EP3923959A1

EP3923959A1 - Stem cell therapy for patients with salivary gland dysfunction

Info

Publication number: EP3923959A1
Application number: EP20703781.3A
Authority: EP
Inventors: Christian GRØNHØJ; Christian VON BUCHWALD; Charlotte Duch LYNGGAARD
Original assignee: Rigshospitalet; Kobenhavns Universitet
Current assignee: Rigshospitalet; Kobenhavns Universitet
Priority date: 2019-02-15
Filing date: 2020-02-14
Publication date: 2021-12-22
Also published as: WO2020165405A1

Abstract

Described herein are compositions comprising allogeneic adipose-derived stem cells and their use in treating or preventing hypofunction or other dysfunction of the salivary glands.

Description

STEM CELL THERAPY FOR PATIENTS WITH SALIVARY 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 hypofunction or other dysfunction of the salivary glands.

BACKGROUND OF THE INVENTION

Hyposalivation, i. e. , reduced production of saliva, can lead to several hampering conditions such as dental decline, recurrent infections, malnutrition, sleep deprivation and loss of social and profession quality of life. Currently, only insufficient symptomatic treatment is available. Several debilitating oral conditions, such as radiation-induced damage, chemotherapy- induced impairment and autoimmune diseases such as Sjogren syndrome, may cause salivary gland dysfunction which in turn can result in hyposalivation.

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 2017/144552 Al ; WO 2017/068140 and WO 2014/203267) . Animal studies testing stem cell therapy for e.g. xerostomia have been reported by Qomi et al. (2017), Wang et al. (2016), Jensen et al. (2014), Lim et al. (2013) and Xu et al. (2012) .

Gronhoj et al. (2017) describes a proposed trial protocol for a Phase I/II clinical trial for treating radiation-induced xerostomia, where autologous ASCs are injected into each submandibular gland of a patient at a maximum total dose per patient of approximately 4.48 x 10⁷ autologous ASCs. Gronhoj et al. (2018) reports that autologous ASCs are safe and significantly improve salivary gland functions and patient-reported outcomes.

Despite these and other advances in the art, however, there remains a need for safe, reproducible and clinically effective treatment options for hyposalivation and other salivary gland dysfunctions in human patients.

It is an object of the invention to provide compositions and methods for such therapies, particularly based on allogeneic ASCs. SUMMARY OF THE INVENTION

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

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

In a second aspect, the present invention relates to a method of treating or preventing dysfunction of a salivary 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 human ASCs.

In one embodiment, the composition is administered at a dosage of at least about 1 x 10⁶, such as at least about 1 x 10⁷, such as at least about 2.5 x 10⁷, such as at least about 5 x 10⁷ allogeneic ASCs to at least one salivary gland in the human subject.

In one embodiment, the composition is administered to at least one parotid gland and/or at least one submandibular gland of the human subject.

In one embodiment, the composition is administered at a dosage of at least about 1 x 10^s allogeneic ASCs per human subject, such as from about 1.5 x 10^s to about 2 x 10^s allogeneic ASCs per human subject.

In one embodiment, at least about one quarter of the dosage, such as between about one quarter and about one third of the dosage, is administered to each of the parotid glands of the human subject, and about half of the remaining dosage is administered to each of the submandibular glands of the human subject.

In some embodiments, about a dose of about 5 x 10⁷ allogeneic ASCs is administered to each of the two parotid and two submandibular glands of the human subject; or a dose of about 5 x 10⁷ allogeneic ASCs is administered to each of the two parotid glands, and a dose of about 2.5 x 10⁷ allogeneic ASCs is administered to each of the two submandibular glands of the human subject.

In one embodiment, the composition is administered at a volume of from about 0. 1 mL to about 2 mL per gland, such as from about 0.5 mL to about 1 mL, via direct intraglandular injection, optionally ultrasound-guided.

In one embodiment, the composition is from a thawed, ready-to-use preparation of cryopreserved allogeneic ASCs free of non-human animal proteins, optionally divided into 2 to 4 separate vials.

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 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 one embodiment, 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.

In one embodiment, at least about 80% of the ASC population in the composition express CD90, CD73, CD 13, CD 105, CD29, CD 166, CD 10, CD140b, CD 160, 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, CD 19, CD 14, CD106, CD31 and CD36.

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

(a) 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;

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

(c) detaching the adherent cells;

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

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

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

(g) thawing the frozen composition.

In one embodiment, the human subject has received prior radiation therapy of the head- and-neck region, has received prior chemotherapy, or suffers from an autoimmune disease or disorder.

In one embodiment, the human subject has received prior radiation therapy for treatment of head-and-neck cancer, has received prior total body radiation, has received radioiodine therapy, or suffers from Sjogren syndrome, diabetes or multiple sclerosis.

In some embodiments, the human subject received prior radiation therapy for treatment of head-and-neck cancer up to 25 years ago.

In some embodiments, the head-and-neck cancer was oropharyngeal cancer, such as oropharyngeal squamous cell carcinoma, optionally pl6-positive and/or p- 16 negative and/or HPV- negative oropharyngeal squamous cell carcinoma.

In some embodiments, the salivary gland dysfunction is hyposalivation, xerostomia or a combination of both.

In one embodiment, the human subject has an unstimulated whole salivary flow rate less than about 0.4 mL/min, such as less than about 0.3 mL/min, such as less than about 0.2 mL/min.

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" salivary gland refers to a salivary gland with an abnormal saliva production, e.g. , a subnormal or insufficient saliva production (hyposalivation) and/or a deficient saliva quality.

As used herein, "hyposalivation" generically refers to a reduction in saliva production, flow, and/or volume as compared to normal saliva production, flow and/or volume according to a predetermined control value. The control value may, for example, be known from scientific or medical literature, measured in healthy control subject(s), or determined in subject(s) prior to a procedure associated with a detrimental effect on salivary gland function, e.g. , radiation treatment, chemotherapy or other medication.

Sialometry for measuring saliva production is known in the art, and include methods measuring the salivary flow rate (SFR) (ml/min) of whole saliva, submandibular/sublingual (SM/SL) glands and/or parotid glands under conditions where the saliva flow is either stimulated or unstimulated .

As used herein, a subject suffers from hyposalivation when the unstimulated whole SFR is at most about 0.2 mL/min, e.g. , between about 0.2 mL/min and about 0.05 mL/min. Preferably, the following method is used for unstimulated whole SFR: A saliva sample is collected between 10 : 00 and 12 : 00 a . m., with a subject having refrained from drinking, eating, chewing gum, wearing lip stick and performing oral hygiene for at least 60 min before sialometry. After 5 minutes of rest, the subject swallows one mouthful of water and unstimulated saliva is collected for 10 minutes, and the unstimulated saliva samples are weighed. For measuring stimulated whole SFR, the patient may receive a tasteless piece of paraffin gum for chewing 1 minute and swallow the newly produced stimulated saliva. During the following 5 minutes, the patient continues chewing and stimulated saliva collected.

Typically, though not necessarily, subjects suffering from hyposalivation also have "xerostomia", i.e. , the sensation of having a dry mouth. Xerostomia may occur with or without hyposalivation. Methods of determining xerostomia include the use of questionnaires.

In some embodiments, a subject who suffers from xerostomia may have an unstimulated whole SFR of up to 0.4 mL/min, up to 0.3 mL/min or up to 0.2 mL/min, e.g. , between about 0.4 mL/min and about 0.05 mL/min. In some embodiments, a subject suffers from hyposalivation and/or xerostomia when the subject's saliva flow is lower after a procedure associated with a detrimental effect on salivary gland function, e.g. , radiation treatment, chemotherapy or other medication.

As used herein, "deficient saliva quality" refers to one or more deviations in saliva properties and/or components important for the dental- or mucosa-protective capacity of the saliva as compared to a predetermined control value. The control value may be, for example, known from scientific or medical literature, or determined in subject(s) prior to a procedure associated with a detrimental effect on salivary gland function, e.g. , radiation treatment, chemotherapy or other medication. The deviations can be identified by chemical analysis of whole saliva or selectively collected saliva from individual glands, i.e. , parotid and submandibular/sublingual saliva as described in Gronhoj et at. (2017), testing, e.g. , for deviations in pH and bicarbonate level by ionic balance estimation (Bardow et at. , Clin Oral Invest 2000 : 245-53) ; the level of sodium, potassium, calcium, phosphate, chloride and/or fluoride (Lajer et a/. , Radiother Oncol 2009;93 : 534-8), the level of total protein, selected proteins (Bruvo et a/. , Caries Res 2009;43 : 163-70), and the level of amylase (Lexner et a/. ; Int J Paediatr Dent [Internet] 2007; 17 : 155- 162) .

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, CD 105 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 encounter 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 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. However, 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 judgment 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 salivary 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. 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 salivary gland in a human subject. The present invention also relates to a method of treating or preventing dysfunction of a salivary gland 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 radiation-induced hyposalivation and xerostomia in previous head and neck cancer patients, described in Examples 1 and 2.

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 human subject in which salivary 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 one or more salivary 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, antifibrotic 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 salivary gland dysfunction to be treated or prevented, the size(s) of the salivary gland(s), 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 at least about 1 x 10⁶ allogeneic ASCs to a salivary gland in a human subject, such as between about 2.5 x 10⁶ cells to 1 x 10⁸ allogeneic ASCs.

In some embodiments, the composition is administered at a dosage of at least about 1 x 10⁷ allogeneic ASCs to a salivary gland in a human subject, such as between about 2 x 10⁷ and about 1 x 10^s, such as about 2 x 10⁷, 2.5 x 10⁷, about 3 x 10⁷, about 4 x 10⁷, about 5 x 10⁷, about 6 x 10⁷, about 7 x 10⁷, about 8 x 10⁷, about 9 x 10⁷ or about 10 x 10⁷ allogeneic ASCs, such as about 2.5 x 10⁷ or about 5 x 10⁷ allogeneic ASCs to a salivary gland .

In some embodiments, the composition is administered at a dosage of at least about 1 x 10^s allogeneic ASCs per human patient, such as about 1 x 10^s, about 1. 1 x 10^s, about 1.2 x 10^s, about 1.3 x 10^s, about 1.4 x 10^s, about 1.5 x 10^s, about 1.6 x 10^s, about 1.8 x 10^s, about 2.0 x 10^s, about 2.2 x 10^s, about 2.4 x 10^s, about 2.6 x 10^s, about 2.8 x 10^s cells, or about 3.0 x 10^s allogeneic ASCs per human subject.

In some embodiments, the composition is administered at a dosage of from about 1.5 x 10⁸ to about 2 x 10^s allogeneic ASCs per human subject, such as about 1.5 x 10^s, about 1.6 x 10^s, about 1.7 x 10^s, about 1.8 x 10^s, about 1.9 x 10^s, or about 1.2 x 10^s allogeneic ASCs per human subject. In one embodiment, the composition is administered at a dosage of about 1.5 x 10⁸ allogeneic ASCs per human subject. In one embodiment, the composition is administered at a dosage of about 2.0 x 10^s allogeneic ASCs per human subject.

In some embodiments, the composition is administered to at least one submandibular gland, a least one parotid gland, or both, of the human subject. In some embodiments, the composition is administered to at least one parotid gland and at least one submandibular gland of the human subject. In one embodiment, the composition is administered to one submandibular gland. In some embodiments, the composition is administered to both submandibular glands. In some embodiments, the composition is administered to one parotid gland. In some embodiments, the composition is administered to both parotid glands. Typically, a salivary gland to which the composition is administered is known or at risk for a dysfunction, such as subnormal salivation, hyposalivation and/or deficient saliva quality.

In some embodiments, the composition is administered to each submandibular gland in a subject, preferably such that the dosage administered to the human subject is divided between the two submandibular glands.

In some embodiments, the composition is administered to each parotid gland in a subject, preferably such that the dosage administered to the human subject is divided between the two parotid glands.

The composition can advantageously be administered to at least one parotid gland and at least one submandibular gland of the human subject, such that the dosage administered to the human subject is divided between the at least one parotid gland and at least one submandibular gland; usually parotid and submandibular glands known or at risk for a dysfunction, such as subnormal salivation, hyposalivation and/or deficient saliva quality. In cases where the composition is administered to only one parotid gland and one submandibular gland in a human subject, the parotid and submandibular glands are typically those located on the same side, e.g. , the right or the left side.

In some embodiments, at least about one quarter of the dosage, such as between about one quarter and about one third of the dosage, is administered to each of the two parotid glands of the human subject, and about half of the remaining dosage is administered to each of the two submandibular glands of the human subject.

In some embodiments, (a) about one quarter of the ASC dosage is administered to each of the two parotid glands and each of the two submandibular glands of the human subject; or (b) about one third of the ASC dosage is administered to each of the two parotid glands and about one sixth the ASC dosage is administered to each of the two submandibular glands of the human subject.

The relative portion of ASCs to be administered to the parotid and submandibular glands may, for example, be determined according to the size of the glands, using, e.g. , ultrasound combined with salivary gland scintigraphy for size estimates. In one embodiment, the relative portion of ASCs administered to the parotid and submandibular glands is decided based on the estimated size of the submandibular glands (see, e.g. , Gronhoj et al. , 2017) ; selecting a higher relative portion (e.g. , option (a)) if the submandibular are deemed large and a lower relative portion (e.g. , option (b)) if they are deemed small. The skilled physician is well familiar with such estimates.

So, at a total dosage of about 2.5 x 10⁷ allogeneic ASCs per human subject, the dosage can, for example, be administered to a submandibular gland.

At a total dosage of about 5 x 10⁷ allogeneic ASCs per human subject, the respective doses can be, for example

(a) about 50 x 10⁶ allogeneic ASCs to one submandibular gland; or

(b) about 25 x 10⁶ allogeneic ASCs to each submandibular gland .

At a total dosage of about 1 x 10^s allogeneic ASCs per human subject, the respective doses can be, for example

(a) about 25 x 10⁶ allogeneic ASCs to each parotid and each submandibular gland ;

(b) about 30 x 10⁶ allogeneic ASCs to each parotid gland and about 20 x 10⁶ allogeneic ASCs to each submandibular gland ;

(c) about 35 x 10⁶ allogeneic ASCs to each parotid gland and about 15 x 10⁶ allogeneic ASCs to each submandibular gland ; or

(d) about 50 x 10⁶ allogeneic ASCs to each submandibular gland .

At a total dosage of about 1.5 x 10^s allogeneic ASCs per human subject, the respective doses can be, for example

(a) about 40 x 10⁶ allogeneic ASCs to each parotid and each submandibular gland ;

(b) about 45 x 10⁶ allogeneic ASCs to each parotid gland and about 30 x 10⁶ allogeneic ASCs to each submandibular gland ;

(c) about 50 x 10⁶ allogeneic ASCs to each parotid gland and 25 x 10⁶ allogeneic ASCs to each submandibular gland.

At a total dosage of about 2 x 10^s allogeneic ASCs per human subject, the respective doses can be, for example

(a) about 50 x 10⁶ allogeneic ASCs to each parotid and each submandibular gland ;

(b) about 60 x 10⁶ allogeneic ASCs to each parotid gland and about 40 x 10⁶ allogeneic human ASCs to each submandibular gland;

(c) about 70 x 10⁶ allogeneic ASCs to each parotid gland and about 30 x 10⁶ allogeneic ASCs to each submandibular gland.

In one particular embodiment, a dose of about 5 x 10⁷ allogeneic ASCs is administered to each of the two parotid and two submandibular glands of the human subject.

In one particular embodiment, a dose of about 5 x 10⁷ allogeneic ASCs is administered to each of the two parotid glands, and a dose of about 2.5 x 10⁷ allogeneic ASCs is administered to each of the two submandibular glands of the human subject.

Administration

The composition comprising ASCs is typically administered parenterally, and preferably locally, i.e. , in proximity to the parotid or submandibular gland to which a dose is to be administered, 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 intraglandularly {i.e. , directly into the gland) or perilg landu la rly {i.e. , near the gland) . 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, the composition is administered intraglandularly, i.e. , directly into the parotid or submandibular gland to which a dose is to be administered . The administration of the composition may be carried out under general or local anaesthesia

The volume of the composition to be administered is adjusted to the concentration of ASCs in the composition so as to administer a therapeutically effective amount of ASCs, i. e. , an amount of ASCs effective in preventing or treating the dysfunctional salivary glands. In some embodiments, the composition is administered at a total volume of from about 0.1 mL to about 4 mL at a given administration event, such as about 0.5 mL to 4 mL at a given administration event. 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, 5 or 6 doses, e.g. , 4 doses, typically where each has a volume of about 0.2 mL to about 2 mL, such as between about 0.3 mL to about 1.5 mL, such as between about 0.5 mL to about 1 mL, such as about 0.2 mL, about 0.3 mL, about 0.5 mL, about 0.7 mL, about 1.0 mL, about 1.2 mL, about 1.5 mL, or about 2.0 mL.

For example, in some embodiments, the composition is administered at a volume of from about 0. 1 mL to about 4 mL to a salivary gland, such as from about 0.5 to about 4 mL to a salivary gland, such as 0.1 mL to about 2 mL per sa livary gland, such as from about 0.5 mL to about 1 mL, via direct intraglandular injection, optionally ultrasound-guided. In one embodiment, the volume administered to a parotid gland is between about 0.5 to about 1.5 mL, such as about 1 mL; and the volume administered to a submandibular gland is about 0.25 mL to about 0.75 mL, such as about 0.5 mL.

The composition may be administered using a delivery device suitable for the present purpose, i. e. , the delivery of cells in proximity or into to the salivary glands. Usually, the cell delivery device is a syringe, such as a sterile, disposable syringe with a volume of 0.5 mL, 1 mL, 1.5 mL, 2 mL or 5 mL and a needle with a suitable gauge and length for injection into the parotid and/or submandibular glands. 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, CD 105, and CD44, and maintaining low or negligible expression levels of CD45 and CD31 (Bourin et a/. , 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, CD 13, CD105, CD29, CD166, CD 10, CD140b, CD 160, 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, CD 19, CD14, CD 106, CD31 and CD36.

In some embodiments, of the ASC population, at least 90% express CD90, CD73, CD 13, CD29 and CD 166; at most 5% express

CD45, CD 19, CD 14 and CD31 ; at most 10% express CD 106; between 2 and 15% express CD36; at least 10% express CD146; at least 80% express CD 105 and at most 40% express CD34; and/or

- at least 90% express CD10, CD 140b, CD 160, CD204, CD272, CD44, CD54, CD9, Galectin 3, Galectin 9, HLA-G and LT3R; at least 80% express CD49a; at least 60% express CD258 and CD270 and at least 5% express CD200; at most 15% express CD 15, 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 M ixed Lymphocyte Reaction (M LR) . 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 CD 10, CD140a, CD160, CD204, CD258, CD270, CD272, CD44, CD49a, CD54, CD9, Galectin 3, Galectin 9, HLA-G, LT3R 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, optionally wherein the composition is divided between several separate vials, e.g. , 2 or 4 separate vials. In a preferred embodiment, the composition comprises a suspension of allogeneic adult human ASCs in a protein-free cryoprotectant 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 with 5% DMSO or 10% DMSO is safe and effective when administered directly into parotid and submandibular 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) . In one specific embodiment, the cryoprotectant comprises about 5% DMSO. In one specific embodiment, the cryoprotectant comprises about 10% DMSO. 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 imM, 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 physiologica l 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⁺, Mg^{2+ 1}Q-, 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-tetrarmethylchrorman-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

(a) 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;

(c) detaching the adherent cells;

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

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

(g) 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. , M inimum 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 (CSCC_ASC(50)) .

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 cells 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 embod iment, 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 salivary gland hypofunction or other dysfunction.

So, in some embodiments, the subject may have xerostomia and/or exhibit other symptoms of salivary gland hypofunction or other dysfunction, or may already be diagnosed as having a salivary gland dysfunction, e.g. , hypofunction or a deficient quality of the saliva . For example, the subject may have a reduced unstimulated whole salivary flow or a reduced stimulated whole salivary flow, or reduced unstimulated or stimulated salivary flow of saliva from the parotid and/or submandibular glands. In some embodiments, the subject has an unstimulated whole salivary flow rate less than about 0.4 mL/min, such as less than about 0.3 mL/min, such as less than about 0.2 mL/min. In one embodiment, the subject has an unstimulated whole salivary flow rate less than about 0.2 rmL/rmin. 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 parotid and/or submandibular glands; saliva production and/or saliva composition; and/or reporting by the patient. Increased whole saliva production (under stimulated and/or unstimulated conditions), increased saliva production by the parotid and/or the submandibular glands (under stimulated and/or unstimulated conditions), and/or saliva composition having normal concentrations/compositions are each indicators that the treatment is effective. In some embodiments, each salivary gland to which the composition is administered is known or at risk for a dysfunction, e.g. subnormal salivation, hyposalivation and/or deficient saliva quality.

In some embodiments, the treatment results in an improvement in one, two, three, four or all of the following parameters : increased unstimulated whole saliva flow; an increased stimulated whole saliva flow, an improved outcome as determined by patient-questionnaires (e.g. , on xerostomia) ; an improved salivary gland function as determined by salivary gland 99mTc scintigraphy, or an improvement of at least one component in the saliva composition. 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%. Preferably, at least the unstimulated and/or stimulated whole saliva flow is increased. In one embodiment, after treatment the unstimulated salivary flow rate is higher than 0.2 mL/min. 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. These parameters may, for example, be evaluated at 1 day after administration, and 1 month, 4 months, 1 year, 2 years, etc. after administration. Preferably, an improvement is observed after, e.g. , 1 month and/or 4 months after administration.

In some embodiments, the subject having xerostomia or diagnosed with salivary gland hypofunction or other dysfunction has received prior radiation therapy of the head- and-neck region and/or prior chemotherapy. In one embodiment, the human subject has received prior radiation therapy for treatment of head-and-neck cancer. For example, the human subject may have received prior radiation therapy up to 25 years ago, such as up to 10 years ago, such as within one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, 8 months, 10 months, one year or at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years ago. In some embodiments, treatment according to the invention may take place during or prior to radiotherapy, e.g. , one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, 8 months, 10 months, one year or at least 1 year, at least 2 years, at least 3 years at least 4 years, or at least 5 years prior to radiotherapy. In some embodiments, the head-and-neck cancer is oropharyngeal cancer, such as oropharyngeal squamous cell carcinoma, such as oral cavity cancer, such hypopharyngeal cancer, such as laryngeal cancer, such as cancer of unknown primary (CUP) of the head and neck. In one embodiment, the head-and-neck cancer was not HPV-positive oropharyngeal squamous cell carcinoma . In some embodiments, the head-and-neck cancer was pl6-positive or pl6-negative and/or HPV- negative oropharyngeal squamous cell carcinoma (see Lewis et al. (2010) for details) .

In some embodiments, the subject has received prior treatment with radioactive iodine or other radionuclide, or total body radiation.

In some embodiments, the subject has an autoimmune disease or disorder. For example, the subject may have an autoimmune disease or disorder that is associated with immune- mediated destruction of salivary glands. Subjects who have or are diagnosed with an autoimmune disease that causes or is associated with detrimental effects on salivary gland function, include, without limitation, those which suffer from rheumatoid arthritis, Sjogren syndrome, multiple sclerosis or diabetes, and are candidates for treatment or prevention of salivary gland dysfunction by administration of compositions comprising ASCs as described herein. Other conditions associated with salivary gland dysfunction include HIV-infection and ageing.

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 salivary glands. 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.

The treatment of a subject or patient according to any aspect or embodiment herein can be repeated if and as needed. For example, a patient having undergone treatment according to the invention may later show a recurrence of symptoms of salivary gland hypofunction or other dysfunction, such as xerostomia, making this patient suitable for a second treatment round . Alternatively, a physician or other person of skill in the art may determine that treatment according to the invention can advantageously be repeated at a predetermined interval, e.g. , at an interval of every 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, year, two years, three years, four years, five years, ten years, or another suitable period . The treatment can be repeated using the same or a different dosage of the ASC composition.

For the purposes of prophylactic (preventive) treatment, the subject may be asymptomatic but have a risk or predisposition to developing a salivary 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 salivary gland hypofunction, e.g. , Sjogren syndrome. Alternatively, the subject may have undergone radiation therapy for head and neck cancer, thereby being at risk for the development of salivary gland dysfunction. In such cases, administration of the compositions described herein may 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 salivary gland hypofunction or other dysfunction.

Sialometrv methods

The following represent alternative sialormetry methods to those described elsewhere herein. Examples of tests include, without limitation, the ones described in Gronhoj et a/. (2017) . In some embodiments, the tests are performed at a predetermined time of day, e.g. , between 2 p. m. and 3 p. m., for all collections to take the sa livary diurnal variation into account, with the subject refraining from eating, drinking, smoking and administering oral hygiene for 2 h prior to collection, and after being seated upright in a chair, the subject relaxes for 5 min and is then instructed to make as few movements as possible, including swallowing, during the collection. Unstimulated whole saliva production :

Unstimulated whole saliva can be collected using the spitting or drooling method (Navazesh et at. , J Dent Res. 1982;61 : 1158-62. ) where the subject transfers their saliva into a collection container over a period of 15 min. The whole salivary flow rate (SFR) (ml/min) SFR is determined as the increase in weight of the container (assuming that 1 g saliva has a volume of 1 mL) divided by the collection time in minutes. In some embodiments, a subject suffers from hyposalivation when the unstimulated whole SFR is at most 0.2 mL/min, e.g. , between about 0.2 mL/min and about 0.05 mL/min. In some embodiments, a subject suffers from hyposalivation when the unstimulated whole SFR is at most 0.1 mL/min (Gronhoj et a/. , 2018) .

Stimulated whole saliva production :

The subject is instructed to chew on 1 g of sterile paraffin wax. The subject chews for 60 s, and clears the oral cavity for saliva. Subsequently, as the glands are now in a stimulated state, the subject continues chewing the paraffin wax and saliva collected for the duration of 5 min. The salivary flow rate (SFR) (ml/min) is determined as the increase in weight of the container (assuming that 1 g saliva has a volume of 1 mL) divided by the collection time in minutes.

Testing of submandibular/sublingual glands:

Saliva is collected directly from the floor of the mouth in an unstimulated and stimulated state. The flow rate of the submandibular/sublingual glands is assessed by the swab method with cotton rolls placed buccally in each maxillary molar region to block the orifices of the parotid ducts and cotton rolls under the tongue in the floor of the mouth to collect submandibular/sublingual saliva . Immediately prior to the start of collection the subject is asked to swallow. Unstimulated saliva testing is performed with neutral cotton rolls and stimulated saliva testing with cotton rolls with 20 mg citric acid. For both measurements, collections takes place during a period of 3 min. Saliva flow rate is determined by weight ( 1 g equals 1 ml of saliva) with cotton rolls weighed before collection and reweighed after. The flow rates are calculated as the increase in weight during collection (assuming that 1 g saliva has a volume of 1 mL) and expressed as mLs per minute. Saliva from the cotton rolls is extracted by centrifugation ( 1500 x g) and analyzed for its composition.

Testing of parotid glands :

Special suction cups are placed over the two small openings in the subject's mouth from which the saliva produced by the parotid glands is released . Saliva produced by the parotid glands is collected in pre-weighed tubes for a predetermined period of 5- 15 minutes. For stimulated parotid saliva production, a sour liquid (such as lemon j uice or a citric acid solution) is applied to the subject's tongue throughout the test to stimulate parotid saliva flow. In some embodiments, a subject suffers from hyposalivation when the stimulated parotid flow rate is at most 0. 10 mL/min and/or the stimulated SM/SL flow rate is at most 0.20 mL/min (Kalk et a/. , Ann Rheum Dis 2001 ;60 : 1110-1116) .

Embodiments

The following particular embodiments are also contemplated.

Al . A composition comprising allogeneic ASCs for use in treating or preventing dysfunction of a salivary gland in a human subject.

A2. The composition for the use according to embodiment Al, wherein the composition is administered at a dosage of at least about 1 x 10^s allogeneic ASCs per human subject, such as from about 1.5 x 10^s to about 2 x 10^s allogeneic ASCs per human subject.

A3. The composition for the use according to any one of the preceding embodiments, wherein the composition is administered to at least one parotid gland and at least one submandibular gland of the human subject.

A4. The composition for the use according to any one of the preceding embodiments, wherein at least about one quarter of the dosage, such as between about one quarter and about one third of the dosage, is administered to each of the parotid glands of the human subject, and about half of the remaining dosage is administered to each of the submandibular glands of the human subject.

A5. The composition for the use according to any one of the preceding embodiments, wherein

(a) a dose of about 5 x 10⁷ allogeneic ASCs is administered to each of the two parotid and two submandibular glands of the human subject; or

(b) a dose of about 5 x 10⁷ allogeneic ASCs is administered to each of the two parotid glands, and about 2.5 x 10⁷ allogeneic ASCs is administered to each of the two submandibular glands of the human subject. A6. 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 2 mL per gland, such as from about 0.5 mL to about 1 mL, via direct intraglandular injection, optionally ultrasound-guided.

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, optionally divided into 2 to 4 separate vials.

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 any one of the preceding embodiments, comprising 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, CD 13, CD 105, CD29, CD166, CD10, CD140b, CD 160, 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, CD 14, 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

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

culture medium supplemented with human platelet lysate;

(c) detaching the adherent cells; (d) freezing the detached cells in a cryoprotectant at a concentration of at least 1 x 10⁶ cells/rmL;

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

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

(g) thawing the frozen composition.

A13. The composition for the use according to any one of the preceding embodiments, wherein the human subject has received prior radiation therapy of the head- and-neck region, has received prior chemotherapy, or suffers from an autoimmune disease.

A14. The composition for the use according to any one of the preceding embodiments, wherein the human subject has received prior radiation therapy for treatment of head-and- neck cancer or suffers from Sjogren syndrome, diabetes or multiple sclerosis.

A15. The composition for the use according to any one of the preceding embodiments, wherein the head-and-neck cancer was not HPV-positive oropharyngeal squamous cell carcinoma.

A16. The composition for the use according to any one of the preceding embodiments, wherein the human subject has an unstimulated whole salivary flow rate less than about 0.2 mL/min.

A17. A method of treating or preventing dysfunction of a salivary 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

The study population is composed of female and male patients with previous head and neck squamous cell carcinoma stage I-II (UICC-8), age 18-70 years, inclusive, with radiation- induced hyposalivation and xerostomia with a minimum of two years of recurrence-free follow-up. The patients are eligible for transplantation of allogeneic stem cells.

Primary Objectives:

- To evaluate the safety and tolerability of treatment with allogeneic adipose-derived mesenchymal stem cells transplanted into both submandibular and parotid glands in patients with radiation-induced hyposalivation and xerostomia.

- To evaluate the efficacy of the treatment with allogeneic adipose-derived mesenchymal stem cells transplanted into both submandibular and parotid glands assessed by change in salivary flow after one and four months compared to baseline in patients with radiation-induced hyposalivation and xerostomia.

Secondary objectives :

• To evaluate the immune response to allogeneic ASCs. Measured by development of de novo human leucocyte antigen antibodies (HLA) .

• Patient-reported outcome measures: Change in quality of life.

• Change in saliva gland function assessed by salivary gland ^99mTc scintigraphy after 4 months

• Change in the composition of saliva after four months.

General Design and Methodology: The study is an investigator-initiated, prospective, non- randomized, open label, single-centre, single dose, Phase 1 study in 10 adults with prior oropharynx cancer and with radiation-induced hyposalivation and xerostomia, receiving transplantation of allogeneic mesenchymal stem cells into both parotid and submandibular glands. The patients receive 50 million ASCs into both parotid glands and 25 million or 50 million ASCs in each submandibular gland according to the size of the glands. The trial compares the safety, tolerability and efficacy of two different formulations of allogeneic adipose tissue-derived mesenchymal stromal/stem cells) as a treatment for radiation-induced hyposalivation and xerostomia for previous oropharyngeal cancer patients.

The study has 3 phases :

• Pre-inclusion Screening, Informed Consent and Eligibility-determination Phase (Day - 60-0)

• Intervention (Day 0) • Follow-up (Day 1-4 months)

Study Drug, Dose and Mode of Administration : The study drug, allogeneic adipose tissue- derived mesenchymal stem cells, are injected ultrasound-guided intraglandular in both the parotid and submandibular glands. The participants receive 50 million ASCs into both parotid glands and 25 million or 50 million ASCs in each submandibular gland according to the size of the glands. This corresponds to an approximate total dose per patient at an average of 150 x 10⁶ - 200 x 10⁶ ASCs when treating the four major salivary glands. The measurements of the salivary gland rely on the size estimates by ultrasound combined with the measures from salivary gland scintigraphy. With ultrasound guidance, the intervention of ASCs is transplanted into the four salivary glands.

Duration of Patient Participation : Patient participation lasts for approximately 5-6 months (including a maximum of 60 day run-in-period and a 16 weeks follow-up period) .

Inclusion Criteria :

• Age between 18-70 years

· Previous radiotherapy +/- chemotherapy for OPSCC stage I- II (UICC-8, 2017) .

• 2 years' follow-up without recurrence

• Clinically reduced salivation and hyposalivation, evaluated by a screening

• Unstimulated salivary flow rate between 0.2mL/min and 0.05mL/min

• Grade 1-3 xerostomia as evaluated by CTCATE

· WHO Performance status (PS) 0- 1 ²

• Informed consent

Exclusion Criteria :

• Any cancer in the previous 4 years (not including OPSCC and basocellular carcinomas)

• Xerogenic medications

· Penicillin or Streptomycin allergy

• Any other diseases of the salivary glands, e.g. , Sjogren syndrome or sialolithiasis

• Previous parotid or submandibular gland surgery

• Previous treatment with any type of stem cells

• Pregnancy or planned pregnancy within the next 2 years

· Breastfeeding

• Smoking within the previous 6 months

• Alcohol abuse

• Any other disease/ condition judged by the investigator to be grounds for exclusion Safety and Tolerability Analysis : All adverse events (AEs) are monitored at the scheduled follow-up (day one, day 5, 1 month and 4 months after the intervention) . AEs are assessed and graded according to Common Terminology Criteria for Adverse Events v5.0 guidelines (CTCAEv5.0) .

Efficacy Analysis: Sialometry (salivary output measurement), which is the most common method for evaluating the salivary gland function and salivary flow rate. A change in the secretion rate of the unstimulated whole saliva in the oral cavity is deemed the most deciding parameter for hyposalivation and xerostomia .

The stored saliva samples are analysed for any change in composition, salivary proteomics or mRNA following the ASCs treatment/intervention. Changes in cytokines in the saliva are analysed for the purpose of detecting any local inflammatory actions in the major saliva glands driven by the intervention

Saliva samples are controlled with samples from healthy control subjects, who have not undergone radiotherapy.

The blood samples are analysed for the potential development of donor specific antibodies.

The primary efficacy endpoint: The results on salivary flow rate is calculated as a percentage change in salivary flow rate (from baseline) in the each participant.

Safety and Tolerability Analysis : All adverse events are monitored at the scheduled follow-up (day one, 1 month and 4 months after the intervention). AEs are assessed and graded according to Common Terminology Criteria for Adverse Events v5.0 guidelines (CTCAEv5.0) .

Control group

In a supplementary control study, a control population composed of ten healthy adults with no prior history of radiotherapy is included and perform a sialometry. The saliva samples from the controls are compared to the results found in the patients.

EXAMPLE 2

Phase I Open Label Study Initial results Patients and Methods

A Phase I clinical study was conducted as described in Example 1, except as indicated or supplemented below.

10 patients between 45 and 70 years of age with hypofunction of the major salivary glands and xerostomia after radiotherapy for oropharynx cancer were enrolled and treated with ASCs in the form of the study drug CSCC_ASC(50), a cryopreserved composition comprising 50 x 10⁶ allogeneic ASCs per mL in either 5% DMSO or 10% DMSO. The patients met inclusion and exclusion criteria described in Example 1. All patients were screened with blood samples for anti-HIV- I and II, syphilis, anti- HBsAg, anti- HBc and anti-HCV, kidney and liver parameters.

At baseline the patients completed quality of life questionnaires, underwent a salivary gland ^99mTc scintigraphy, performed a sialometry and had a battery of baseline blood samples take. The patients received ultrasound-guided injections of 50 x 10⁶ ASCs in both parotid glands and 25 x 10⁶ ASCs in both submandibular glands. Patients 1-5 received ASCs cryopreserved in DMSO 5% and patients 6- 10 received ASCs cryopreserved in 10% DMSO. Patients 1-2 and 6-7 had ASCs injected in the right parotid gland and right submandibular gland on day 0 and on day 5 they were injected into the left parotid and left submandibular gland. Patients 3-5 and 8- 10 were injected in all four glands, both parotid and submandibular glands, performed at day 0.

The patients returned for follow-up on 1 day, 5 days, 30 days and 120 days after the intervention to register adverse events and for collection of saliva and blood samples. Change in saliva gland function was assessed by sialometry after 1 month and 4 months, and by salivary gland 99mTc scintigraphy after 4 months.

Saliva collection was performed by sialometry. In more detail, the patients were informed to drink a minimum of 2 L of water the day before a sialometry. Saliva samples at baseline, after one and four months were collected between 10 : 00 and 12 : 00 a . m. and all performed in the same room at the ENT department, University Hospital of Copenhagen, Rigshospitalet, Denmark. The patients were refrained from drinking, eating, chewing gum, wearing lip stick and performing oral hygiene for at least 60 min before sialometry. After 5 minutes of rest, the patients swallowed one mouthful of water and collected unstimulated saliva for 10 minutes. The unstimulated saliva samples were immediately after weighing submerged into liquid nitrogen for snap freezing. Subsequently, the patients got a tasteless piece of paraffin gum and chewed for 1 minute and then swallowed the newly produced stimulated saliva . The following 5 minutes the patients chewed and stimulated saliva were collected. The stimulated saliva was after weighing divided into cryotubes. All the stimulated saliva samples, apart from the tube with RNAprotect agent, were immediately submerged in liquid nitrogen. The saliva samples were stored at -80°C within 15 min from collection and are stored until processing . Initial Results

Ten ( 10) patients (seven men and three women, median age 64) were included in the study. Patients had undergone radiotherapy for a pl6-positive oropharyngeal squamous cell carcinoma between two and nine years before receiving the intervention. Patient

characteristics are presented in Table 1. One-month data are presented for all 10 patients and four months data for the first five patients.

Table 1. Patient demographics

Safety: One patient had a serious adverse event (SAE) with a stroke 82 days after the ACSs treatment. The patient had a medical history with several transient ischemic attacks (TIA) and the SAE was judged not related to the therapy/intervention. One patient had an adverse event with vasovagal syncope (VVS) in response to the ASCs injections. Patient had a history with VVS numerous times during hospital visits.

Change in saliva gland function :

Table 2 shows alterations in unstimulated flow rate (UWS) and stimulated flow rate (SWS) after one ( 10 patients) and four months (5 patients) . The median change in unstimulated flow rate after 4 months for the first five patients were 64% (range -3- 120) . For stimulated flow rate, the median change after 4 months was 14 % (range 1-27) .

Table 2. Initial results of change in unstimulated (UWS SRF) and stimulated whole salivary flow rate (^'SI/I/S SRF)

EXAMPLE 3 Phase II/ III study

The following is a single-centre, randomised, placebo- controlled phase II-III study to investigate the safety and efficacy of mesenchymal stem cells for radiation-induced hyposalivation in patients having undergone radiation therapy for head and neck cancer. The study population includes patients in the age range 18-75 years with a minimum of two years of recurrence-free period after the radiotherapy. Patients with and without pl6 overexpression are eligible.

Primary objectives

• To evaluate efficacy and safety of with allogeneic adipose-derived mesenchymal stem cells transplanted into both submandibular and parotid glands in patients with radiation-induced hyposalivation and xerostomia assessed by change in unstimulated and stimulated salivary flow rates after 4 months.

Secondary objectives

• Change in health-related quality of life, Patient reported outcome measures.

• Safety measured by SAE and SARs

• Development of donor specific antibodies

• Change in saliva composition

• Change in saliva proteomics and gene expression

General Design and Methodology: The trial is an investigator-initiated, randomized, placebo- controlled phase II-III study to assess efficacy and safety of allogeneic adipose-derived mesenchymal stem cells in patients with hyposalivation and xerostomia after a radiotherapy for a head and neck cancer. The patients receive ultrasound-guided injections of 25 x 10⁶ ASCs into each submandibular gland and 50 x 10⁶ ASCs into each parotid gland.

The study has three phases

• Intervention (Day 0)

• Follow-up (Day 1-4 months)

Study Drug, Dose and Mode of Administration : The study drug, allogeneic adipose tissue- derived mesenchymal stem cells are injected ultrasound-guided intraglandular in both the parotid and submandibular glands. The participants receive 50 million ASCs into both parotid glands and 25 million ASCs in each submandibular gland . This corresponds to an approximate total dose per patient at an average of 150x10^® ASCs when treating the four major salivary glands. Duration of Patient Participation : Patient participation lasts for approximately 5-6 months (including a maximum of 60 day run-in-period and a 4-month follow-up period).

Inclusion Criteria :

• Age between 18-75 years

• Previous radiotherapy +/- chemotherapy for a head and neck cancer.

• 2 years' follow-up without recurrence

• Clinically reduced salivation and hyposalivation, evaluated by a screening

• Unstimulated salivary flow rate between 0.29 mL/min and 0.04 mL/min

• WHO Performance status (PS) 0-1 ²

• Informed consent

Exclusion Criteria :

• Xerogenic medications

• Penicillin or Streptomycin allergy

• Previous parotid or submandibular gland surgery

• Previous treatment with any type of stem cells

• Breastfeeding, pregnancy or planned pregnancy within the next 2 years

• Smoking within the previous 6 months

• Alcohol abuse

• Any other disease/ condition judged by the investigator to be grounds for exclusion

Safety and Tolerability Analysis: All adverse events (AEs) are registered at the follow-up visit. Adverse events are assessed and graded according to Common Terminology Criteria for Adverse Events v5.0 guidelines (CTCAEv5.0).

The primary efficacy endpoint: The results on salivary flow rate is calculated as a percentage change in salivary flow rate (from baseline) in the group of participants given ASCs compared to in the control group. LIST OF REFERENCES

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

Gronhoj etal.. Radiation Oncology, vol. 101, no.3, pp. 581-592, 2018 (Epub 6 March 2018). Gronhoj eta/. Trials, vol. 18, p. 108, 2017 (Pub 7 March 2017).

Qomi eta/.. World J Stem cells, vol.9, no.8, pp. 107-117, 2017 (Pub 26 Aug 2017).

Wang et a/. J Oral Maxillofac Surg, vol.75, no.8, pp. 1784-1790, 2017(Epub 16 Aug 2016). Xu eta/. Blood, vol. 120, no. 15, pp.3142-3151, 2012.

Jensen etal. Oral Surg Oral Med Oral Pathol Oral Radiol, vol. 117, pp.335-342, 2014.

Lirm eta/. PLOS ONE, vol.8, issue 8, e71167 (1 August 2013).

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

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

Lewis etal., Am J Surg Pathol.2010;34(8). doi: 10.1097/PAS.0b013e3181e84652

Cancer Institute N. Common Terminology Criteria for Adverse Events (CTCAE) v5.0

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

EU Clinical Trials Register (EudraCT) Number: 2018-003856-19

ClinicalTrials.gov Identifier: NCT03874572

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-derived mesenchymal stems cells (ASCs) for use in treating or preventing dysfunction of a salivary gland in a human subject.

2. In one embodiment, the composition is administered at a dosage of at least about 1 x 10⁶, such as at least about 1 x 10⁷, such as at least about 2.5 x 10⁷, such as at least about 5 x 10⁷ allogeneic ASCs to at least one salivary gland in the human subject.

3. The composition for the use according to any one of the preceding claims, wherein the composition is administered to at least one parotid gland and/or at least one submandibular gland of the human subject.

4. The composition for the use according to any one of the preceding claims, wherein the composition is administered at a dosage of at least about 1 x 10^s allogeneic ASCs per human subject, such as from about 1.5 x 10^s to about 2 x 10^s allogeneic ASCs per human subject.

5. The composition for the use according to any one of the preceding claims, wherein at least about one quarter of the dosage, such as between about one quarter and about one third of the dosage, is administered to each of the parotid glands of the human subject, and about half of the remaining dosage is administered to each of the submandibular glands of the human subject.

6. The composition for the use according to any one of the preceding claims, wherein

(b) a dose of about 5 x 10⁷ allogeneic ASCs is administered to each of the two parotid glands, and about 2.5 x 10⁷ allogeneic ASCs is administered to each of the two submandibular glands of the human subject.

7. 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 2 mL per gland, such as from about 0.5 mL to about 1 mL, via direct intra-glandular injection, optionally ultrasound-guided.

8 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, optionally divided into 2 to 4 separate vials.

9. 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.

10. The composition for the use according to any one of the preceding claims, comprising dimethylsulphoxide (DMSO) at a concentration of about 5% to about 15% (v/v), such as about 5% or about 10% (v/v) .

11. The composition for the use according to claim 10, 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.

12. 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, CD 10, CD 140b, 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, CD 14, CD 106, CD31 and CD36.

13. 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

(c) detaching the adherent cells;

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

(g) thawing the frozen composition.

14. The composition for the use according to any one of the preceding claims, wherein the human subject has received prior radiation therapy of the head-and-neck region, has received prior chemotherapy, has received prior total body radiation, has received radioiodine therapy, or suffers from an autoimmune disease or disorder.

15. The composition for the use according to any one of the preceding claims, wherein the human subject has received prior radiation therapy for treatment of head-and-neck cancer or suffers from Sjogren syndrome, diabetes, multiple sclerosis or HIV.

16. The composition for the use according to any one of the preceding claims, wherein

(a) the human subject received prior radiation therapy for treatment of head-and- neck cancer up to 25 years ago; and/or

(b) the head-and-neck cancer was oropharyngeal cancer, such as oropharyngeal squamous cell carcinoma, optionally pl6 and/or HPV-negative oropharyngeal squamous cell carcinoma .

17. The composition for the use according to any one of the preceding claims, wherein the salivary gland dysfunction is hyposalivation, xerostomia or a combination of both.

18. The composition for the use according to any one of the preceding claims, wherein the human subject has an unstimulated whole salivary flow rate less than about 0.4 mL/min, such as less than about 0.3 mL/min, such as less than about 0.2 mL/min.

19. A method of treating or preventing dysfunction of a salivary 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.