein-free cryoprotectant comprising about 5% (v/v) DMSO and, optionally, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl-, H₂P0₄-, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine and glutathione.

In one specific embodiment, the composition comprises a suspension of allogeneic adult human ASCs at a concentration of about 5 x 10⁷ per mL, the composition prepared by suspending allogeneic adult human ASCs in a protein-free cryoprotectant comprising about 5% (v/v) DMSO and, optionally, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl-, H₂P0₄-, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine and glutathione.

Alternatively, in any one of the preceding embodiments, the cryoprotectant is DMSO in a v/v concentration between about 1% to about 15%, such as about 5% or about 10%, in a chemically-defined serum-free and xeno-free media suitable for ASCs, such as, e.g. , Eagles Basal Medium or Dulbecco's Modified Eagle's Medium (DMEM). Such cryoprotectants are described in WO 2017/144552 Al.

Preferably, the compositions for use as described herein is obtained or obtainable by a process comprising the steps of (i) adding the stromal vascular fraction (SVF) of a lipoaspirate collected from a human donor to a bioreactor wherein at least one surface is pre-treated to promote adhesion of adult human stem cells;

(ii) in the bioreactor, cultivating adherent cells to confluence in a serum-free culture medium supplemented with human platelet lysate;

(iii) detaching the adherent cells;

(v) thawing the frozen cells and repeating steps (ii) and (iii) at least once,

(vi) freezing the detached cells in a cryoprotectant at a concentration of at least 1.5 x 10⁷ cells/mL; and

(vii) thawing the frozen composition.

In one embodiment, the lipoaspirate in step (i) is obtained from abdominal adipose tissue from the donor.

In one embodiment, in step (ii), at least one surface of the bioreactor protein is pre-treated with a composition comprising or consisting of cryoprecipitate. Cryoprecipitate is a well- known blood product prepared from plasma, e.g. , where fresh plasma is frozen and thawed and the precipitate collected. The product typically contains fibrinogen and Factor VIII, as well as e.g. von Willebrand factor, Factor XIII and fibronectin. In some embodiments, the cryoprecipitate contains at least 140 mg or more of fibrinogen per 70 IU of Factor VIII, optionally prepared from either AB or low-titer A blood donors.

In one embodiment, in step (ii), the serum-free culture medium comprises about 5% human platelet lysate. In a specific embodiment, the serum-free culture medium is a minimal essential medium (e.g. , Minimum Essential Medium, MEM Alpha (aMEM) without

Ribonucleosides and Deoxyribonucleosides, (Gibco, Life Technologies)) supplemented with 1% Penicillin/Streptomycin (e.g. , Gibco, Life Technologies) and about 5% human platelet lysate (e.g. , Stemulate, Cook General Biotechnology). In step (v), if steps (ii) and (iii) are repeated more than once, step (iv) may be conducted in between each round, i.e. , so that there is a freezing step after each detaching step.

In a specific embodiment, the composition is prepared as described in Example 1 of WO 2017/068140 Al, except that the total volume of the final CSCC_ASC may be less than 5 mL, such as between 0.25 mL and 1.5 mL, and/or the concentration of the ASCs may be higher, such as about 2 x 10⁷ per mL or higher, e.g. , up to about 5 x 10⁷ per mL.

Also provided are the compositions obtained when thawing the frozen ASC compositions. The frozen ASC compositions may, for example, be thawed in a 37°C water bath or

thawed/stored in room temperature in the operation room.

In some embodiments, when determined immediately after thawing of the composition, at least about 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% of the ASCs are viable, as determined by dye exclusion methods known in the art. For example, the DNA-binding fluorescent dye propidium iodide can be added to the cells, and an image cytometer used to determine the proportion of non-fluorescent cells. Preferably, at least 90% of the cells are viable.

As for proliferation capacity, in some embodiments, when placed in culture immediately after thawing of the composition, the ASCs are characterized by a population doubling (PD) of at least 1, such as at least 1.3, such as at least 1.5, such as at least 1.7, such as at least 2, when cultured in tissue culture flasks for 48h (e.g. , according to the method in Example 3 of WO 2017/068140 Al). Preferably, the ASCs have a PD of at least 1, such as at least 1.5. PD is calculated as Ln (N)/Ln 2, where N = Cell harvested/Cel I seeded.

Preferred are compositions where, immediately after thawing

(a) at least 85% of the ASC population are viable cells, and the viability after storage in room temperature for 2 hours is at least 80%;

(b) the ASC population has a proliferation capacity providing for a PD of at least 1 when cultured for 48 hours;

(c) the ASC population is capable of suppressing dendritic cell maturation and activation;

(d) the recovery after thawing is over 95%, and the recovery of cells after storage at room temperature for 2 h after thawing is at least 85%

(e) the ASC population has an in vitro cell adherence such that at least 60%, such as at least 65%, such as at least 70% of the total number of cells are adherent after 5h in cultivation. Additional components may also be present in the composition. For example, the

pharmaceutical composition may further comprise a soluble biomaterial or hydrogel containing natural or synthetic biopolymers such as extracellular matrix proteins, -peptides or -glycosaminoglycans and/or alginate. For example, the pharmaceutical composition may comprise sterile and endotoxin free Alginate (Sodium alginate VLVG, Novamatrix, FMC

Biopolymers, Norway), particularly calcium cross-linked with D-gluconic acid and hemicalcium salt (Follin et a/. , Cytotherapy. 2015 Aug; 17(8) : 1104- 18) . In one embodiment, the alginate is mixed with ASCs and cryoprotectant to a final concentration of 1 % (w/v) partially cross- linked alginate before the final cryopreservation step. In another embodiment partially cross- linked alginate is stored at RT and mixed with the final product to a final concentration of 1% (w/v) alginate, e.g. , by injecting the ASC preparation into the alginate container before the final suspension is aspirated into a e cell-delivery device as described herein.

Treatment methods

Patients amenable to treatment include subjects at risk of disease but not showing symptoms, as well as patients presently showing symptoms of lacrimal gland and/or meibomian gland dysfunction. The lacrimal gland and/or meibomian gland dysfunction may, for example, be a dry eye disease (DED), such as an aqueous-deficient dry eye disease (ADDE) or an evaporative dry eye disease (EDE) as described herein, or have

components/symptoms of both an ADDE and an EDE. In some embodiments, the ADDE is severe ADDE.

In various embodiments, the subject may already exhibit symptoms of DED or may already be diagnosed as having DED. For example, the subject may be exhibiting one or more symptoms of DED, ADDE and/or severe EDE as described herein. In such cases,

administration of compositions comprising ASCs as described herein can reverse or delay progression of, and or reduce the severity of, the disease symptoms. The effectiveness of treatment can be determined by comparing a baseline measure of a parameter of disease before administration of the composition to the same parameter one or more time-points after ASCs have been administered. Illustrative parameters that can be measured include without limitation stabilization and/or increase in functionality of the lacrimal gland and/or the meibomian glands; tear production and/or tear composition; examination of the corneal and/or conj unctival tissues; and/or reporting by the patient. Increased functionality of the lacrimal gland and/or the meibomian glands, increased tear production and tear composition having normal salt and/or lipid concentrations/compositions, and/or stabilized and/or improved corneal and/or conjunctival tissues is an indicator that the treatment is effective. In some embodiments, the treatment results in an improvement in one, two, three or all of the following parameters: increased ocular comfort as assessed with the OSDI questionnaire pre and post treatment, increased tear production as evaluated with the Schirmer's I test, reduced tear osmolarity (e.g. , as evaluated with TearLab™ osmolarity test), and reduction in objective signs of DED as evaluated with the Ocular SICCA Grading Score. Preferably, the improvement is at least about 5%, such as at least about 10%, such as at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 70%, such as at least about 100%. More preferably, the improvement in one, two, three or all of the parameters is statistically significant at a predetermined level. For example, in one embodiment, differences are considered statistically significant if the two- sided p-value is less than 0.10, such as less than 0.05, according to standard statistical methods known in the art, e.g. , R Statistics. In one embodiment, at least an improved OSDI score is observed. In one embodiment, at least one of increased tear production, reduced tear osmolarity, and improved ocular staining, are observed. These parameters may, for example, be evaluated at 1 week, 4 weeks, 4 months, 1 year and/or 3 years after administration. Preferably, an improvement is observed after at least one of 4 months and 1 year from administration.

In some embodiments, the treatment is found safe in that one, two or three of the following treatment outcomes are observed at an average of Grade 2 or less (as defined in Example 1) : eyelid function disorder (e.g. ptosis), periorbital edema, ocular discomfort, and flu-like symptoms. In some embodiments, the treatment is found safe in that all of the following treatment outcomes are observed at an average of Grade 2 or less (as defined in Example 1) : eyelid function disorder (e.g. ptosis), periorbital edema, ocular discomfort, and flu-like symptoms. In some embodiments, the treatment is found safe in that one, two or three of the following treatment outcomes are observed at an average of Grade 1 or less (as defined in Example 1) : eyelid function disorder (e.g. ptosis), periorbital edema, ocular discomfort, and flu-like symptoms. In some embodiments, the treatment is found safe in that all of the following treatment outcomes are observed at an average of Grade 1 or less (as defined in Example 1) : eyelid function disorder (e.g. ptosis), periorbital edema, ocular discomfort, and flu-like symptoms. These parameters may, for example, be evaluated at 1 week, 4 weeks, 4 months, 1 year and 3 years after administration.

For the purposes of prophylactic (preventive) treatment, the subject may be asymptomatic but have a risk or predisposition to developing a lacrimal and/or meibomian gland dysfunction. For example, the subject may have an autoimmune disease that causes or is associated with the development of an exocrine gland dysfunction such as DED. In such cases, administration of the compositions described herein can prevent or delay onset of dysfunction/disease or progression of dysfunction/disease into later stages of disease, and/or reduce the severity of the disease once present. So, for example, administration of the compositions described herein can prevent or delay onset of DED, prevent or delay onset of ADDE, prevent or delay onset of EDE, or prevent or delay onset of severe ADDE in a subject suffering from mild ADDE.

In some embodiments, the subject has an autoimmune disease. For example, the subject may have an autoimmune disease that is associated with immune-mediated destruction of the lacrimal gland. Subjects who have or are diagnosed with an autoimmune disease that causes or is associated with symptoms of dry eye disease, include, without limitation, those suffering from rheumatoid arthritis, polyarteritis nodosa, Wegener's granulomatosis, systemic lupus erythematosus (SLE), Sjogren syndrome, scleroderma, primary biliary cirrhosis, diabetes or Vogt-Koyanagi-Harada Syndrome (VKH Syndrome), and are candidates for treatment or prevention of dry eye disease by administration of compositions comprising ASCs as described herein. Other conditions include congenital alacrima, Triple A syndrome, sarcoidosis, lymphoma, viral infection, radiation injury, graft-versus-host disease with involvement of the lacrimal gland. The subject may or may not exhibit symptoms of dysfunctional lacrimal and/or meibomian glands.

In some embodiments, the subject has Sjogren syndrome. Sjogren syndrome is a chronic autoimmune disorder of the exocrine glands, with associated lymphocytic infiltrates of the affected glands. Primary symptoms are dry mouth and dry eyes, resulting from involvement of the salivary and lacrimal glands. The exocrinopathy can be encountered alone (primary Sjogren syndrome) or in the presence of another autoimmune disorder such as rheumatoid arthritis (secondary Sjogren syndrome). In both salivary and lacrimal glands, inflammation causes acinar and ductal epithelial cell dysfunction and/or destruction. Diagnosis is by biopsy of glands and blood tests looking for specific antibodies. The subject suffering from Sjogren syndrome may or may not exhibit symptoms of dysfunctional lacrimal glands.

So, in one particular embodiment, the subject has, or is at risk for, DED. In one embodiment, the subject exhibits symptoms of DED. In one embodiment, the subject does not exhibit symptoms of DED but is at risk for DED.

In one particular embodiment, the subject has, or is at risk for, ADDE, EDE, or a combination thereof. In one embodiment, the subject exhibits symptoms of ADDE. In one embodiment, the subject exhibits primarily symptoms of ADDE. In one embodiment, the subject exhibits primarily symptoms of EDE. In one embodiment, the subject exhibits symptoms of both ADDE and EDE. In one embodiment, the subject is at risk for ADDE, EDE, or for a

combination thereof. In one particular embodiment, the subject has, or is at risk for, severe ADDE. In one preferred, embodiment, the subject exhibits symptoms of severe ADDE. In one embodiment, the subject is at risk for severe ADDE. For example, the subject has symptoms of mild ADDE.

In one particular embodiment, the subject has primary or secondary Sjogren syndrome. In one embodiment, the subject has primary Sjogren syndrome. In one embodiment, the subject has secondary Sjogren syndrome.

Embodiments

The following specific embodiments are also contemplated.

Al . A composition comprising allogeneic adipose tissue-derived mesenchymal stem cells (ASCs) for use in treating or preventing dysfunction of a lacrimal gland in a human subject.

A2. The composition for the use according to embodiment Al, wherein the composition is administered at a dosage of from about 1 x 10⁶ to about 5 x 10⁷ allogeneic ASCs.

A3. The composition for the use according to any one of the preceding embodiments, wherein the composition is administered at a dosage of from about 7 x 10⁶ to about 3 x 10⁷, such as from about 9 x 10⁶ to about 13 x 10⁶, such as between about 10 x 10⁶ to about 12 x 10⁶, such as about 11 x 10⁶ allogeneic ASCs.

A4. The composition for the use according to any one of the preceding embodiments, wherein the composition is administered at a volume of from about 0.1 mL to about 1 mL, such as about 0.2 mL or about 0.5 mL, directly into the lacrimal gland.

A5. The composition for the use according to any one of the preceding embodiments, wherein the composition is administered by transconjunctival injection into the palpebral lobe of the lacrimal gland or by transcutaneous injection into the orbital lobe of the lacrimal gland, optionally by injecting approximately equal volumes at two separate injection sites on the lobe.

A6. The composition for the use according to any one of the preceding embodiments, wherein the administration is carried out under topical (eye drop) anesthesia . A7. The composition for the use according to any one of the preceding embodiments, wherein the composition is from a thawed, ready-to-use preparation of cryopreserved allogeneic ASCs free of non-human animal proteins.

A8. The composition for the use according to any one of the preceding embodiments, wherein the composition comprises a suspension of allogeneic adult ASCs in a protein-free cryoprotectant at a concentration of at least about 1.5 x 10⁷ allogeneic adult ASCs per mL, such as from about 2 x 10⁷ to about 5 x 10⁷ allogeneic adult ASCs per mL.

A9. The composition for the use according to embodiment A8, wherein the protein-free cryoprotectant comprises dimethylsulphoxide (DMSO) at a concentration of about 5% to about 15% (v/v), such as about 5% or about 10% (v/v).

A10. The composition for the use according to embodiment A9, wherein the composition further comprises Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl-, H₂P0₄ ^_, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine and glutathione.

Al l. The composition for the use of any one of the preceding embodiments, wherein at least about 80% of the ASC population express CD90, CD73, CD13, CD105, CD29, CD166, CD10, CD140b, CD160, CD204, CD272, CD44, CD49a, CD54, CD9, Galectin 3, Galectin 9, HLA-G and LT3R and at most about 15% of the ASC population express CD45, CD19, CD14, CD106, CD31 and CD36.

A12. The composition for the use according to any one of the preceding embodiments, wherein the composition is obtained or obtainable by a process comprising the steps of

(i) adding the stromal vascular fraction (SVF) of a lipoaspirate collected from a human donor to a bioreactor wherein at least one surface is pre-treated to promote adhesion of adult human stem cells;

(ii) in the bioreactor, cultivating adherent cells to confluence in a serum-free

culture medium supplemented with human platelet lysate;

(iii) detaching the adherent cells;

(v) thawing the frozen cells and repeating steps (ii) and (iii) at least once,

(vi) freezing the detached cells at a concentration of at least 1.5 x 10⁷ cells/mL; and (vii) thawing the frozen composition.

A13. The composition for the use according to any one of the preceding embodiments, wherein the subject has, or is at risk for, dry eye disease (DED) .

A14. The composition for the use according to embodiment A13, wherein the DED is aqueous-deficient dry eye disease (ADDE), evaporative dry eye disease, (EDE), or a combination thereof.

A15. The composition for the use according to any one of the preceding embodiments, wherein the human subject has, or is at risk for, severe ADDE.

A16. The composition for the use according to any one of the preceding embodiments, wherein the human subject has primary or secondary Sjogren syndrome.

A17. A method of treating or preventing dysfunction of a lacrimal gland in a human subject, comprising administering a composition comprising allogeneic ASCs to the human subject, optionally wherein the composition comprises a therapeutically effective amount of allogeneic ASCs and/or wherein the method comprises the features of any one or more of the preceding embodiments.

The invention is further illustrated by the following Examples, which are not to be construed as limiting .

EXAMPLE 1

Treatment with Allogeneic Adipose-derived Mesenchymal Stem Cells in Patients with Aqueous Deficient Dry Eye Disease - First Human Trial

Trial design

Patients with severe ADDE are recruited if they are eligible for the study and the informed consent form is signed .

At inclusion the participants fill out the Ocular Surface Disease Index (OSDI) questionnaire (described in Schiffman et al. , 2000) and undergo an eye examination in the following order: measurement of tear osmolarity (TearLab™), tear break-up time (TBUT), ocular surface staining according to the Ocular SICCA Grading Score (Whitcher et al. , 2010), and Schirmer's I test (Wolffsohn et al. , 2017).

Measurement of tear osmolarity is performed as follows:

A single-use Test Card containing a microfluidic channel is gently placed in the inferior lateral tear meniscus, and via passive capillary action, less than 50-nanoliters of tear sample is instantly and automatically collected when it comes in contact with tear fluid. The Test Card is held by the Osmolarity Test Pen, for safe collection. The Osmolarity Test Pen is then connected to the docking station where the TearLab Osmolarity Test is performed utilizing a temperature-corrected impedance measurement to provide an indirect assessment of osmolarity. After applying a lot-specific calibration curve, osmolarity is calculated and displayed as a quantitative numerical value in mOsms/L

TBUT is performed as follows:One uses a fluoroscein strip. One drop of sterile saline (0.9%) is applied to the strip and the strip is then applied in the fornix inferior by contacting the mucosa with the strip with a brushing motion at a range of approx. 1 cm. The patient blinks a few times, after which the score is read in the slit lamp. The time from the last complete flash to the tear film begins to break is the tear break up time.

Ocular surface staining according to the Ocular SICCA Grading Score is performed as follows:

With fluoroscein in the eye from the measurement of TBUT, the patient blinks a few times and the number of staining dots are counted on both corneas.

A lissamine green strip (Lissaver-Plus) containing 1.5 mg of color is applied with one drop of sterile saline (0.9%) to the strip and the strip is then applied in the fornix inferior by contacting the mucosa with the strip with a brushing motion at a range of approx. 1 cm. The patient blinks a few times, after which the score is read in the slit lamp on the conjunctiva.

The scoring system used is Van Bijesterveld, which divides the eye's surface into 3 zones: the nasal bulbar conjunctiva, the temporal bulbar conjunctiva and the cornea. Each zone is scored with a score of 0 to 3, where 0 indicates that there is no staining and 3 indicates confluent staining. In addition, one can get up to "3 extra points" with fluorescein. The maximum staining score is 12 per eye. A score of 5 or more is pathological.

Schirmer's I test is performed as follows: A strip of standardized filter paper is bent corresponding to the 0 line. The paper is laid over the lower eyelid margin of the lateral 1/3 of the eyelid with the tip toward the fornix inferior on both eyes. The patient sits with closed eyes. After 5 minutes, remove the filter paper and measure how far out the tear fluid has wetted the paper. Under or equal to 5 mm of wetting the paper is compatible with reduced tear secretion on that eye.

After a maximum of 14 days from screening all participants receive an injection with ASCs. 1 week (±2 days), 4 weeks (±4 days), and 4 months (± 7 days) after intervention the participants are followed up with eye examination as described above, OSDI questionnaire, and blood test, see Table 1. At 4 months the primary outcomes of safety are evaluated.

Table 1

a: If indicated after screening via phone call Eligibility criteria

Evaluation of eligibility criteria and Consent Forms are collected by the investigator.

Inclusion criteria : Age > 18 years; OSDI-score > 30; Schirrmer's test 2-5 m in 5 minutes; TBUT < 10 sec.

If both eyes fulfil the eligibility criteria the most affected eye with the lowest tear production assessed with the Schirrmer's test will be the study eye; the contralateral eye will not be treated but examined according to the same protocol as the study eye at each follow-up.

Procedure

Approximately 11 x 10^® ASCs in the form of a CSCC_ASC preparation are administered per lacrimal gland (LG). Using the method described by Nava-Castaneda et a/. (2006), a transconjunctival injection of ASCs is performed using anesthetic eye drops (Oxybuprocain Minims 0,4 %) and sterile technique in an outpatient setting.

The ASCs are delivered frozen in a CellSeal vial with a total volume of 1.3 mL. Preparing for treatment, CellSeal vials are thawed in a 37°C water bath. Within 1 hour from thawing 0.5 mL of the cell suspension is aspirated with a needle into a sterile syringe. After receiving the cell suspension, the surgeon everts the upper eyelid, identifies the LG and injects the ASC suspension directly into the palpebral lobe of the LG using two separate injection sites; 0.25 mL at each site. The participant is administered over-the-counter pain relief.

End-points

Adverse events (AEs) and severe adverse events (SAEs) are recorded and graded according to Common Terminology Criteria for Adverse Events (CTCAE). However, adverse events (AEs) and serious adverse events (SAEs) with causality linked to either the injection procedure or the treatment with ASCs will be a primary outcome.

The primary safety measures are:

Pain at injection site (grade 1 : mild pain, grade 2: moderate pain; limiting instrumental activities of daily living (ADL), grade 3: severe pain; limiting self-care ADL) Infection at injection site (grade 1 : localized; local intervention indicated, grade 2: oral intervention indicated (antibiotic, antifungal, antiviral), grade 3: intravenously administered (IV) antibiotic, antifungal, or antiviral agent indicated; or operative intervention indicated, grade 4: life-threatening consequences; urgent intervention needed)

Bleeding at injection site (Grade 1 : Mild bleeding; intervention not indicated; Grade 2:

Moderate; minimal, local or noninvasive intervention indicated; Grade 3: Severe or medically significant but not immediately life-threatening; hospitalization indicated)

Eyelid function disorder e.g. ptosis (grade 1 : Asymptomatic; clinical or diagnostic

observations only; intervention not indicated; Grade 2: Symptomatic; nonoperative intervention indicated; limiting instrumental ADL; Grade 3: Limiting self care ADL; operative intervention indicated)

Periorbital edema (Grade 1 : Soft or non-pitting; Grade 2: Indurated or pitting edema; topical intervention indicated; Grade 3: Edema associated with visual disturbance; increased intraocular pressure, glaucoma or retinal hemorrhage; optic neuritis; diuretics indicated; operative intervention indicated)

Ocular discomfort (grade 1 : mild discomfort, grade 2: moderate pain, grade 3: disabling pain)

Flu-like symptoms (grade 1 : Mild flu-like symptoms present; grade 2: Moderate flu-like symptoms, limiting self care ADL; grade 3: Severe flu-like symptoms, limiting self care ADL)

Participants will be informed to measure their body temperature if they experience any grade of flu-like symptoms:

Fever (grade 1 : 38.0-39.0 C°; grade 2: >39.0-40.0 C°; grade 3: >40.0 C° for < 24 hours; grade 4: >40.0 C° for > 24 hours)

Secondary outcomes are increased ocular comfort as assessed with the OSDI questionnaire pre and post treatment, increased tear production as evaluated with the Schirmer's test, reduced tear osmolarity, and reduction in objective signs of DED as evaluated with the Ocular SICCA Grading Score. Immune response to allogeneic ASCs are evaluated as development of circulating anti-FILA antibodies. Follow-up

At follow-up an assessment of AEs, the OSDI questionnaire, visual acuity, intraocular pressure and findings and measurements of the objective eye examination are recorded.

Blood tests: To assess the immunological reaction to the treatment in the study participants a blood test to measure the level of anti-HLA antibodies in serum are performed before injection on the day of treatment, at 1 week, 4 weeks, and 4 months follow-up. A total om 20 milliliter blood is drawn at each time point.

The LABScreen HLA class I and II single antigen bead assay on a Luminex 100 (One Lambda, Inc., Thermo Fisher, Canoga Park, CA, USA) are used for analyses. Tests are performed in accordance with the manufacturer's instructions and laboratory standard operating procedures for clinical samples. Trimmed mean values are normalized for background and expressed as mean fluorescence intensity (MFI). Cut-off for positivity are defined as MFI 1.000. The laboratory performance of the analyses is accredited by the European Federation of Immunogenetics. Statistical analyses

Pseudo anonymized data is exported for statistical analyses using R Statistics (R for Windows 3.4.1; https://cran. rstudio.com/index. html. The results addressing secondary outcomes: tear production, ocular surface staining, OSDI, and tear osmolarity are calculated as a percentage change in tear production from baseline to each follow-up. Whether a variable is considered normally distributed will be evaluated by the Shapiro-Wilks test. If the conditions for parametric tests are not present, a nonparametric test is performed. Differences are considered statistically significant if the two-sided p-value is less than 0.05.

EXAMPLE 2

Safety and feasibility of injecting allogeneic adipose-derived mesenchymal stem cells into the lacrimal gland in patients with aqueous deficient dry eye disease - Initial results from the first human trial

Patients & Methods

The trial was conducted as described in Example 1, except as indicated or supplemented below.

This trial was conducted according to the principles of the Declaration of Helsinki and the ICH-GCP Guideline and was monitored by the GCP unit in the Capital Region of Denmark. The trial is approved by the National Ethics Committee (protocol no. 1810172), Danish Medicines Agency (EudraCT no. 2018-003387-31), and is registered as a clinical trial at

ClinicalTrials.gov (NCT03878628) .

Trial design : In this open-label prospective, intervention study 5 study participants with severe aqueous deficient dry eye disease (ADDE) due to either primary or secondary

Sjogrens Syndrome received one transconj unctival injection of CSCC_ASC (22 x 10⁶ allogeneic adipose-derived mesenchymal stem cells (ASCs) per mL in 10% DMSO) into the lacrimal gland (LG) in one eye.

Inclusion criteria were : age > 18 years, Ocular Surface Disease Index (OSDI)-score > 30, Schirmer's I test 2-5 mm/5 minutes, and tear film breakup time (TBUT) < 10 sec. These parameters, tear osmolarity (TearLab®), and corneal staining according to the Oxford classification (not the Ocular Sicca Score as outlined in Example 1 due to simplification of the study visit protocol) was evaluated at each follow-up. The study participants were examined at baseline, at 1 week, and 4 weeks after treatment while the first study participant was also examined 4 months after treatment. Immune response to allogeneic ASCs was evaluated as development of circulating anti-HLA antibodies in peripheral blood samples. In addition to the description of the trial in Example 1 out of safety concerns each study participant received an M RI scan to measure the volume of the LG before treatment in order to inject a maximal volume corresponding to 50% of the LG volume.

The MRI of the lacrimal gland was performed as follows: The images were obtained using a 3 Tesla MRI machine. The imaging protocol contained a 3D T1 TFE sequence without fat saturation and the patient did not receive any contrast material. Both lacrimal glands were identified in each study participant and the volume was calculated by measuring the dimensions of the lacrimal gland in the coronal and anterior-posterior projection.

If both eyes fulfilled the inclusion criteria the study eye was determined as the eye with the lowest Schirmer's I test. The concentration of ASCs was 22 x 10⁶ per mL in all treatments while the injection volume varied corresponding to a maximum of 50% of the LG volume as assessed on MRI.

The transconjunctival injection of ASCs was performed by the same experienced consultant eye surgeon in all cases using sterile technique in an outpatient setting. The ocular surface was prepared for injection using two drops of anaesthetic eye drops (Oxybuprocain Minims 0,4%) with 30 sec. intervals, then 2 drops of Povidone-iodine 5% with 30 sec. intervals, and finally the LG was identified by the surgeon by retracting the upper eyelid and the

conjunctival injection site was anaesthetized in sterile conditions using a cotton swap soaked in a ***e 10% solution for a minimum of two minutes. The designated volume of the thawed ASC solution (0.1 or 0.2 mL) was extracted into a 1 mL syringe with luer lock and was then injected directly into the lacrimal gland using a 10 mm long 30G needle. The injection was administered slowly over the course of approximately 10 seconds. By the end of each injection the needle was kept in the lacrimal gland for 10 seconds with slight pressure on the syringe piston to avoid backflow of the injected solution.

The primary outcome measure was safety by evaluating any adverse reactions to the study treatment according to the Common Terminology Criteria for Adverse Events (CTCAE).

Secondary outcome measures were changes in ocular comfort as assessed with the Ocular Surface Disease Index (OSDI) questionnaire, changes in tear production as evaluated with the Schirmer's I test, changes in tear osmolarity (TearLab®), changes in tear film break-up time (TBUT), and changes in corneal staining according to the Oxford classification. Immune response to allogeneic ASCs was evaluated as development of circulating anti-HLA

antibodies.

HLA antibodies:

To assess the immunological reaction to the treatment in the study participants a blood test to measure the level of anti-HLA antibodies in serum was performed before injection on the day of treatment, at 1 week, 4 weeks, and 4 months follow-up. The HLA class I and II assays were performed on Lurminex mixed flow beads (Labscreen Mixed, One Lambda, Inc., Thermo Fisher, Canoga Park, CA), used for initial analyses. If this test was positive, a test using Luminex single antigen flow beads (Labscreen Mixed, One Lambda, Inc., Thermo Fisher, Canoga Park, CA) was performed. Tests were performed in accordance with the manufacturer's instructions and laboratory standard operating procedures for clinical samples. The laboratory performance of the analyses is accredited by the European Federation of Immunogenetics.

Results:

Within a mean follow-up of 49 days after treatment, no adverse reactions to the study treatment presented. All study participants showed improvements in more than one of the secondary outcome measures, see Tables 1, 2 and 3.

The LG volumes on the study eyes had a mean value of 0.31 ± 0.12 mL. The ASC doses were either 0.1 mL (3/5 study participants) or 0.2 mL (2/5 study participants) corresponding to a dose of 2.2 x 10⁶ or 4.4 x 10⁶ ASCs per LG, respectively, and a range from 8.46 x 10⁶ to 11 x 10⁶ ASCs per LG volume (cells per mL of LG).

The mean change in OSDI score decreased from 58.4± 16.6 at baseline to 32.3± 11 at latest follow-up (Table 2). In the study eye, mean TBUT increased from 4± 1.7 to 6.6± 1.9 sec., mean Oxford score decreased from 2.6±0.8 to 1.2±0.7, mean Schirmer's I test increased from 4.6±0.8 to 6.0±0.9 mm/5min., while mean tear osmolarity remained unchanged from 316.4±9 to 316.8± 10.6 mosm/L (Table 3). In the fellow eye, mean TBUT was 3.8± 1 sec. at baseline and 3.4±0,8 sec. at follow-up, mean Oxford score decreased from 2.4± 1 to

1.8±0.8, mean Schirmer's I test varied more than the study eyes, but showed a tendency towards an increase from 3.6±2.7 to 6.2 ± 5.6 mm/5min., while mean tear osmolarity (TearLab®) showed an increase from 313.5±4.2 to 342.6±22.7 mosm/L (Table 4).

4/5 study participants had no HLA class I or II antibodies on the Labscreen test at baseline and none of them developed HLA class I or II antibodies during the follow-up period. One participant (study ID 3) had HLA class I and II antibodies at baseline (HLA class I : A33, B8, B18, B59, B64, B65. HLA class II : DR13, DR17, DR18, DR52) which did not change within the 26-day follow-up period.

In conclusion, based on our results, injection of ASCs in the form of CSCC_ASC into the LG seemed to be a safe and feasible treatment of severe ADDE. Table 2:

Table 3:

STUDY EYE

Table 4:

FELLOW EYE

EXAMPLE 3

Efficacy of Treatment with Allogeneic Adipose-derived Mesenchymal Stem Cells in Patients with Severe Aqueous Deficient Dry Eye Disease

The purpose of the trial is to evaluate the efficacy of injection of ASCs into the lacrimal gland as a treatment of Severe Aqueous Deficient Dry Eye Disease compared to placebo/sham.

Trial design :

In this double-blinded, randomized phase II study a total of 40 study participants are allocated to two groups in a 1 : 1 ratio : ASCs (at a concentration of 22 x 10⁶ or 50 x 10⁶ cells/mL) and placebo/sham procedure, see Figure 1. All study participants receive treatment on both eyes if both fulfil inclusion criteria, see below. The participants receive an M RI scan at baseline to measure LG volume on both eyes. The injected volume of ASC suspension in the ASC group is a maximum of 50% of the LG volume determined on the M RI scan, adj usted to 0. 1, 0.2, 0.3, 0.4 or 0.5 mL. All participants are examined at baseline, 1 week, 4 weeks, 4 months, and 12 months after intervention. At 1 year the primary outcome is evaluated. End of trial is defined as last participant's last visit (LPLV) at 1 year's follow-up.

Inclusion criteria : age > 18 years, Ocular Surface Disease Index (OSDI)-score > 30, Schirmer's I test 1-5 mm/5 minutes in minimum 1 eye, and tear film breakup time (TBUT) < 10 sec. in both eyes.

Exclusion criteria : Previously established allergies to Oxybuprocain or DMSO (rare) ; Reduced immune response (e.g. HIV positive) ; Pregnancy or planned pregnancy within the next 2 years; Breastfeeding; Treatment with an anticoagulant that cannot be stopped during the intervention period; Treatment with systemic medication known to reduce tear production (with an odds ratio >2,0) : anxiolytics, antipsychotics, and inhaled steroids; Topical treatment with eye drops other than to treat their dry eye disease; Any other disease/condition judged by the investigator to be grounds for exclusion, such as infection in or around the eye.

Criteria for withdrawal of subjects during study: Pregnancy ascertained in the time from enrolment to treatment; Withdrawal of consent from participants. The injection of ASCs in the ASC groups is performed as described in Example 2.

Sham procedure :

The study participants allocated to the sham procedure group will be examined according to the same protocol as the ASC groups. The sham procedure is designed to be as closed to the injection of the active study treatment as possible and will be performed by an experienced consultant eye surgeon using sterile technique in an outpatient setting. The ocular surface will be prepared for sham injection using two drops of anaesthetic eye drops (Oxybuprocain M inims 0.4 %) with 30 sec. intervals, then 2 drops of Povidone-iodine 5 % with 30 sec. intervals, and finally the LG will be identified by the surgeon by retracting the upper eyelid and the injection site will be anaesthetized in sterile conditions using a cotton swap soaked in a ***e 10% solution for a minimum of two minutes. The empty 1 mL syringe without a needle will then be held in contact with the conjunctiva above the LG for approximately 20 seconds to resemble the procedure in the ASC groups.

The primary outcome is change in the subjective dry eye symptoms assessed with the OSDI questionnaire. Secondary outcomes are changes in Schirmer's I test, changes in tear osmolarity, changes in TBUT, changes in Oxford Score, and safety assessed as any adverse reactions to the study treatment within the 12 months follow-up.

LIST OF REFERENCES

Each reference cited below or elsewhere herein is incorporated by reference in its entirety.

Stapleton F, et a/. Ocul Surf [Internet]. 2017 Jul; 15(3) : 334—65.

Gomes JAP, et a/. Ocul Surf [Internet]. 2017 Jul; 15(3) : 511— 38.

Craig JP, et a/. Ocul Surf [Internet]. 2017, http://dx.doi.Org/10.1016/j .jtos.2017.08.003. Jones L, et al. Ocul Surf. [Internet]. 2017 Jul; 15(3) : 575-628.

Nelson JD, et a/. Ocul Surf [Internet]. 2017 Jul; 15(3) : 269—75.

Wolffsohn JS, et a/. Ocul Surf [Internet]. 2017 Jul; 15(3) : 539-74.

Villatoro AJ, et al. Biormed Res Int [Internet]. Volume 2015, Article ID 527926, pp. 1-10. Bittencourt MKW, et al. Cell Med [Internet]. 2016;8: 63-77.

Wood JA, et al. J Ocular Pharmacol Ther. 2012;28(3) : 307-17.

Beyazyildiz E, et al. Stem Cells Int [Internet]. 2014;2014: 1-9.

Bourin P, et al. Cytotherapy. 2013 June; 15(6) : 641-648.

Le Blanc K, Davies LC. Cytotherapy [Internet]. 2018 Mar;20(3) : 273-8.

Nava-Castaneda A, et al. Ophthalmic Plast Reconstr Surg [Internet]. 2006 Nov;22(6) :453-6. Cancer Institute N. Common Terminology Criteria for Adverse Events (CTCAE) v5.0

[Internet]. 2017. Available from: https://www.meddra.org/

WO 2012/166932 A2 (The Regents of the University of California)

WO 2014/203267 (Kasiak Research PVT Ltd)

WO 2017/068140 A1 (Rigshospitalet)

WO 2017/144552 A1 (Centauri Biotech)

Claims

1. A composition comprising allogeneic adipose tissue-derived mesenchymal stem cells (ASCs) for use in treating or preventing dysfunction of a lacrimal gland in a human subject.

2. The composition for the use according to claim 1, wherein the composition is administered at a dosage of from about 1 x 10⁶ to about 5 x 10⁷ allogeneic ASCs.

3. The composition for the use according to any one of the preceding claims, wherein the composition is administered at a dosage of from about 1 x 10⁶ to about 1 x 10⁷ allogeneic ASCs, such as about 2 x 10⁶, about 3 x 10⁶, about 4 x 10⁶, about 5 x 10⁶, about 7 x 10⁶, about 9 x 10⁶, or about 10 x 10⁶ allogeneic ASCs.

4. The composition for the use according to any one of the preceding claims, wherein the composition is administered at a volume of from about 0.1 mL to about 0.5 mL, such as about 0. 1 mL, about 0.2 mL or 0.5 mL, directly into the lacrimal gland .

5. The composition for the use according to any one of the preceding claims, wherein the composition is administered by transconjunctival injection into the palpebral lobe of the lacrimal gland or by transcutaneous injection into the orbital lobe of the lacrimal gland, optionally by injecting approximately equal volumes at two separate injection sites on the lobe.

6. The composition for the use according to any one of the preceding claims, wherein the administration is carried out under topical (eye drop) anesthesia .

7. The composition for the use according to any one of the preceding claims, wherein the composition is from a thawed, ready-to-use preparation of cryopreserved allogeneic ASCs free of non-human animal proteins.

8. The composition for the use according to any one of the preceding claims, wherein the composition comprises a suspension of allogeneic adult ASCs in a protein-free cryoprotectant at a concentration of at least about 1.5 x 10⁷ allogeneic adult ASCs per mL, such as from about 2 x 10⁷ to about 5 x 10⁷ allogeneic adult ASCs per mL.

9. The composition for the use according to claim 8, wherein the protein-free

cryoprotectant comprises dimethylsulphoxide (DMSO) at a concentration of about 5% to about 15% (v/v), such as about 5% or about 10% (v/v).

10. The composition for the use according to claim 9, wherein the composition further comprises Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl-, H₂P0₄ ^_, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine and glutathione.

11. The composition for the use of any one of the preceding claims, wherein at least about 80% of the ASC population express CD90, CD73, CD13, CD105, CD29, CD166, CD10, CD140b, CD160, CD204, CD272, CD44, CD49a, CD54, CD9, Galectin 3, Galectin 9, HLA-G and LTPR and at most about 15% of the ASC population express CD45, CD19, CD14, CD106, CD31 and CD36.

12. The composition for the use according to any one of the preceding claims, wherein the composition is obtained or obtainable by a process comprising the steps of

culture medium supplemented with human platelet lysate;

(iii) detaching the adherent cells;

(v) thawing the frozen cells and repeating steps (ii) and (iii) at least once,

(vi) freezing the detached cells at a concentration of at least 1.5 x 10⁷ cells/mL; and

(vii) thawing the frozen composition.

13. The composition for the use according to any one of the preceding claims, wherein the subject has, or is at risk for, dry eye disease (DED).

14. The composition for the use according to claim 13, wherein the DED is aqueous- deficient dry eye disease (ADDE), evaporative dry eye disease, (EDE), or a combination thereof.

15. The composition for the use according to any one of the preceding claims, wherein the human subject has, or is at risk for, severe ADDE.

16. The composition for the use according to any one of the preceding claims, wherein the human subject has primary or secondary Sjogren syndrome.

17. A method of treating or preventing dysfunction of a lacrimal gland in a human subject, comprising administering a composition comprising allogeneic ASCs to the human subject, optionally wherein the composition comprises a therapeutically effective amount of allogeneic ASCs and/or wherein the method comprises the features of any one or more of the preceding claims.