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EP3923960A1 - Stammzelltherapie für tränendrüsenfunktionsstörungen - Google Patents

Stammzelltherapie für tränendrüsenfunktionsstörungen

Info

Publication number
EP3923960A1
EP3923960A1 EP20704038.7A EP20704038A EP3923960A1 EP 3923960 A1 EP3923960 A1 EP 3923960A1 EP 20704038 A EP20704038 A EP 20704038A EP 3923960 A1 EP3923960 A1 EP 3923960A1
Authority
EP
European Patent Office
Prior art keywords
composition
ascs
allogeneic
cells
use according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20704038.7A
Other languages
English (en)
French (fr)
Inventor
Michael MØLLER-HANSEN
Steffen HEEGAARD
Ann-Cathrine LARSEN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Københavns Universitet
Rigshospitalet Copenhagen University Hospital
Original Assignee
Københavns Universitet
Rigshospitalet Copenhagen University Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Københavns Universitet, Rigshospitalet Copenhagen University Hospital filed Critical Københavns Universitet
Publication of EP3923960A1 publication Critical patent/EP3923960A1/de
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents

Definitions

  • 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.
  • ASCs adipose tissue-derived mesenchymal stem cells
  • 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.
  • ADDE aqueous-deficient dry eye disease
  • 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) .
  • trachoma ocular chemical burns
  • graft-versus-host disease Gomes et a/. , 2017
  • 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.
  • 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.
  • tear conservation e.g. punctal occlusion
  • topical and systemic secretagogues e.g. punctal occlusion
  • autologous serum drops e.g. autologous serum drops
  • surgical interventions such as tarsorrhaphy.
  • a curative treatment of severe ADDE does not, however, exist (Jones et al. , 2017) .
  • MSCs Mesenchymal stem cells
  • ASCs adipose tissue
  • compositions and methods for such therapies particularly based on ASCs.
  • allogeneic ASCs are safe and effective for treating and/or preventing lacrimal gland dysfunction in human subjects.
  • 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.
  • ASCs allogeneic adipose tissue-derived mesenchymal stem cells
  • the invention in a second aspect, 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.
  • the composition is administered at a dosage of from about 1 x 10 6 to about 5 x 10 7 allogeneic ASCs.
  • the composition is administered at a dosage of from about 1 x 10 6 to about 1 x 10 7 allogeneic ASCs, such as about 2 x 10 6 , about 3 x 10 6 , about 4 x 10 6 , about 5 x 10 6 , about 7 x 10 6 , about 9 x 10 6 , or about 10 x 10 6 allogeneic ASCs.
  • 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.
  • 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.
  • the administration is carried out under topical (eye drop) anesthesia.
  • the composition is from a thawed, ready-to-use preparation of cryopreserved allogeneic human ASCs free of non-human animal proteins.
  • the composition comprises a suspension of allogeneic adult ASCs in a protein-free cryoprotectant at a concentration of at least about 1.5 x 10 7 allogeneic adult ASCs per mL, such as from about 2 x 10 7 to about 5 x 10 7 allogeneic adult ASCs per mL.
  • 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).
  • the composition further comprises Trolox (6-hydroxy-2, 5,7,8- tetramethylchroman-2-carboxylic acid), Na + , K + , Ca 2+ , Mg 2+ , Cl-, H 2 P0 4- , HEPES,
  • lactobionate sucrose, mannitol, glucose, dextran-40, adenosine and glutathione.
  • 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.
  • composition is obtained or obtainable by a process comprising the steps of
  • the subject has, or is at risk for, dry eye disease (DED) .
  • DED dry eye disease
  • the DED is aqueous-deficient dry eye disease (ADDE), evaporative dry eye disease, (EDE), or a combination thereof.
  • ADAE aqueous-deficient dry eye disease
  • EEE evaporative dry eye disease
  • the subject has, or is at risk for, severe ADDE.
  • the human subject has primary or secondary Sjogren syndrome.
  • 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.
  • ASCs allogeneic adipose tissue-derived mesenchymal stem cells
  • the invention in a fourth aspect, 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.
  • 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.
  • DED dry eye disease
  • DES dry eye syndrome
  • KCS keratokonj unctivitis sicca
  • 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.
  • DED dry eye disease
  • DES dry eye syndrome
  • KCS keratokonj unctivitis sicca
  • 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.
  • DED dry eye disease
  • ADAE aqueous-deficient dry eye disease
  • EBE evaporative dry eye disease
  • OSDI Ocular Surface Disease Index
  • a score of 0- 100 is calculated : 0- 12 is normal, 13-22 mild, 23-32 moderate, and > 33 severe DED.
  • 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) .
  • TBUT tear breakup time
  • Ocular Surface Staining > 5 corneal spots
  • > 9 conj unctival spots and/or lid margin >2 mm length & >25% width
  • ADDE is a condition in which the tear production in the lacrimal gland (LG) is impaired, resulting in lacrimal hyposecretion.
  • LG lacrimal gland
  • 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.
  • ADDE can be diagnosed using the Schirmer's I test without applying anaesthetic eye drops.
  • 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) .
  • ADDE causes scarring and vascularization of the cornea leading to visual impairment
  • 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.
  • 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.
  • ADDE tear hyperosmolarity results when lacrimal secretion is reduced, in conditions of normal evaporation from the eye.
  • 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.
  • MSD Meibomian gland dysfunction
  • 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
  • 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.
  • CTS crocodile tear syndrome
  • gusto- lacrimal reflex usually following a Bell's palsy or a traumatic facial paralysis.
  • ASCs adipose tissue-derived mesenchymal stem cells
  • mesenchymal stem cells multipotent stromal cells
  • 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”.
  • ASCs mesenchymal stem cells, multipotent stromal cells, multipotent stem cells, and mesenchymal stromal/stem cells
  • 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.
  • they can be distinguished from bone-marrow-derived MSCs by their positivity for CD36 and negativity for CD 106.
  • cryopreserve refers to preserving cells for storage in a cryoprotectant at sub-zero temperatures.
  • cryovials containing the cells and cryoprotectant are usually placed in liquid nitrogen.
  • cryoprotectant 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.
  • impermeant dye e.g. , Trypan Blue, FVS-780, SYTOX blue, propidium iodide
  • Proliferative capacity 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.
  • 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.
  • treatment means 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 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.
  • 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.
  • the pharmaceutical composition may additionally include other pharmaceutically acceptable, non-cellular components, such as pharmaceutically acceptable carriers.
  • 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.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, or excipient, which are known in the art.
  • 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) .
  • terapéuticaally 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
  • 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.
  • “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 10 8 to 2.4 x 10 8 cells, such as from 1.8 x 10 8 to 2.2 x 10 8 cells, such as from 1.9 x 10 8 to 2. 1 x 10 8 cells.
  • 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.
  • 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 .
  • the ASC composition is administered locally, i.e. , in proximity to or directly into the lacrimal and/or meibomian glands.
  • 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.
  • 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.
  • the composition is administered at a dosage of from about 1 x 10 6 to about 5 x 10 7 allogeneic ASCs, such as about 1 x 10 6 cells, about 2 x 10 6 cells, about 3 x 10 6 cells, about 4 x 10 6 cells, about 5 x 10 6 cells, about 7 x 10 6 cells, about 9 x 10 6 cells, about 10 x 10 6 cells, about 11 x 10 6 cells, about 13 x 10 6 cells, about 15 x 10 6 cells, about 17 x 10 6 cells, about 20 x 10 6 cells, about 22 x 10 6 cells, about 25 x 10 6 cells, about 30 x 10 6 cells, about 35 x 10 6 cells, about 40 x 10 6 cells, about 45 x 10 6 cells, or about 50 x 10 6 cells.
  • allogeneic ASCs such as about 1 x 10 6 cells, about 2 x 10 6 cells, about 3 x 10 6 cells, about 4 x 10 6 cells, about 5 x 10 6 cells, about 7 x 10 6 cells, about
  • the composition is administered at a dosage of from about 4 x 10 6 to about 3 x 10 7 allogeneic ASCs.
  • the composition is administered at a dosage of from about 9 x 10 6 to about 13 x 10 6 , such as between about 10 x 10 6 to about 12 x 10 6 allogeneic ASCs.
  • the composition is administered at a dosage of about 10 x 10 6 allogeneic ASCs. In one particular embodiment, the composition is administered at a dosage of about 11 x 10 6 allogeneic ASCs.
  • the composition is administered at a dosage of from about 1 x 10 6 to about 5 x 10 6 , such as between about 2 x 10 6 to about 4 x 10 6 allogeneic ASCs. In a particular embodiment, the composition is administered at a dosage of about 2.2 x 10 6 allogeneic ASCs.
  • the composition is administered at a dosage of from about 2 x 10 6 to about 7 x 10 6 , such as between about 3 x 10 6 to about 6 x 10 6 allogeneic ASCs. In a particular embodiment, the composition is administered at a dosage of about 4.4 x 10 6 allogeneic ASCs.
  • the composition is administered at a dosage of from about 20 x 10 6 to about 30 x 10 6 , such as between about 23 x 10 6 to about 27 x 10 6 allogeneic ASCs. In a particular embodiment, the composition is administered at a dosage of about 25 x 10 6 allogeneic ASCs.
  • 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.
  • 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.
  • the ASCs may be administered transconj unctivally or trancutaneously into the lacrimal gland .
  • 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.
  • 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 .
  • the composition is administered directly into the lacrimal gland, by transconj unctival or transcutaneous administration.
  • both lacrimal glands in a subject are dysfunctional.
  • 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.
  • 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,
  • eye drops containing oxybuprocaine hydrochloride e.g. , at a concentration of 0.4% w/v, is used for providing topical anaesthesia prior to
  • 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.
  • 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.
  • 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.
  • 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.
  • 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) .
  • MRI magnetic resonance imaging
  • 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) .
  • 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.
  • the predetermined volume selected form this list is the volume closest to 50% of the estimated volume of the lacrimal gland.
  • 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.
  • 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 .
  • 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.
  • 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.
  • the about 0.5 mL composition administered comprises from about 5 x 10 6 to about 20 x 10 6 , such as from about 7 x 10 6 to about 15 x 10 6 , such as about 9 x 10 6 to about 13 x 10 6 , such as from about 10 x 10 6 to about 12 x 10 6 allogeneic ASCs, such as about 11 x 10 6 ASCs.
  • the about 0.5 mL composition administered comprises from about 20 x 10 6 to about 25 x 10 6 , such as from about 23 x 10 6 to about 25 x 10 6 allogeneic ASCs, such as about 25 x 10 6 allogeneic ASCs.
  • 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.
  • the about 0.2 mL 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.
  • the about 0.2 mL 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.
  • the about 0.2 mL composition injected transconj un
  • the about 0.2 mL composition administered comprises from about 8 x 10 6 to about 10 x 10 6 , such as from about 9 x 10 6 to about 10 x 10 6 , such as about 10 x 10 6 allogeneic ASCs.
  • 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 10 6 to about 3 x 10 6 , such as from about 1.5 x 10 6 to about 2.7 x 10 6 , such as about 2 x 10 6 to about 2.5 x 10 6 , such as about 2.2 x 10 6 ASCs.
  • the about 0. 1 mL composition administered comprises from about 2 x 10 6 to about 10 x 10 6 , such as from about 3 x 10 6 to about 6 x 10 6 , such as about 4 x 10 6 to about 6 x 10 6 , such as about 5 x 10 6 ASCs.
  • 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.
  • an about 0.05 mL composition administered comprises from about 1 x 10 6 to about 2.5 x 10 6 ASCs, such as about 1 x 10 6 , about 1.5 x 10 6 , about 2 x 10 6 , or about 2.5 x 10 6 ASCs.
  • an about 0. 1 mL composition administered comprises from about 2 x 10 6 to about 5 x 10 6 ASCs, such as about 2 x 10 6 , about 3 x 10 6 , about 4 x 10 6 , or about 5 x 10 6 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.
  • a delivery device suitable for the present purpose i. e. , the delivery of cells in proximity to the lacrimal and/or meibomian glands.
  • 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 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.
  • ASCs i.e. , ASCs that are obtained or isolated from a healthy donor.
  • 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.
  • the concentration of the ASCs in the composition is at least about 1.5 x 10 7 , such as at least about 2 x 10 7 , such as at least about 3 x 10 7 , such as at least about 5 x 10 7 of allogeneic ASCs per mL. In one embodiment, the concentration is from about 1.8 x 10 7 to about 2.6 x 10 7 ASCs per mL, such as from about 2.0 x 10 7 to about 2.4 x 10 7 ASCs per mL, such as about 2.2 x 10 7 ASCs per mL.
  • the concentration is from about 4 x 10 7 to about 5 x 10 7 ASCs per mL, such as from about 45 x 10 6 to about 5 x 10 7 ASCs per mL, such as about 50 x 10 6 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.
  • 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.
  • 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.
  • 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
  • the ASCs may further be characterized by their immunosuppressive properties.
  • 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
  • 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).
  • 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.
  • LPS lipopolysaccharide
  • the ASCs of the invention suppress the proliferation of PBMCs, e.g. , as determined in a Mixed Lymphocyte Reaction (MLR).
  • MLR Mixed Lymphocyte Reaction
  • 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.
  • MLR Mixed Lymphocyte Reaction
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a pro-inflammatory cytokine such as interferon-gamma.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • the composition is from a thawed, ready-to-use preparation of cryopreserved allogeneic adult human ASCs free of non-human animal proteins.
  • 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 10 7 allogeneic adult human ASCs per mL. In some embodiments, the concentration is from about 2 x 10 7 to about 5 x 10 7 allogeneic adult human ASCs per mL in a protein-free cryoprotectant.
  • the concentration is from about 1.8 x 10 7 to about 2.6 x 10 7 , such as from about 2.0 x 10 7 to about 2.4 x 10 7 , such as about 2.2 x 10 7 ASCs per mL in a protein-free cryoprotectant. In one embodiment, the concentration is from about 4 x 10 7 to about 5 x 10 7 , such as from about 45 x 10 6 to about 5 x 10 7 , such as about 50 x 10 6 ASCs per mL in a protein-free cryoprotectant.
  • 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.
  • the cryoprotectant comprises DMSO, preferably at a concentration of about 5% to about 15% (v/v).
  • the cryoprotectant comprises about 5%, about 6%, about 8%, about 10%, about 12% or about 15% DMSO.
  • the cryoprotectant comprises about 5% DMSO (v/v) or about 10% DMSO (v/v).
  • 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.
  • the cryoprotectant comprises a 1 : 10 to about 1 : 20 mixture of DMSO and an aqueous solution comprising
  • 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;
  • 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
  • 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;
  • 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;
  • 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
  • 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
  • At least one agent which regulates apoptotic induced cell death at least one agent which regulates apoptotic induced cell death.
  • cryoprotectants 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.
  • 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 + , Ca 2+ , Mg 2+1 Q-, H 2 P0 4 -, 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
  • WO 2000/002572 Al and WO 2010/064054 Al are hereby incorporated by reference in their entireties.
  • 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 + , Ca 2+ , Mg 2+ , Cl-, H 2 P0 4 -, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine and glutathione.
  • DMSO at a concentration of about 5% to about 15% (v/v), such as about 5% (v/v) or 10% (v/v)
  • Trolox 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
  • Na + , K + , Ca 2+ , Mg 2+ , Cl-, H 2 P0 4 -, HEPES lactobionate
  • the composition comprises a suspension of allogeneic adult human ASCs at a concentration of about 2.2 x 10 7 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 + , Ca 2+ , Mg 2+ , Cl-, H 2 P0 4 -, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine and glutathione.
  • 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 + , Ca 2+ , Mg 2+ , Cl-, H 2 P0 4 -, HEPES
  • the composition comprises a suspension of allogeneic adult human ASCs at a concentration of about 5 x 10 7 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 + , Ca 2+ , Mg 2+ , Cl-, H 2 P0 4 -, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine and glutathione.
  • 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 + , Ca 2+ , Mg 2+ , Cl-, H 2 P0 4 -, HEPES,
  • the composition comprises a suspension of allogeneic adult human ASCs at a concentration of about 2.2 x 10 7 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 + , Ca 2+ , Mg 2+ , Cl-, H 2 P0 4 -, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine and glutathione.
  • 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 + , Ca 2+ , Mg 2+ , Cl-, H 2 P0 4 -
  • the composition comprises a suspension of allogeneic adult human ASCs at a concentration of about 5 x 10 7 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 + , Ca 2+ , Mg 2+ , Cl-, H 2 P0 4 -, HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine and glutathione.
  • 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 + , Ca 2+ , Mg 2+ , Cl-, H 2 P0 4 -,
  • 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).
  • DMEM Dulbecco's Modified Eagle's Medium
  • 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;
  • SVF stromal vascular fraction
  • the lipoaspirate in step (i) is obtained from abdominal adipose tissue from the donor.
  • 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.
  • 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.
  • the serum-free culture medium comprises about 5% human platelet lysate.
  • the serum-free culture medium is a minimal essential medium (e.g. , Minimum Essential Medium, MEM Alpha (aMEM) without
  • 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.
  • 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 10 7 per mL or higher, e.g. , up to about 5 x 10 7 per mL.
  • compositions obtained when thawing the frozen ASC compositions are also provided.
  • the frozen ASC compositions may, for example, be thawed in a 37°C water bath or
  • 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.
  • 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.
  • at least 90% of the cells are viable.
  • the ASCs when placed in culture immediately after thawing of the composition, 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).
  • the ASCs Preferably, have a PD of at least 1, such as at least 1.5.
  • 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%;
  • the ASC population has a proliferation capacity providing for a PD of at least 1 when cultured for 48 hours;
  • the ASC population is capable of suppressing dendritic cell maturation and activation
  • 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.
  • the composition may also be present in the composition.
  • 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.
  • the pharmaceutical composition may comprise sterile and endotoxin free Alginate (Sodium alginate VLVG, Novamatrix, FMC
  • 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.
  • 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.
  • ADAE aqueous-deficient dry eye disease
  • EEE evaporative dry eye disease
  • the ADDE is severe ADDE.
  • the subject may already exhibit symptoms of DED or may already be diagnosed as having DED.
  • the subject may be exhibiting one or more symptoms of DED, ADDE and/or severe EDE as described herein. In such cases,
  • 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.
  • 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 TearLabTM osmolarity test), and reduction in objective signs of DED as evaluated with the Ocular SICCA Grading Score.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the subject may be asymptomatic but have a risk or predisposition to developing a lacrimal and/or meibomian gland dysfunction.
  • the subject may have an autoimmune disease that causes or is associated with the development of an exocrine gland dysfunction such as DED.
  • 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.
  • the subject has an autoimmune disease.
  • 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.
  • 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).
  • 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.
  • the subject has, or is at risk for, DED.
  • the subject exhibits symptoms of DED.
  • the subject does not exhibit symptoms of DED but is at risk for DED.
  • the subject has, or is at risk for, ADDE, EDE, or a combination thereof.
  • the subject exhibits symptoms of ADDE.
  • the subject exhibits primarily symptoms of ADDE.
  • the subject exhibits primarily symptoms of EDE.
  • the subject exhibits symptoms of both ADDE and EDE.
  • the subject is at risk for ADDE, EDE, or for a
  • the subject has, or is at risk for, severe ADDE.
  • the subject exhibits symptoms of severe ADDE.
  • the subject is at risk for severe ADDE.
  • the subject has symptoms of mild ADDE.
  • 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.
  • 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.
  • ASCs allogeneic adipose tissue-derived mesenchymal stem cells
  • composition for the use according to embodiment Al wherein the composition is administered at a dosage of from about 1 x 10 6 to about 5 x 10 7 allogeneic ASCs.
  • 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 10 6 to about 3 x 10 7 , such as from about 9 x 10 6 to about 13 x 10 6 , such as between about 10 x 10 6 to about 12 x 10 6 , such as about 11 x 10 6 allogeneic ASCs.
  • 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.
  • 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 10 7 allogeneic adult ASCs per mL, such as from about 2 x 10 7 to about 5 x 10 7 allogeneic adult ASCs per mL.
  • 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).
  • DMSO dimethylsulphoxide
  • Trolox 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
  • Na + , K + , Ca 2+ , Mg 2+ , Cl-, H 2 P0 4 _ HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine and glutathione.
  • 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.
  • DED dry eye disease
  • 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.
  • DED aqueous-deficient dry eye disease
  • EAE evaporative dry eye disease
  • 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.
  • 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 (TearLabTM), 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).
  • OSDI Ocular Surface Disease Index
  • 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:
  • 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.
  • Inclusion criteria Age > 18 years; OSDI-score > 30; Schirrmer's test 2-5 m in 5 minutes; TBUT ⁇ 10 sec.
  • Approximately 11 x 10 ® ASCs in the form of a CSCC_ASC preparation are administered per lacrimal gland (LG).
  • LG lacrimal gland
  • 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.
  • CellSeal vials 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.
  • AEs Adverse events
  • SAEs severe adverse events
  • CCAE Common Terminology Criteria for Adverse Events
  • AEs adverse events
  • SAEs serious adverse events
  • 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)
  • Eyelid function disorder e.g. ptosis (grade 1 : Asymptomatic; clinical or diagnostic
  • 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)
  • 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 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.
  • MFI mean fluorescence intensity
  • CSCC_ASC 22 x 10 6 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.
  • OSDI Ocular Surface Disease Index
  • TBUT tear film breakup time
  • 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.
  • the study eye was determined as the eye with the lowest Schirmer's I test.
  • the concentration of ASCs was 22 x 10 6 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 cocaine 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.
  • CCAE Common Terminology Criteria for Adverse Events
  • 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.
  • 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 10 6 or 4.4 x 10 6 ASCs per LG, respectively, and a range from 8.46 x 10 6 to 11 x 10 6 ASCs per LG volume (cells per mL of LG).
  • HLA class I 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.
  • 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.
  • 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.
  • OSDI Ocular Surface Disease Index
  • TBUT tear film breakup time
  • 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.
  • 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 cocaine 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.

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