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US-12624343-B2 - Extracellular vesicles (EVs) derived from mesenchymal stromal cells and method for obtaining said EVs

US12624343B2US 12624343 B2US12624343 B2US 12624343B2US-12624343-B2

Abstract

The current invention relates to a process for the manufacturing of a composition of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs), said method comprises: culturing and expanding MSCs in a serum-free and xeno-free medium comprising purified human serum albumin and human transferrin; —collecting cell supernatant, said cell supernatant comprises EVs; —filtering said cell supernatant to obtain EVs; and concentrating said EVs, preferably by means of ultrafiltration. In a second and further aspect, said invention is directed to compositions comprising EVs and their clinical use.

Inventors

  • Marcin JURGA

Assignees

  • EXO BIOLOGICS SA

Dates

Publication Date
20260512
Application Date
20210708
Priority Date
20200709

Claims (12)

  1. 1 . A process for the manufacturing of a pharmaceutical composition of Extracellular Vesicles (EVs) derived from mesenchymal stromal cells (MSCs), said method comprising: culturing and expanding MSCs in a serum-free and xeno-free medium comprising human albumin at a concentration between 1 g/l and 5 g/l and human transferrin at a concentration between 55 mg/l and 100 mg/l; collecting a cell supernatant from said MSCs, wherein said cell supernatant comprises EVs; filtering said cell supernatant to obtain EVs, wherein said filtering is a two-step filtration process comprising filtering said cell supernatant with a filter having a mesh size of between 1 and 5 μm and bringing a filtrate of the first filtration step over a second filter having a mesh size of below 1 μm; and concentrating a filtrate of the filtering using a tangential flow filtration (TFF) step with a cut-off of 100 kDa.
  2. 2 . The process according to claim 1 , wherein said human albumin and said human transferrin are purified from human plasma or are recombinant albumin and recombinant transferrin.
  3. 3 . The process according to claim 1 , wherein at least 0.25×10 9 particles/ml medium are produced in a time span of 18 to 24 hours of culturing said MSCs, wherein at least 90% of said particles with particle size of between 0.05 and 0.22 micron are EVs.
  4. 4 . A pharmaceutical composition comprising Extracellular Vesicles (EVs) derived from mesenchymal stromal cells (MSCs), wherein said composition has at least 1×10 11 particles per ml, wherein said particles have a particle size between 0.05 and 0.22 micron, and wherein said composition has a human albumin concentration of between 10 and 30 g/l, and wherein at least 90% of said human albumin present in said composition is associated with said EVs.
  5. 5 . The pharmaceutical composition according claim 4 , wherein said composition comprises human transferrin in a concentration of between 60 mg/l to 600 mg/l.
  6. 6 . The pharmaceutical composition according to claim 4 , wherein said composition comprises human transferrin and human albumin at a ratio of between 2 and 60 mg transferrin per g albumin.
  7. 7 . The pharmaceutical composition according to claim 4 for therapeutic or prophylactic use.
  8. 8 . The pharmaceutical composition according to claim 4 for use in the prevention or treatment of lung disorders, wherein said lung disorder is an inflammatory lung disease, lung vascular disease, or acute lung injury.
  9. 9 . The composition for use of claim 3 , wherein said inflammatory lung disease is pulmonary hypertension, asthma, bronchopulmonary dysplasia (BPD), allergy, pneumonia, or idiopathic pulmonary fibrosis, and wherein said acute lung injury is associated with sepsis or is acute respiratory distress syndrome (ARDS).
  10. 10 . The pharmaceutical composition according to claim 4 for use in the treatment of COVID-19 induced or acute pneumonia.
  11. 11 . The pharmaceutical composition according to claim 4 for use in the prevention or treatment of inflammatory bowel disease.
  12. 12 . The pharmaceutical composition according to claim 4 wherein said a therapeutically effective amount of said composition is administered to a patient, said patient being an adult, an infant or a neonate, wherein said composition is administered at a dose of 10 9 EVs/kg to 10 12 EVs/kg of said patient or for each administration.

Description

FIELD OF THE INVENTION The present invention relates to extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) and compositions comprising said vesicles as well as methods for obtaining the latter. BACKGROUND EVs are lipid bilayer-delimited particles that are naturally released from a cell and, unlike a cell, cannot replicate. EVs range in diameter from near the size of the smallest physically possible unilamellar liposome (around 20-30 nanometers) to as large as 10 microns or more, although the vast majority of EVs are smaller than 200 nm. They carry a cargo of proteins, nucleic acids, lipids, metabolites, and even organelles from the parent cell. Most cells that have been studied to date are thought to release EVs, including some bacterial, fungal, and plant cells that are surrounded by cell walls. A wide variety of EV subtypes have been proposed, defined variously by size, biogenesis pathway, cargo, cellular source, and function, leading to a historically heterogenous nomenclature including terms like exosomes, microvesicles and ectosomes. EVs could be used for therapeutic purposes, such as delivering nucleic acids or other cargos to diseased tissue and cells. This growing interest was paralleled by formation of companies and funding programs focused on development of EVs as biomarkers or therapies of disease, the founding of an International Society for Extracellular Vesicles (ISEV), and establishment of a scientific journal devoted to the field, the Journal of Extracellular Vesicles. With the growing interest in EVs for therapeutic use, also the need for clinical-grade EVs has risen. In order to be useful in the clinic, EVs need to be produced in a reproducible and controlled manner. Good Manufacturing Practice (GMP) for the manufacturing of compositions comprising EVs is crucial, in order to assure that various batches produced are uniform and of consistent quality. GMP is particularly difficult when dealing with cell-derived therapeutics. Production processes for exosomes derived from mesenchymal stromal cells (MSCs) are known from, for example, Reka Agnes Haraszti et al., 2018. In addition, Diem Huong Huang at al., 2020, describes MSC-derived exosomes in the context of their regenerative potential for cutaneous tissue, the exosomes produced under specific conditions. Various media may be used to culture MSCs, amongst which a serum-free and xeno-free culture medium as disclosed in Lucas G. Chase et al., 2012. Antoine Monsel et al., 2016, further, describe the use of MSC secretome and extracellular vesicles (EVs) in the treatment of lung diseases. The presented data for their use in humans is mostly speculative and highlight several problems in the state of the art, such as low reproducibility and low yield. In addition, the document warns about potential problems with the use of bioreactors for large scale culture of MSCs for the production of EVs. Finally, Youngja Park et al., 2011, refer to the use of albumin in the treatment of lung diseases. Several problems, however, still need to be solved before the clinical use of EVs can become widespread. It remains a challenge to develop platforms for the production, storage and handling of clinical grade EVs in a reliable and reproducibly quantifiable manner. MSC-derived EV-based pharmaceuticals and subsequent clinical trials demand the resolution of several scientific, regulatory, technological and mechanistic issues prior to bringing MSC-derived EV-based therapies to the clinic. There is thus need for a protocol that enables the production of clinical-grade MSC-derived EVs in a stable and reproducible manner, under GMP conditions, and at cost effective large scale. Moreover, there is a need for an EV-based product that has good stability and commercially attractive shelf life. SUMMARY OF THE INVENTION The current invention provides a method that enables the production of EVs from MSCs in a stable and repeatable manner, under GMP conditions according to the Internal Conference on Harmonization (ICH) 2020 GMP quality guidelines. The EV composition obtained is a clinical-grade, cGMP product that is readily useable in clinical trials and in patients and has good stability. To that purpose, the current invention provides a manufacturing process and a composition. Preferred embodiments are described herein. Said product may have various clinical applications and therapeutic effects. Uses of said product are described herein. Due to the long-term stability of said composition, the product can be used in various clinical applications. DESCRIPTION OF FIGURES FIG. 1 shows an embodiment of a MSC expansion unit and EV processing unit that can be used to produce EVs of the current invention. DEFINITIONS Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidan