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What is a Therapeutic Dose for Extracellular Vesicles?

Extracellular vesicles derived from MSCs (MSC-EVs) are promising therapeutic candidates that have been used in several clinical trials for indications including graft versus host disease (GvHD), kidney disease, and diabetes, among many others [1]. The number of clinical trials investigating MSC-EVs as therapeutics has increased dramatically from 2006 (2 trials) to 2020 (74 trials). To learn more about MSC-EVs journey as therapeutic, check out a recent blog post that answers the question: What Are Extracellular Vesicles, Exosomes, & Microvesicles & Why Are They En Route to the Clinic?

The growing number of clinical trials demonstrates the exciting therapeutic promise of extracellular vesicles. However, the dose of MSC-EVs applied to patients across these studies has varied considerably in terms of the method of MSC-EV quantification and the amount of applied MSC-EVs. A single dose is a unit and amount of a therapeutic compound to be applied to a patient. Experimental medicine that occurs in clinical trials typically involves dose regimens, which include multiple doses of the therapeutic compound over a period of time, with each dose occurring at a specific frequency [2]. The total dose is the sum of all doses in a dose regimen. Being novel therapeutic candidates, given their heterogeneity and often limited in vivo retention, MSC-EVs challenge the conventional dose regimen framework. Consensus MSC-EV dose definitions are still in development across scientific and medical fields. In this blog article, we will explore the current state of MSC-EV dosing in completed and ongoing clinical trials, discuss the factors that affect MSC-EV dose, including MSC-EV quantification methods, and establish a target ‘working range’ for MSC-EV dose in humans.

The current consensus affirms that extracellular vesicles are small particles 50-1000 nm in diameter, comprised of a lipid bilayer containing a variety of biological cargoes, including nucleic acids, proteins, and lipids. Thus, an ‘amount’ of extracellular vesicles in a preparation can be determined by either quantifying the number of EVs in the preparation (using nanoparticle tracking analysis) or by quantifying the number of nucleic acids, proteins, or lipids (using colorimetric or fluorometric assays) in the preparation. An alternative, simpler yet indirect method to estimate the amount of extracellular vesicles in a preparation is by counting the number of cells cultured in a given medium to produce extracellular vesicles. Crucially, any of these three methods can be used in a dose escalation/response study for researchers to claim that a specific unit and amount of extracellular vesicles achieved the desired therapeutic effect. However, although used previously, the cell counting method does not provide a quantitative measurement of extracellular vesicles and is thus not recommended for industry applications.

To date, MSC-EV doses in clinical trials have been reported using all three above methods. However, these methods are non-standardized, which can lead to variability across trials. In one clinical study, conditioned medium containing MSC-EVs derived from 40 million cells was administered intravenously to a patient every 2-3 days, totaling 4 doses per regimen, and thus requiring 160M cells in total [3]. Another clinical study used intravenous administration of 100 µg MSC-EV protein per kg patient body weight [4], where protein content is measured using Bradford assay. In a more recent trial targeting pneumonia resulting from COVID-19 infection, patients inhaled 1.25×10¹⁰ EVs twice per day over 10 days, totaling 2.5×10¹¹ EVs per dose regimen [5]. These studies differ dramatically by route of administration, EV quantification method, and dose regimen, demonstrating the variation in EV administration in humans on a trial-by-trial basis, which motivates the need to better standardize reported EV dose descriptions.

Ultimately, the effective extracellular vesicle dose will depend on the specific disease indication. However, determining effective EV dose stands to benefit from ongoing work that can provide as much information as possible for researchers to understand typical quantities of EVs being administered to patients – a ‘working range’ of EV dose in humans. To evaluate this ‘working range’ of EV dose, we must consider the administration route, the EV quantification method, and the number of doses occurring per dose regimen.

How Does Administration Route Affect Dose?

For novel therapeutics, the route of administration is important since it directly precedes how therapeutics interact with the body; a concept known broadly as pharmacokinetics. This field uses ADME (Absorption, Distribution, Metabolism, Elimination) to describe the process that occurs after therapeutic administration [6].

• Absorption: Different classes of therapeutics show different absorption profiles depending on administration route, and thus some administration routes for certain therapeutics require a significantly lower or higher dose [6].
• Distribution: Distribution of EVs throughout the body is reported to be heterogeneous and depends on parent cell type, source, EV composition, and administration route [7]. Recent studies have used sophisticated biological labeling strategies to track EV distribution in animals [8], likely to be translated to humans in the near future.
• Metabolism: Whether EVs are metabolized by specific organs or within the bloodstream is likely to depend on administration route [9]. Some evidence shows that patrolling macrophages in the bloodstream take up EVs efficiently in zebrafish [10], but this is yet to be observed in humans.
• Elimination: Many studies report that EVs are cleared rapidly (within hours) after administration [11]. These results will drive clinical work into establishing prolonged dosing regimens involving multiple doses over time.

In summary, administration route is likely to affect the effective EV dose, and thus should be considered carefully. Most preclinical studies in animals used intravenous administration, direct (site-specific) injection, or subcutaneous injection [12].

The Extracellular Vesicle Quantification Challenge

Early reporting of extracellular vesicle quantity in preclinical studies focused on the number of cells used in producing a given quantity of medium over a given time interval. As preclinical studies evaluating extracellular vesicles commenced in animal models, investigators began reporting EV quantity in terms of protein or lipid content, a method typically used for other preclinical biologic compounds. Though this method is acceptable, it is problematic because extracellular vesicles are not made up entirely of protein or lipid, as they are a mix of several biological components. This is further complicated by the fact that non-EV-specific proteins and lipids exist within the conditioned medium, either in a soluble form or in complex with the EV surface. These contaminants require purification to remove, and studies sometimes do not report or otherwise incompletely define the purification methods used. Thus, reports of EV quantity in terms of total protein or lipid content are dependent on many factors and are unlikely to be comparable across other studies without additional information.

To potentially solve this problem, nanoparticle tracking analysis (NTA) was developed and used [13] to measure the number of extracellular vesicles and their size distribution. While this information is critical, extracellular vesicles can range variably in size between 50-1000 nm in diameter. It stands to reason, for example, that larger extracellular vesicles probably contain more biological material, and thus the contents within larger EVs are likely overrepresented for the total sample. Furthermore, the components within extracellular vesicles mediating therapeutic effects are biological cargoes, unless EVs are loaded artificially with small molecule drugs or metabolites [14]. Information about these cargoes is not necessarily contained within the data for number of vesicles or their size distribution. Thus, the combination of measurements for EV number, size, and content is necessary to best describe an EV population for dosing purposes.

To attempt to resolve the above issues, the International Society of Extracellular Vesicles (ISEV) lays out the minimal information required for studies of extracellular vesicles (MISEV) which includes the recommendation above. According to MISEV, a description of a given extracellular vesicle’s composition should include the parent cell number and source, the EV number and size, and the EV protein content, lipid content, RNA content, and presence of surface markers on EVs [15]. In the near future, better technologies such as flow cytometry or single particle interferometric imaging will be utilized to characterize individual EVs within a population to better describe the entire population [16]. However, as it stands currently, dosing information remains varied across studies, making it necessary to evaluate studies individually to make more general conclusions.

Establishing a Working Range for Effective EV Dose

In general, preclinical work done in rodents has typically reported MSC-EV dose in terms of micrograms (µg) of protein [12]. After reviewing 18 preclinical studies* performed across different disease models in rodents, it was found that MSC-EV dose ranges between 0.10 and 250 µg of protein with a mean value of 70 µg. Assuming 1 µg equates to around 2 x 10⁹ MSC-EVs [17], this yields a rough estimate of 1.4 x 10¹⁰ MSC-EVs per dose being used on average in preclinical rodent studies. Given that humans are much larger than rodents, it is probably safe to assume that a dose of 1.4 x 10¹⁰ MSC-EVs is close to an absolute minimum dose for humans [18]. As pointed out by Sverdlov, the basal concentration of EVs in human blood is roughly 1.2 x 10⁹ per mL [17]. Assuming the average human has 5L of blood in circulation (according to redcrossblood.org), this means there are roughly 6 x 10¹² EVs circulating in the average human bloodstream at any given time. It seems unreasonable to supply a patient with more EVs than currently exist in their bloodstream, so this value is probably a safe upper limit. Thus, a working total MSC-EV dose likely falls somewhere within the broader range of 1 x 10¹⁰-6 x 10¹² MSC-EVs.

Table 1**. Therapeutic extracellular vesicle / exosome studies in humans reporting dose. Search used clinicaltrials.org as of 09Nov2021 with terms “MSC extracellular vesicles” or “MSC exosomes”. Protein content to EV number conversion assumes 2 x 10⁹ MSC-EVs per 1 µg protein. eq. = equivalent. For (6) and (14), assuming an average adult human measures 66 kg.

After performing a search of human studies involving extracellular vesicles/exosomes as the therapeutic compound, 23 human studies and clinical trials were found with only 18 of them reporting the MSC-EV dose used (see Table 1**). Of these studies, 11 report dose based on MSC-EV number, 5 report dose based on protein content, and 2 report dose based on cell number. Notably, 44% of studies use intravenous administration and 36% use inhalation, with the remainder using topical or local injection. Furthermore, 11 of 18 (61%) use a dose regimen versus a single dose. Excluding the two studies reporting cell number, the total MSC-EV dose ranges from 1.7 x 10⁸ to 4.2 x 10¹⁵, with a median value of 4.3 x 10¹⁰. Importantly, this value exists squarely within the broader range determined above, indicating an agreement between this theoretical prediction and the reality occurring in ongoing clinical trials. Thus, the working range of EV dose exists within 1 x 10¹⁰-6 x 10¹² with a target dose around 4 x 10¹⁰.

Nailing the Number: Large-scale Extracellular Vesicle Production

Now that a working range for extracellular vesicle doses in humans has been established, an important question arises — how feasibly can batches of extracellular vesicles be produced to this scale? Though total dose will vary by indication, given a conservative estimate of 1000 EVs produced per cell per production process [17,19], it is likely that 100-1000M MSCs will be required to create a single dose regimen in the working range, which is in line with predictions reported previously [20]. To meet the growing demand for MSC-EV therapeutic investigations, existing manufacturing processes will require significant improvements. Furthermore, since EV properties can differ according to production conditions, it is imperative to develop scalable methods for EV production to ensure consistent potency in the desired therapeutic indication before a significant investment in clinical translation of EV products. Our goal at RoosterBio is to be at the forefront of establishing processes and products that seek to maximize the productivity of hMSCs, and the extracellular vesicles that they produce, to meet this growing demand. We will address extracellular vesicle manufacturing processes and variables that drive productivity and yield in future blog posts.

References

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15. Théry, C. et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 7, 1535750, doi:10.1080/20013078.2018.1535750 (2018).
16. Bordanaba-Florit, G., Royo, F., Kruglik, S. G. & Falcón-Pérez, J. M. Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles. Nature Protocols 16, 3163-3185, doi:10.1038/s41596-021-00551-z (2021).
17. Sverdlov, E. D. Amedeo Avogadro’s cry: What is 1 µg of exosomes? BioEssays 34, 873-875, doi:https://doi.org/10.1002/bies.201200045 (2012).
18. Nair, A. B. & Jacob, S. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm 7, 27-31, doi:10.4103/0976-0105.177703 (2016).
19. Record, M., Subra, C., Silvente-Poirot, S. & Poirot, M. Exosomes as intercellular signalosomes and pharmacological effectors. Biochemical Pharmacology 81, 1171-1182, doi:10.1016/j.bcp.2011.02.011 (2011).
20. Olsen, T. R., Ng, K. S., Lock, L. T., Ahsan, T. & Rowley, J. A. Peak MSC—Are We There Yet? Frontiers in Medicine 5, doi:10.3389/fmed.2018.00178 (2018).

* Studies used in this calculation: