Potency assay matrix for establishing consistency of manufactured cells

Abstract

Disclosed herein is a potency assay matrix developed for use in screening factors involved in overall manufactured cell quality. Non-limiting example screening factors include activated lymphocyte suppression, macrophage polarization, and regulatory T cell (Treg) stimulation. Also disclosed is an application of the potency assay matrix for developing an allogeneic Mesenchymal Stem / Stromal Cells (MSCs) therapy. The matrix also has use in controlling the manufacturing processes for cells, and for measuring inter-batch consistency.

Description

OSIUM.016WO PATENT POTENCY ASSAY MATRIX FOR ESTABLISHING CONSISTENCY OF MANUFACTURED CELLSRELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This application claims the benefit of U.S. Provisional Ser. No. 63 / 755125, filed February 6, 2025, which is hereby incorporated by reference in its entirety.FIELD

[0002] The present disclosure generally relates to a mathematical matrix used as a standard for manufactured cell quality. In some embodiments, the disclosure relates to a potency assay matrix that includes screening factors to monitor cell quality. Also disclosed are uses of such a matrix, such as in the development of cell therapy and in controlling the manufacturing processes for cells.BACKGROUND

[0003] Mesenchymal stromal cells (MSCs) are widely utilized in clinical trials based on proven in vitro and animal studies of their immunomodulatory and regenerative properties. While clinical trials have demonstrated clear safety of utilizing MSCs, the overwhelmingly positive results obtained through in vitro and preclinical studies have not been realized in clinical efficacy at-scale. Clinical use of MSCs typically relies on the effective storage (e.g., cryopreservation) of the intermediary and final products, which greatly facilitates distribution. However, cryopreservation has been attributed to impaired function of cryopreserved MSCs.SUMMARY

[0004] Disclosed herein are methods for developing a potency assay matrix for use in monitoring the quality of a manufactured cell. In some embodiments, the methods include testing the manufactured cell for activity in one or more screening factors and assigning a value to the activity for each screening factor. In some embodiments, the value for each screening factor is combined to form the potency assay matrix. In some embodiments, the one or morescreening factors include at least one of immune cell migration, immune cell chemotaxis, phagocytosis, leukocyte mediated immunity, activated lymphocyte suppression, macrophage polarization, and / or regulator}' T cell stimulation, or any combination thereof. In some embodiments, the one or more screening factors include activated lymphocyte suppression, macrophage polarization, and / or regulatory T cell stimulation, or any combination thereof. In some embodiments, the one or more screening factors include at least one of leukocyte migration, leukocyte chemotaxis, myeloid leukocyte migration, granulocyte migration, neutrophil chemotaxis, neutrophil migration, mononuclear cell migration, and / or lymphocyte migration, or any combination thereof. In some embodiments, the manufactured cell is a stem cell or stromal cell. In some embodiments, the manufactured cell is a mesenchymal stem / stromal cell (MSC). In some embodiments, the value is assigned a numerical scale, for example, 1 to 3, 1 to 5, or 1 to 10. In some embodiments, one end number on the scale represents the least amount of change in activity, and the other end number on the scale represents the most amount of change in activity. As a nonlimiting example, a protein with no change in expression and / or activity level may be assigned “1”, a protein with some change in expression and / or activity level may be assigned “2,” and a protein with significant change in expression and / or activity level may be assigned “3.”

[0005] Also disclosed herein are methods of insuring inter-batch consistency across a group of manufactured cells in a cell batch. In some embodiments, the methods include administering the potency assay matrix of any one of the embodiments of the present disclosure, and removing from the cell batch any manufactured cell that does not reach a suitable activity value in at least one screening factor.

[0006] Also disclosed herein are methods of determining the quality of a stored cell. In some embodiments, the methods include measuring the expression level of an at least one gene involved in an at least one activity pathway in a fresh cell, and measuring the expression level of the at least one gene in the stored cell. In some embodiments, no significance in expression levels between the fresh and stored cell is indicative of a good quality of the stored cell, and a significant difference in expression level is indicative of a poor quality of the stored cell. In some embodiments, the stored cell is cryopreserved. In some embodiments, the stored cell is a stem cell or a stromal cell. In some embodiments, the stored cell is an MSC. In some embodiments, the at least one activity pathway includes at least oneof immune cell migration, immune cell chemotaxis, phagocytosis, leukocyte mediated immunity, activated lymphocyte suppression, macrophage polarization, and / or regulatory T cell stimulation, or any combination thereof. In some embodiments, the at least one activity pathway includes at least one of: activated lymphocyte suppression, macrophage polarization, and / or regulatory T cell stimulation, or any combination thereof. In some embodiments, the at least one activity pathway includes at least one of leukocyte migration, leukocyte chemotaxis, myeloid leukocyte migration, granulocyte migration, neutrophil chemotaxis, neutrophil migration, mononuclear cell migration, and / or lymphocyte migration, or any combination thereof. In some embodiments, the at least one gene includes at least one of VAV1, IDO-1, TREM1, S1PR1, MY01G, ITGA7, EDNRB, CCL18, ADAM8, CCL2, CSF1, and / or CD63, or any combination thereof. In some embodiments, the at least one gene includes 1, 2, 3, or 4 of: IDO1, CSF1, CD63, and / or CCL2, or any combination thereof. In some embodiments, when the stored cell is of poor quality, the expression level of the at least one gene is significantly reduced in the stored cell compared to the fresh cell. In some embodiments, when the stored cell is of good quality, the expression level of the at least one gene is not significantly altered in the stored cell compared to the fresh cell. In some embodiments, when the stored cell is of poor quality, the overall activity level of the at least one activity pathway is significantly reduced in the stored cell compared to the fresh cell. In some embodiments, when the stored cell is of good quality, the overall activity level of the at least one activity pathway is not significantly reduced, or is increased, in the stored cell compared to the fresh cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] In addition to the features described above, additional features and variations will be readily apparent from the descriptions of the drawings and embodiments provided herein. It is to be understood that these drawings depict embodiments and are not intended to be limiting in scope.

[0008] FIGs. 1A-1D depict a nonlimiting example quantification of MSCs and their potency factors. FIG. 1A shows interferon gamma (IFN-y)-induced IDO-1 expression levels. FIG. IB correlates IDO-1 expression with T cell suppression potency. FIG. 1C depicts a larger panel of donors, varying from 10-90% IFN-y-induced IDO-1 expression. FIG. ID depicts the correlation (r = -0.9; P < 0.001) between IDO-1 and suppression.

[0009] FIGs. 2A-2H depict a nonlimiting example quantification of MSC-derived CSF1 (M-CSF) potently polarizes macrophages to an M2 anti-inflammatory phenotype. FIGs.2A-2C depict the neutralization of CSF1 secreted by MSC in co-culture with monocytes / macrophages prevented polarization, as determined by CD163 MFI and secreted IL-10 levels. FIGs. 2D-2E depict the inhibition of CSF1 receptor (CSFR1) also reduced M2 polarization in co-culture with MSCs. FIG. 2F depicts the CSF1 expression varies with donor MSCs. FIGs.2G-2H depict MSC expressed CSF1 levels and M2 polarization of 2 donor monocytes were correlated.

[0010] FIGs. 3A-3E depict a nonlimiting example quantification of MSC secreted chemokine CCL2 attracted monos / macros, which were then polarized. FIGs. 3A-3B depict Bulk RNA-seq analysis of monos / macros after culturing with MSC conditioned medium (CM) suggested upregulation of migratory signaling pathways. FIG. 3C depict levels of CCL2 secretion varied with MSC donor. FIG. 3D depict MSC-induced migration of monocytes was dependent on CCL2. FIG. 3E depicts migration toward MSC-CM induced M2 polarization.

[0011] FIGs. 4A-4H depict a nonlimiting example quantification of MSC extracellular vesicles (EVs) induced Tregs and macrophage M2 polarization. FIG. 4A depicts macrophages engulfed MSC EVs labeled with cell bright orange (CBO). FIG. 4B depicts EVs induced M2 polarization (increased CD163 MFI). FIG. 4C depicts EV uptake was MerTK dependent. FIG. 4D depicts EV-mediated M2 polarization is dose-dependent. FIG. 4E depicts neutralizing the latent form of TGFpi (LAP) reduced EV polarization. FIGs. 4F-4H depict EVs dose-dependently induced Tregs.

[0012] FIGs. 5A-5B depict a nonlimiting example quantification of development of a weighted matrix score to select MSCs with optimal potency. FIG. 5A depicts the range in levels of each potency factor expressed by MSCs isolated from 8 donors was quantitated. The coefficients of variation were: IDO1 (47.3%. top left panel); CSF1 (25.8%. top right panel); CD63 (20.5%, bottom left panel); and CCL2 (60.5%, bottom right panel). FIG. 5B depicts a weighted matrix score that was assigned to each factor based on the quartile of expression, with lowest assigned a score of 0 highest assigned 3. The score for each factor was adjusted based on fold effects on target cells observed in vitro. The points assigned to each factor were summed to yield a relative score by which the therapeutic fitness of MSCs is graded.

[0013] Figure 6 demonstrates Mesenchymal stem cells inhibit effector T cell proliferation and promote Treg and M2 macrophage polarization. Panel A shows a representative flow cytometry plots showing dilution of CFSE in CD4+and CD8+T cells during co-culture of MSCs with peripheral blood mononuclear cells (PBMCs) for 4 days. Panel B shows a normalized replication index for CD4+and CD8+T cells with increasing cell numbers of MSCs for MSC donor 063 (black dots) and 043 (grey squares). Data are means ± SD. n = 3 technical replicates using one representative PMBC donor. The number of MSCs that inhibited proliferation by 50% (IC50) was determined using a nonlinear fit model Panel C shows a Flow cytometry data showing the relative frequency of FOXP3+CD25+Tregs among CD4+T cells stimulated with aCD3 / aCD28 beads + IL-2 and co-cultured with MSCs (+MSCs), without MSCs (Stim) or cultured without aCD3 / aCD28 beads (Unstim) for 4 days. Data are means ± SD. n = 3 technical replicates using one PBMC donor. Panel D shows Flow cytometry data showing the frequency of FOXP3+CD25+CD4+T cells stimulated with aCD3 / aCD28 beads + IL-2 and cultured with 50% or 12.5% MSC-CM, without MSC-CM (Stim) or cultured without aCD3 / aCD28 beads (Unstim) for 4 days. Data are means ± SD. n = 3 technical replicates using one PBMC donor. Panel D shows IL- 10 levels in the supernatant of T cells stimulated with aCD3 / aCD28 beads + IL-2 and cultured with 50% or 12.5% MSC-CM, without MSC-CM (Stim) or cultured without aCD3 / aCD28 beads (Unstim) for 3 days. Data are means ± SD. n = 3 technical replicates using one PBMC donor. Panels F and G show Flow cytometry data showing the expression levels of CD 163 (F) and CD80 (G) in monocytes differentiated to Ml macrophages, M2 macrophages, co-cultured with MSCs, or left untreated for 3 days. Data are means ± SD. n = 4 independent replicates using four different PBMC donors. Monocytes were differentiated to Ml macrophages with 50ng / mL GM-CSF and lOOng / mL IFNy and differentiated to M2 macrophages using 50 ng / mL M-CSF and lOng / mL IL- 10. Panel H shows IL- 10 levels in the supernatant of monocytes differentiated to Ml macrophages, co-cultured with MSCs or left untreated and IL-10 levels in MSCs cultured without monocytes for 3 days. Data are means ± SD. n > 4 independent replicates using three different PBMC donors. Panel I shows Flow cytometry data showing the expression levels of CD163 monocytes differentiated to Ml macrophages, M2 macrophages, cultured with 50% MSC-CM, or left untreated for 3 days. Data are means ± SD. n = 4 independent replicates using four different PBMC donors. Panel J shows IL- 10 levels in the supernatant of monocytes co-cultured with MSCs, cultured with 50% MSC-CM or left untreated for 3 days. Data are means ± SD. n = 4 independent replicates using four different PBMC donors. Panel K shows Relative RNA expression levels of IL10, TNF, and IL1B in monocytes differentiated to Ml macrophages, M2 macrophages, cultured with 50% MSC-CM, or left untreated for 24hrs. Data are means ± SD. n = 4 independent replicates using > 3 different PMBC donors. Statistical significance was determined using one-way ANOVA with Tukey’s HSD test (C, D, E, F, G, H, I, J, K). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0014] Figure 7 demonstrates the development of a robust T cell suppression surrogate assay. Panel A shows representative flow plots of intracellular IDO1 expression in MSCs co-cultured with T cells ± 3 pg / mL alFNy. Panel B shows quantification of IDO1-positive MSCs co-cultured with T cells plus varying concentrations of c / .IFNy antibody. The experiment was replicated once using the same PMBC donor and yielded similar results. Panel C shows representative flow cytometry plots of CFSE-dilution in CD8+T cells co-cultured with MSCs ± 3 pg / mL alFNy. Panel D shows replication index of CD8+ T cells co-cultured with MSCs plus varying concentrations of alFNy antibody, n > 2 technical replicates using one PBMC donor. MSC donor 063 was used for all experiments. Panel E shows percentage of proliferated CD4+T cells co-cultured with MSCs alone or additionally supplemented with an IDO1 inhibitor or tryptophan, n = 3 technical replicates using one PBMC donor. Panel F shows T cells isolated from mouse spleen were co-cultured with MSCs ± recombinant human IFNy. Representative CFSE-dilution of CD4+T cells (top) and IDO1 expression in MSCs is shown (bottom). The experiment was repeated once, yielding similar results (Statistical significance was determined using one-way ANOVA with Tukey’s HSD test (E). Pearson r was calculated, and the statistical significance was determined using a two-tailed t-test. ****P < 0.0001.

[0015] Figure 8 demonstrates that M-CSF secreted by MSCs drives M2 macrophage polarization. Panel A shows a chord diagram showing CSF1 - CSF1R interaction from publicly available scRNA-seq data set (GSE253355). The thickness of the line corresponds to the strength of the predicted interaction. GMP = granulocyte monocyte progenitor; CLP = common lymphoid progenitor; AEC = arterial endothelial cell; VSMC = vascular smooth muscle cell; SEC = sinusoidal endothelial cell. Panel B shows violin plots showing the relative expression of CSF1 in stromal cell populations (top) and CSF1R in myeloid populations (bottom). Panel C shows M-CSF levels in the supernatant of threedifferent MSC donors. Data are means ± SD. n = 5 independent replicates using four different PBMC donors. Data are means ± SD. n = 5 independent replicates using five different PBMC donors. Dot shading corresponds to monocytes derived from each donor. Pearson r was calculated, and the statistical significance was determined using a two-tailed t-test.

[0016] Figure 9 demonstrates that MSCs secrete CCL2 necessary for monocyte recruitment. Panel A shows the heatmap of the log2 fold-change of genes included in the “leukocyte migration” pathway between Ctrl, MSC-CM, Ml, and M2 macrophages. Panel B shows rrepresentative flow plots of lower chamber monocytes as in (Fig. 3E). Data are means ± SD. n = 4 independent replicates using four PBMC donors. Panels C-D show MFI of CD163, MerTK and CD80 on monocytes migrated (lower chamber) or not migrated (upper chamber) and cultured for 3 days. Data are means ± SD. n = 2 independent replicates using two PBMC donors. Statistical significance was determined using one-way ANOVA with Tukey’s HSD test (D). ***P < 0.001; ****P < 0.0001.

[0017] Figure 10 demonstrates that MSCs produce high levels of EVs that drive M2 polarization. Panel A is a heatmap showing the log2 fold-change in expression of genes included in the “phagocytosis” pathway between Ctrl, MSC-CM, Ml, and M2 macrophages. Panel B shows Flow cytometry data showing the expression levels of MerTK on monocytes differentiated to Ml macrophages, M2 macrophages, co-cultured with MSCs, cultured with 50% MSC-CM, or left untreated for 3 days. Data are means ± SD. n = 4 independent replicates using four different PBMC donors. Panel C shows Time course of CBO-labeled EV uptake and expression of CD163, MerTK, CD80, and CD14 on monocytes. Data are means ± SD. n = 2 technical replicates using one PBMC donors. Panel D shows CD163 and MerTK expression on monocytes treated with EVs isolated from MSC-CM (EVs MSC-CM) or cRPMI (EVs RPMI) for 3 days. Data are means ± SD. n = 4 independent EV preparation from one MSC donor (donor 063), using four different PBMC donors. Panel E shows IL-10 levels in the supernatant of monocytes treated with EV isolated from MSC-CM or cRPMI for 3 days. Data are means ± SD. n = 4 independent EV preparation from one MSC donor (donor 063) and using four different PBMC donors. Panel F shows CD 163 and MerTK expression and IL- 10 levels at different concentrations of EVs. Data are means + SD. n = 2 independent experiments using two different PBMC donors. Panel G shows M-CSF ELISA levels of EV FT or EVs from MSCs. Data are means ± SD. n = 4 independent experiments using four different PBMCdonors. Panel H shows CD163 MFT of monocytes incubated with EVs isolated from cRPMT, MSC-EVs (Ctrl) or MSC-EVs plus an aM-CSF antibody, CSFIRi, an aMerTK antibody, or MerTKi. Data are means ± SD. n = 4 independent experiments using four different PBMC donors. Statistical significance was determined using one-way ANOVA with Tukey’s HSD test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0018] Figure 11 demonstrates that MSC-derived EVs drive Treg polarization. Panel A shows IE- 10 levels in the supernatant of T cells incubating with a range of EV numbers or without EVs. Data are means ± SD. n = 3 technical replicates using one PBMC donors. Panel B shows Replication index ratio of FoxP3-positive vs. FoxP3-negative CD4+ T cells treated with different numbers of EVs or without EVs. Data are means ± SD. n = 3 technical replicates using one PBMC donors. Panel C shows Representative imaging flow cytometry images of CD4+ and CD8+ T cells incubated with CBO-labeled EVs for 24 hrs. Panel D shows ELISA TGFpi levels on EVs (4 x 1011EVs used for ELISA) from MSC-CM derived from three different MSC donors. Data are means ± SD. n = 3 technical replicates using three independent EVs preparations per MSC donor. Panel E shows the Frequency of FoxP3+ cells among CD4+ T cells treated with 4 x 1011EVs for 3 days in combination with 5 pM SB431542 or 1 pM A83-01 or vehicle control (DMSO) or incubated without EVs (Ctrl). Data are means ± SD. n = 3 technical replicates using one PBMC donors. Panel F shows the Frequency of FoxP3+ cells among CD4+ T cells treated with or without 4 x 1011EVs for 3 days ± 5 pg / mL anti-TGFpi antibody or isotype control. Data are means ± SD. n > 3 technical replicates using > 3 different PMBC donors. Statistical significance was determined using one-way ANOVA with Tukey’s HSD test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0019] Figure 12 demonstrates the development of a potency matrix to enhance consistency of MSCs as cell therapy product. Panel A shows the potency matrix whereby 0 to 3 points are assigned to each MSC donor for each potency factor based on quartiles. Panel B shows the Weighted potency matrix whereby 2 points per quartile are assigned for IDO1 and EV scores and V2 point per quartile for CCL2 scores.DETAILED DESCRIPTION

[0020] Reference is made to particular features and / or non-limiting embodiments of the disclosure. It is to be understood that the disclosure in this specification includes allpossible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment, or a particular claim, that feature may also be used, to the extent possible, in combination with and / or in the context of other particular aspects and embodiments, generally.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by those of skill in the art. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety unless stated otherwise. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

[0022] Low or variable control of manufacturing parameters and inconsistency of primary seed stocks have been identified as major factors driving variability between cellular therapy clinical trial lots. Provided herein are allogeneic MSC therapies using a carefully engineered and controlled manufacturing processes that maintain enhanced potency. A key driver for controlling variability is the development of a matrix of potency factor assays to measure inter-batch consistency.

[0023] One objective of the present disclosure is to develop a panel of potency assays to measure factors important for activated lymphocyte suppression. In some embodiments, the panel of potency assays to measure factors associated with suppression of activated lymphocytes includes polarization of macrophages toward anti-inflammatory phenotypes, and stimulation or expansion of regulatory T cells (Tregs). For example, indoleamine 2,3-dioxygenase-l (IDO-1) expression or enzymatic activity may be assessed as a correlate of T-cell suppression; macrophage-colony stimulating factor (M-CSF) secretion levels may be quantified as an indicator of macrophage polarization capacity; and extracellular vesicle (EV) production, composition, or functional activity may be evaluated as a surrogate for Treg induction. Assays may include, without limitation, quantitative PCR, immunoassays, flow cytometry-based functional co-culture systems, enzyme activity measurements, proteomic profiling, and nanoparticle tracking analysis of vesicle populations. Another objective is to establish threshold levels for each of the factors which allows grading different sources of MSCs as well as end of production lots for the appropriate potency levels.

[0024] Mesenchymal stem / stromal cells (MSCs) are multipotent progenitor cells capable of differentiating into a variety of cell types, including osteoblasts, chondrocytes, andadipocytes. Originally identified in the bone marrow, MSCs have since been isolated from a wide range of tissues such as adipose tissue, umbilical cord, and dental pulp. Beyond their regenerative potential, MSCs possess robust immunomodulatory properties, influencing both innate and adaptive immune responses through the secretion of cytokines, growth factors, and by direct cell-cell interactions. These characteristics have positioned MSCs as promising therapeutic agents in a variety of inflammatory and immune-mediated disorders. While the plethora of immunoregulatory properties possessed by MSCs suggests therapeutic potential as a cellular therapy, it is important to validate the exact mechanism of action in each disease context.

[0025] A landmark for MSC therapies occurred in 2004 with the clinical demonstration that bone marrow-derived MSCs (BM-MSCs) were effective in treating steroid-refractory acute Graft- versus-Host Disease (SR-aGvHD). Over the last two decades, the immunomodulatory and tissue repair properties of MSCs have been explored therapeutically in a range of diseases characterized by extreme immune system dysregulation causing pathological tissue damage. Multiple early phase clinical trials with small numbers of patients have demonstrated a strong safety profile and favorable response rates for a variety of indications. However, larger studies have produced mixed results. The variation in outcomes is likely driven by multiple factors, including differences in MSC expansion protocols, cell dose, patient populations, donor and source tissue variability, and whether the cells were cryopreserved or freshly cultured at the time of administration.

[0026] The general lack of predictive disease biomarkers and disease-relevant potency assays remain major obstacles, underscoring the need for further research into the mechanism driving clinical responses. Furthermore, the U.S. Food and Drug Administration (FDA) requires the implementation of validated potency assays for cell-based therapies to demonstrate biological activity and ensure consistent manufacturing quality. These assays should measure cellular functions related to the mechanism of action for addressing the intended clinical indication and incorporate orthogonal assays to measure different dimensions of potency. The lack of clear and correlative potency assays has plagued previous late-stage trials - most notably Mesoblast’s SR-aGVHD trial with remestemcel-L. These issues were eventually resolved, but at the expense of much time and. presumably, money, with FDAclearance in December 2024 of a biological license application for remestemcel-L for treatment of SR-aGVHD in pediatric patients.

[0027] Aspects of the present disclosure relate to BM-MSCs derived from deceased organ donor vertebral body bone marrow as a cellular therapy for inflammatory diseases. This BM-MSC product has been optimized for high post-thaw viability through cell cycle synchronization prior to cryopreservation which prevents lethal DNA damage. Furthermore, immediate post-thaw immunomodulatory function is enhanced by priming the cells with interferon-' / (IFNy) prior to cryopreservation. To facilitate clinical development, initially for GvHD, immunomodulatory properties were first evaluated and then assays that are compatible with a Good Manufacturing Practices (GMP)-compliant manufacturing and testing environment were developed.

[0028] In this study, it is demonstrated that bone marrow-derived MSCs from deceased donors robustly suppress T cell proliferation, promote regulatory T cell (Treg) differentiation, and induce anti-inflammatory M2 macrophage polarization. These effects were mediated through multiple distinct mechanisms. Surrogate potency assays were further identified and validated based on these immunomodulatory mechanisms, establishing a quantitative matrix to evaluate lot-to-lot consistency. This matrix provides a foundation for correlating in vitro potency with clinical outcomes in future trials.Mesenchymal stromal cells (MSCs)

[0029] Mesenchymal stromal cells (MSCs) are cells of non-hematopoietic stromal origin that reside in bone marrow as well as in a variety of tissues including adipose and placenta. MSCs have potent immunosuppressive activity mediated via a variety of cell-to-cell contacts and soluble factors such as Indoleamine 2,3 -dioxygenase (IDO), PGE2, TSG-6, CCL-2 and PD-L1, and TGFp. These mediators inhibit T and B lymphocyte. NK cell and dendritic cell activation and function. MSCs also facilitate endogenous tissue repair and regeneration through secretion of cytokines such as VEGF, IL-6, IL-11, GM-CSF and SCF. As MSCs seem to be hypoimmunogenic and do not express costimulatory molecules, they can be infused into major histocompatibility complex disparate recipients.

[0030] MSCs have been widely exploited for their potent immunosuppressive activity in a variety of therapeutic indications, including inflammatory conditions, includingimmune -modulating conditions like Graft-versus-Host-Disease (GvHD). Particularly GvHD following an organ and / or cellular transplant.

[0031] Effective cryopreservation would enable more robust cellular therapies utilizing MSCs. Previous reports of attenuated fitness of MSCs post cryopreservation and thaw have speculated that intrinsic properties of MSCs were likely modified by the cryoprotectant system. Historically dimethyl sulfoxide (DMSO) has been the most widely used cryoprotectant for MSCs, and several reports have focused on potential direct effects of the DMSO on post thaw outcomes . To some degree, these reports have pointed to improved intrinsic functions of MSCs and viability post -thaw, however interactions with immune responders and functional fitness has not been demonstrated, and delayed onset cell death due to apoptosis, is still observed.

[0032] Recent work evaluating proliferating cell lines has pointed to DNA defects and alterations of higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. These studies pointed out that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance and can lead to apoptosis. Thus, it is hypothesized that such DNA damage could be the primary driver of the delayed onset death which was observed in post thaw culture.

[0033] Thus, modifying the cell cycle process immediately prior to cryopreservation to prevent cells from entering the S phase would mitigate cryoinjury. Serum starvation is a mechanism for blocking the cell cycle at the G1 to S phase transition for studying cell cycle. The process involves transferring cells grown in complex medium containing blood components to provide growth factors to a medium that substantially lacks these factors. Here, culture medium containing fetal bovine serum (FBS) or equivalent is removed and replaced with a medium containing no or very low levels (e.g., 1 % or less) of FBS. The cells are not able to progress past G1 to S phase and are thus effectively blocked at this transition point. For this reason, cells in the culture are synchronized at the same phase.

[0034] The compositions and methods provided herein dramatically reduced the percentage of MSCs in S phase from 25% to <2% (FIGs. 4A-4H). These techniques can greatly improve current cryopreservation methods for storage MSCs for pharmaceutical use.

[0035] Further, the immunoregulatory properties of MSCs are also impacted by inflammatory processes. For example, immunosuppression by MSCs is enhanced after exposure to cytokines such as IFN-y and TNFa. For this reason, a number of priming approaches have been employed to improve the efficacy of MSC therapies for clinical application. For example, in certain embodiments, multi-cytokine priming of human MSCs with TNF-a, IL-ip, and IFN-y enhances NF-KB, STAT5, and p38-MAPK signaling pathways and augments recruitment of polymorphonuclear granulocytes. Others have similarly shown that priming of MSCs with cytokines enhances the downregulation of proinflammatory cytokines, induces regulatory T cells, inhibits T-cell proliferation, and polarizes M2 -type macrophages.

[0036] MSCs have potent immune modulatory activity, including prophylactic activity of asthma, which is markedly enhanced by exposure to IFN-y. In vivo, MSCs are stimulated in response to abnormal conditions, e.g., caused by infection, cancer, and injury. The presence of circulating IFN-y can be a signal to MSCs which identifies an abnormal condition. In response to the circulating IFN-y, the MSCs are primed and secrete, at least, factors and proteins that help remedy the abnormal condition. In various aspects of the present disclosure, MSCs are primed in vitro or ex vivo by contact with IFN-y to transform the cells into yMSCs. As used herein, IFN-y -primed MSCs are referred to as “yMSCs”.

[0037] Although several MSC priming approaches have been used, priming with IFN-y has been widely studied and enhances the immunosuppressive properties of the MSCs. yMSCs upregulate expression of immunosuppressive factors such as IDO and secrete PGE2, HGF, TGFp and CCL2. yMSCs also inhibit T cell effector functions and suppress NK activation more efficiently than non-primed MSCs. In an experimental animal model of colitis, yMSCs also had a higher rate of migration to sites of inflammation and showed greater efficacy in the reduction of mucosal damage and inflammatory responses compared to non-primed bone marrow MSCs.

[0038] In the present disclosure, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in this disclosure, including the drawings and claims, are not meant to be limiting. Some embodiments may be utilized, and other changes may be made, without departing from the spirit or scope ofthe subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs when read in light of the current disclosure.

[0040] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0041] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby. Although certain dependent claims may be described as depending from a single preceding claim, it is to be understood that each such dependent claim may alternatively be construed as depending from any one of the preceding claims, whether independent or dependent, unless expressly stated otherwise. All such alternative dependencies and combinations are expressly contemplated as part of the disclosure.

[0042] Use of absolute or sequential terms, for example, “will,” “will not,” “shall,” “shall not,” “must,” “must not,” “first,” “initially,” “next,” “subsequently,” “before,” “after,” “lastly,” and “finally,” are not meant to limit scope of the present embodiments disclosed herein but as illustrative.

[0043] The terms “first,” “second,” and “third” used in combination with substances are intended to designate distinguishable features to similar substances and do not imply any particular order unless otherwise specified.

[0044] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereofare used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0045] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0046] As used herein, the phrases “at least one”, “one or more”, and “and / or” are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and / or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

[0047] As used herein, “or” may refer to “and”, “or,” or “and / or” and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning.

[0048] The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and the number or numerical range may vary from, for example, from 1% to 15% of the stated number or numerical range. In examples, the term “about” refers to ±10% of a stated number or value. In some instances, the term “about” refers to one standard deviation greater or less than the stated number or value.

[0049] The term “from” as in “from 1 to 10” includes the initial and final number recited. Therefore, “from 1 to 10” includes the whole numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and includes fractions thereof, (e.g., about .1, .2, .3. .4, .5, .6, .7. .8, and about .9).

[0050] The terms “increased”, “increasing”, or “increase” are used herein to generally mean an increase by a statically significant amount. In some aspects, the terms “increased.” or “increase.” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least ab out 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase from about 10 to about 100% as compared to a reference level, standard, or control. Other examples of “increase” include an increase of at least 2 -fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.

[0051] The terms “decreased”, “decreasing”, or “decrease” are used herein generally to mean a decrease by a statistically significant amount. In some aspects, “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease e.g., absent level or non-detectable level as compared to a reference level), or any decrease from about 10 to about 100% as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%. at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.

[0052] The terms “individual”, “subject”, or “patient” as used herein have their plain and ordinary meaning as understood in light of the specification, and may include a human or a non-human mammal, e.g., a dog. a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate, or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate. The term “mammal” is used in its usual biological sense. Thus, it includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or the like.

[0053] As used herein, the term “isolated” has its plain and ordinary meaning as understood in light of the specification, and may include a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from equal to, about, at least, at least about, not more than, or not more than about, 10%. about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated (or ranges including and / or spanning the aforementioned values). In some embodiments, isolated agents are, are about, are at least, are at least about, are not more than, or are not more than about 80%, about 85%. about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure (or ranges including and / or spanning the aforementioned values). As used herein, a substance that is “isolated” may be “pure” (e.g., substantially free of other components). As used herein, the term “isolated cell” may refer to a cell not contained in a multi-cellular organism or tissue.

[0054] As used herein, “in vivo” has its plain and ordinary meaning as understood in light of the specification and may include to the performance of a method inside living organisms, usually animals, mammals, including humans, and plants, as opposed to a tissue extract or dead organism.

[0055] As used herein, “ex vivo” has its plain and ordinary meaning as understood in light of the specification and may include to the performance of a method outside a living organism with little alteration of natural conditions.

[0056] As used herein, “in vitro” has its plain and ordinary meaning as understood in light of the specification and may include to the performance of a method outside of biological conditions, e.g., in a petri dish or test tube.

[0057] The term “purity” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and may include an actual abundance of the substance, compound, or material relative to the expected abundance. For example, the substance, compound, or material may be at least 80. 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between. Purity may beaffected by unwanted impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membrane, cell debris, small molecules, degradation products, solvent, carrier, vehicle, or contaminants, or any combination thereof. In some embodiments, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious agents. Purity can be measured using technologies including but not limited to electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectrometry, infrared spectrometry, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.

[0058] The term “yield” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and may include an actual overall amount of the substance, compound, or material relative to the expected overall amount. For example, the yield of the substance, compound, or material is, is about, is at least, is at least about, is not more than, or is not more than about, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected overall amount, including all decimals in between. Yield may be affected by the efficiency of a reaction or process, unwanted side reactions, degradation, quality of the input substances, compounds, or materials, or loss of the desired substance, compound, or material during any step of the production.

[0059] The terms “function” and “functional” as used herein have their plain and ordinary meaning as understood in light of the specification, and may include a biological, enzymatic, or therapeutic function.

[0060] The term “inhibit” as used herein has its plain and ordinary meaning as understood in light of the specification, and may include the reduction or prevention of a biological activity. The reduction can be by a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount that is within a range defined by any two of the aforementioned values. As used herein, the term “delay” has its plain and ordinary meaning as understood in light of the specification, and refers to a slowing, postponement, or defermentof a biological event, to a time which is later than would otherwise be expected. The delay can be a delay of a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 10%, 20%, 30%. 40%, 50%, 60%. 70%, 80%, 90%, 100%. or an amount within a range defined by any two of the aforementioned values. The terms inhibit and delay may not necessarily indicate a 100% inhibition or delay. A partial inhibition or delay may be realized.

[0061] As used herein, the terms “treating” or “treatment” have their plain and ordinary meaning as understood in light of the specification, and may include an approach for obtaining beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (e.g., not worsening) the state of disease, prevention of a disease's transmission or spread, delaying or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the recurrence of disease, and remission, whether partial or total and whether detectable or undetectable. “Treating” and “treatment” as used herein also include prophylactic treatment. Treatment methods include administering to a subject a therapeutically effective amount of an active agent. The administering step may include a single administration or may include a series of administrations. The compositions are administered to the subject in an amount and for a duration sufficient to treat the subject. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age and genetic profile of the subject, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required.

[0062] The term “administering” may include oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intra-tumoral, intrathecal, intranasal, or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal,vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intra-tumoral, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “coadminister” it is meant that a first compound described herein is administered at the same time, just prior to, or just after the administration of a second compound described herein.

[0063] The term “% w / w” or “% wt / wt” as used herein has its plain and ordinary meaning as understood in light of the specification and may include a percentage expressed in terms of the weight of the ingredient or agent over the total weight of the composition multiplied by 100. The term “% v / v” or “% vol / vol” as used herein has its plain and ordinary meaning as understood in the light of the specification and refers to a percentage expressed in terms of the liquid volume of the compound, substance, ingredient, or agent over the total liquid volume of the composition multiplied by 100.

[0064] As used herein, IFN-y -primed MSCs are referred to as “yMSCs”.

[0065] Some embodiments provided herein may be described in the following enumerated alternatives.

[0066] 1. A method of developing a potency assay matrix for use in monitoring the quality of a manufactured cell, the method comprising: testing the manufactured cell for activity in one or more screening factors; and assigning a value to the activity for each screening factor; wherein the value for each screening factor is combined to form the potency assay matrix.

[0067] 2. The method of alternative 1, wherein the one or more screening factors comprise at least one of: immune cell migration, immune cell chemotaxis, phagocytosis, leukocyte mediated immunity, activated lymphocyte suppression, macrophage polarization, regulatory T cell stimulation, or any combination thereof.

[0068] 3. The method of alternative 2, wherein the one or more screening factors comprise at least one of: activated lymphocyte suppression, macrophage polarization, regulatory T cell stimulation, or any combination thereof.

[0069] 4. The method of alternative 2 or 3, wherein the one or more screening factors comprise at least one of: leukocyte migration, leukocyte chemotaxis, myeloid leukocytemigration, granulocyte migration, neutrophil chemotaxis, neutrophil migration, mononuclear cell migration, lymphocyte migration, or any combination thereof.

[0070] 5. The method of any one of alternatives 1-4, wherein the manufactured cell is a stem cell or a stromal cell.

[0071] 6. The method of alternative 5, wherein the manufactured cell is a Mesenchymal Stem / Stromal Cell (MSC).

[0072] 7. A method of insuring inter-batch consistency across a group of manufactured cells in a cell batch, the method comprising: administering the potency assay matrix of any one of alternatives 1-6; and removing from the cell batch any manufactured cell that does not reach a suitable activity value in at least one screening factor.

[0073] 8. A method of determining the quality of a stored cell, the method comprising: measuring the expression level of an at least one gene involved in an at least one activity pathway in a fresh cell; and measuring the expression level of the at least one gene in the stored cell; wherein no significance in expression levels between the fresh and stored cell is indicative of a good quality of the stored cell, and a significant difference in expression level is indicative of a poor quality of the stored cell.

[0074] 9. The method of alternative 8, wherein the stored cell is cryopreserved.

[0075] 10. The method of alternative 8 or 9, wherein the stored cell is a stem cell or a stromal cell.

[0076] 11. The method of alternative 10, wherein the stored cell is an MSC.

[0077] 12. The method of any one of alternatives 8-11, wherein the at least one activity pathway comprises at least one of: immune cell migration, immune cell chemotaxis, phagocytosis, leukocyte mediated immunity, activated lymphocyte suppression, macrophage polarization, regulatory T cell stimulation, or any combination thereof.

[0078] 13. The method of any one of alternative 8-12, wherein the at least one gene comprises at least one of: VAV1, IDO-1, TREM1, S1PR1, MY01G, ITGA7, EDNRB, CCL18, ADAM8, CCL2, CSF1, CD63, or any combination thereof.

[0079] 14. The method of alternative 13, wherein the at least one gene comprises 1, 2, 3, or 4 of: IDO1, CSF1, CD63, CCL2, or any combination thereof.

[0080] 15. The method of any one of alternative 8-14, wherein when the stored cell is of poor quality, the expression level of the at least one gene is significantly reduced in the stored cell compared to the fresh cell.EXAMPLES

[0081] The following illustrative examples are representative of embodiments of the methods and compositions described herein and are not meant to be limiting in any way.Example 1: Development of Potency Assay Matrix

[0082] MSCs were recovered from deceased human organ donor vertebral bone marrow and cultured. T cells and monocytes were isolated from Ficoll-purified peripheral blood mononuclear cells (PBMCs) using Stem Cell Technologies EasySep human cell isolation kits. T cell suppression assays were performed by CSFE-labeled total T cells that were activated with CD3 / CD28 Dynabeads. Monocytes (80x103) were cultured in RPMI / 10%FBS, differentiated to Ml macrophages using 50ng / mL GM-CSF and lOOng / mL IFN-y, or differentiated to M2 macrophages using 50ng / mL CSF1 and lOng / mL IL-10 or cultured with 40xl03MSCs or 50% MSC-CM for 3 days.

[0083] For EV production, MSCs were cultured at a seeding cell density of 4.5x105cells per well of a 6-well plate for 72hrs. 6mL of cleared supernatant was loaded onto a Vivaspin 6 Centrifugal Concentrator with a pore size of 300,000 MWCO (Vivaproducts, #VS0652) and centrifuged for 50 mins at 4,500g at RT. A sample from the flow-through was taken and 3mL PBS were added to the top to wash EV and the sample was centrifuged again at 4,500g for 30mins at RT. EVs were resuspended to a final volume of 400pL in RPMF10%FBS and stored at -80°C until further processing. EV numbers were determined using the ZetaView and a CD63 ELISA. Single cell suspensions were stained with the appropriate antibodies. DAPI was added immediately before acquisition. For intracellular staining, cells were stained for viability using Ghost Dye Violet 450 or Fixable Viability Dye eFluo 780, followed by surface antibody staining. Next, cells were fixed with IC Fixation Buffer, followed by permeabilization and stained with antibodies.

[0084] For bulk RNA-seq of monocytes, freshly thawed cells were cultured at a concentration of 106cells per well of a 6-well plate in 2 mL for 24hrs. Monocytes were eithercultured with 50% MSC-CM, 50 ng / mL GM-CSF plus 100 ng / mL IFN-y, 50ng / mL CSF1 plus 10 ng / mL IL-10, or left untreated. RNA was isolated processed by poly-A-enrichment-based library preparation for sequencing. Aligned FPKM reads were loaded into R. Differentially expressed genes were identified using the DESeq2 package. Gene ontology analysis was performed using clusterProfiler.

[0085] Isolated MSCs from different donors showed significant variation in potency factor expressing (FIGs. 1A-1D). The data were used to establish threshold levels of IDO-1 required for potency. It was also observed that MSC-derived CSF1 (M-CSF) potently polarizes macrophages to an M2 anti-inflammatory phenotype, and that M2 polarization of 2 donor monocytes were correlated (FIGs. 2A-2H). MSCs were also shown to secrete chemokine CCL2 attracted monos / macros. which were then polarized (FIGs. 3A-3E). MSC extracellular vesicles (EVs) induced Tregs and macrophage M2 polarization (FIGs. 4A-4H).

[0086] From this data, a weighted matrix score was developed to select MSCs with optimal potency (FIGs. 5A-5B). A weighted matrix score was assigned to each factor based on the quartile of expression, with lowest assigned a score of 0 highest assigned 3. The score for each factor was adjusted based on fold effects on target cells observed in vitro. The points assigned to each factor were summed to yield a relative score by which the therapeutic fitness of MSCs is graded.

[0087] As disclosed above, a robust potency matrix for grading MSC functionality has been generated. The factors comprising the matrix have been shown to directly modulate inflammatory processes involved in relevant diseases. This potency matrix is suitable as a release criterion for manufactured MSCs to ensure inter-batch consistency. The matrix can also be used to screen MSCs isolated from potential donors.

[0088] It is envisioned that the matrix can be further refined by adding additional potency factors as they are discovered and validated. Ideally, the factor chosen would be relevant for full MSC immunomodulation in clinically relevant disease models. It is also envisioned that the potency assay of the present disclosure can be incorporated during manufacture of a cell for use in therapy. For example, the assay can be used in the manufacturing of individual cell lots for MSC therapy.Example 2: Development and Validation of a Potency Assay Matrix for Optimized and Consistent Manufacture of Clinical Mesenchymal Stem Cells

[0089] Introduction: Mesenchymal stem / stromal cells (MSCs) are being evaluated as cell-based therapies for inflammatory and immune-mediated disorders. However, variability in clinical efficacy and a lack of validated potency assays have impeded regulatory approval for commercialization. Disclosed herein is the development of a matrix of potency assays for evaluating the therapeutic fitness of bone marrow-derived MSCs and demonstrate that the cells consistently suppress T cell proliferation, induce regulatory T cell differentiation, and polarize monocytes into anti-inflammatory M2 macrophages.

[0090] Methods: Vertebrae were recovered from consented and screened organ donors by Organ Procurement Organizations and shipped on ice to a central processing facility for isolation of vertebral body bone marrow. MSCs were cultured in a xenogeneic-free medium and characterized based on established markers and expanded for 4 passages. Modulation of immune cells isolated from peripheral blood was evaluated using T cell suppression assays, macrophage polarization, regulatory T cell (Treg) induction and monocyte / macrophage chemoattraction assays.

[0091] Results: Mechanistic studies revealed that potency is mediated by MSC-secreted immunoregulatory molecules, including macrophage colony- stimulating factor (M-CSF), transforming growth factor-pi (TGFpi), and the chemokine CCL2, as well as by tryptophan depletion via the cytoplasmic protein indoleamine 3,4 dioxygenase- 1 (IDO1). Additionally, it was shown that MSCs secrete high levels of extracellular vesicles which potently induce an anti-inflammatory phenotype in T cells and monocytes. These findings were employed to develop a matrix of surrogate potency assays which consistently demonstrated predicted in vitro functionality of MSCs derived from 10 donors.

[0092] Discussion: This potency assay platform provides a critical tool for ensuring the quality and consistency of MSC products and will facilitate clinical translation by demonstrating comparability between MSC donors as well as manufactured lots and potentially predicting therapeutic efficacy in clinical trials.Materials and MethodsCells and cell culture

[0093] Deceased donor-derived vertebral bone marrow MSCs were isolated, cultured and expanded utilizing published methods (Johnstone et al., 2020). Individual MSC donor characteristics are presented in Supplemental Table 1. Conformance to the International Society of Cellular Therapy (ISCT) standards for MSC characterization (Dominici et al., 2006) (e.g., surface marker expression, colony forming capacity and trilineage differentiation) was previously established (Johnstone et al„ 2020) and presented again here for the two main donor MSCs (identified as 046 and 063) used in the present studies. Unless stated otherwise, experiments were performed with MSCs derived from donor 063.

[0094] MSC-CM was produced by culturing freshly thawed MSCs for 3 days in RPMI / 10%FBS at a seeding cell density of 450 x 103cells per well of a 6-well plate (VWR, 820050-844). To remove cells and cell debris, the supernatant was spun at 2,000 g for 10 mins. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats (Versiti Blood Center, Indianapolis, IN, USA) using Ficoll-Paque® (Cytiva, Marlborough, MA, USA) density gradient centrifugation. To account for biological variability, peripheral blood from a total of 22 donors was collected for isolation of PBMCs used in this study (supplemental Table 1). T cells were isolated from PBMCs using the EasySep Human T Cell Isolation Kit (SCT, 17951) according to manufacturer’s instructions. Monocytes were isolated from PBMCs using the EasySep Human Monocyte Isolation Kit (SCT, 19359) according to manufacturer’s instructions. CD4+ T cells were isolated using the EasySep Human CD4+ T cells isolation kit (SCT, 17952). PBMCs and T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE; BD Bioscience) at a concentration of 5 pM per 20 x 106cells for PBMCs and 2.5 pM per 20 x 106cells for T cells. Cells were incubated for 15 mins at 37 °C, washed twice with access RPMI / 10%FBS, followed by cryopreservation in 90% FBS / 10% Me2SO.

[0095] Spleens from C57BL / 6 mice were mechanically dissociated through a 40 pm pore size nylon cell strainer to obtain single-cell suspensions. Cells were labeled with CFSE and cryopreserved in freezing medium containing 10% DMSO and 90% FBS for later use.

[0096] For co-culture studies, thawed MSCs were seeded in RPMI / 10% FBS at the indicated cell densities using either a 96- or 48-well plate (VWR, 734-2327 and 10062-898, respectively). PBMCs were activated using an aCD3c antibody (Bio X Cell, Lebanon, NH, USA) at a concentration of 150 ng / mL and an aCD28 antibody (InVivoMab, BE0248) at aconcentration of 1 pg / mL (see supplemental Table 2 for a complete list of antibodies used in this study). Purified T cells were activated using Dynabeads™ Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (ThermoFisher, 1116 ID) at a ratio of one conjugated bead per cell. IL-2 (PeproTech, 200-02- 10UG) was added where indicated.

[0097] For experiments with purified EVs, purified T cells were plated and stimulated as above and then EVs or cRPMI control at the indicated concentrations were added. For Treg induction experiments, cells were harvested after 3 days and stained for intracellular (FoxP3) or extracellular (CD25 and C127) protein expression as quantified by flow cytometry (see supplemental Table S2 for a list of conjugated antibodies). Cell-free supernatants were also collected for ELISA.

[0098] For activation of mouse T cells, isolated splenocytes were stimulated with Dynabeads® Mouse T-Activator CD3 / CD28 (Gibco, 11452D) at a cell-to-bead ratio of 2:1. A total of 2 x 105splenocytes per well were cultured in 96-well plates in RPMI-1640 supplemented with 10% FBS and 50 pM 2-mercaptoethanol for 4 days.

[0099] Monocytes were cultured by seeding 80 x 103cells in RPMI / 10%FBS in 96-well plates and differentiated to either Ml macrophages using 50 ng / mL GM-CSF (Thermo Fisher, 300-03-50UG) and 100 ng / mL IFNy (R&D Systems, 285-IF-100 / CF), or M2 macrophages using 50 ng / mL M-CSF (ThermoFisher, PHC9504) and 10 ng / mL IL-10 (ThermoFisher, 200-10-50UG) or cultured with 40 x 103MSCs or 50% MSC-CM for 3 days using 96-well plates. Where indicated, 2 pg / mL M-CSF neutralizing antibody (R&D systems, MAB216-100), 2 pg / mL MerTK antibody (R&D Systems, AF891), 50 nM CSF1R inhibitor (Selleckchem, S7725) or 2.5 pM MerTKi (Selleckchem, S7342) was added.T cell suppression assays

[0100] MSCs were co-cultured with PBMCs or purified T cells for at 37°C for 3 or 4 days at the indicated cell densities using either 96- or 48-well plates. Cell proliferation was determined by CSFE dilution by flow cytometry and the replication index was calculated (see Source Data for formula). For IFNy neutralization, an IFNy-neutralizing antibody (BioXcell, BE0235, clone B 133.5) was added to the co-culture system at various concentrations, as indicated in the Results section. IDO1 inhibitor 1-MethyLL-tryp tophan (Sigma- Aldrich,447439) were added at a concentration of 500 pM and Trp (Sigma-Aldrich, T0254) was supplemented at a concentration of 5 mg / L.Flow cytometry

[0101] Single cell suspensions were stained with the appropriate antibodies (Supplemental Table S2) in MACS buffer (0.5% BSA, 2mM EDTA, PBS) for 15-30 mins on ice in the dark. DAPI was added immediately before acquisition. For intracellular staining, cells were stained for viability using Ghost Dye Violet 450 (Cytek, 13-0863-T100) or Fixable Viability Dye eFluo 780 (ThermoFisher, 65-0865-14) for 10 mins on ice in the dark, followed by surface antibody staining for 30mins on ice in the dark. Next, cells were fixed with IC Fixation Buffer (ThermoFisher, 88-8824-00) for 15mins at RT, followed by permeabilization with Perm Buffer (ThermoFisher, 88-8824-00) for 5mins at RT and stained for Ihr with antibodies at RT. A NovoCyte 2060R flow cytometer (Agilent Technologies, Santa Clara, CA, USA) was used for flow cytometry data acquisition. Data was analyzed using NovoExpress software (version 1.5.6).

[0102] For intracellular IDO-1 assessment, MSC were fixed and permeabilized using eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (ThermoFisher Scientific) and stained with APC conjugated anti-IDO-1 antibody (clone eyedio; Invitrogen) according to manufacturer’s instruction. Labelled cells were analyzed via flow cytometry.

[0103] Flow cytometry data were pre-gated to exclude debris and doublets using forward and side scatter parameters (FSC-H vs SSC-H; FSC-A vs FSC-H; SSC-A vs SSC-H), and non-viable events were excluded using a viability dye where applicable. Compensation and gating were established using single- stained controls and fluorescence-minus-one (FMO) controls for each fluorochrome in combination with appropriate controls (e.g. nondifferentiation or non-stimulated cells), as well as isotype controls for intracellular stainings. Data were acquired under identical instrument settings across experiments, with minimum event thresholds applied per sample.Enzyme-Linked Immunosorbent Assay

[0104] IL- 10 concentration in the supernatants was evaluated using the IL- 10 ELISA kit (Invitrogen, 88-7106-88) following manufacturer’s instructions. M-CFSconcentration in the supernatants was evaluated using the M-CSF (CSF1) ELISA kit (Invitrogen, EHCSF1). CCL2 concentration in the supernatants was evaluated using the MCP-1 / CCL2 ELISA kit (Invitrogen, 88-7399-22). CD63 concentration in the supernatants and extracellular vesicles (EVs) was evaluated using the CD63 ELISA kit (Invitrogen, EH95RB). TGFpi concentration was evaluated using the TGFpi ELISA kit (R&D Systems, DB100C) following manufacturer’s instructions. TGFpi was activated by adding 20 pL IM HC1 to 200 pL pre-diluted samples and an incubation for 1 hour at RT, followed by neutralization with 20 pL IM NaOH. Where indicated, samples were not activated (data not shown). For measurement of M-CSF and TGFP on EVs were isolated and enriched as described below.Transwell migration assay

[0105] 16hrs before the assay, 300,000 MSCs were seeded per well of a 24- well plate (Coming, 3398, 3 pm pore size) without the insert in 500 pL cRPMI or 500 pL MSC-CM was added per well without the insert. Ihr before the addition of monocytes 2 pg / mL anti-CCL2 (R&D Systems, MAB679) or 75 ng / mL recombinant CCL2 (ThermoFisher, 300-04) were added where indicated. Insert was carefully inserted to the well plates and 120,000 monocytes were added to the insert in 200 pL cRPMI. A control of untreated cells was included. After Ihr, the insert was carefully removed, the cells in the well plate were vigorously resuspended, followed by rinsing of the well plate and processed via flow cytometry to determine the number of migrated monocytes. To determine polarization, the lower well cells (migrated cells) were placed back into the incubator. The monocytes attached to the insert (none-migrated cells) were vigorously resuspended, and the transferred into a new well of a 24-well plate and cultured in 500 pl cRPMI. After 48hrs, the cells were harvested and processed via flow cytometry to determine monocyte polarization.Isolation and quantification of EVs

[0106] MSCs were cultured at a seeding cell density of 4.5 x 105cells per well of a 6-well plate for 72hrs. The supernatant was carefully harvested and centrifuged at 1,000 g for 10 mins to remove cells and cell debris. 6 mL of cleared supernatant was loaded onto a Vivaspin 6 Centrifugal Concentrator with a pore size of 300,000 MW cut-off (Vivaproducts, VS0652) and centrifuged for 50 mins at 4,500 g at RT. A sample from the flow-through wastaken and 3 mL PBS were added to the top to wash EVs and the sample was centrifuged again at 4,500 g for 30 mins at RT. EVs were resuspended to a final volume of 400 uL in RPMI / 10%FBS and stored at -80°C until further processing. EV numbers were determined using the ZetaView (Particle Metrix) and a CD63 ELISA (ThermoFisher, EH95RB). Acquisition settings or the ZetaView were sensitivity: 85; shutter speed: 70; frame rate: 30 fps. Additionally, for flow cytometry staining, EVs were incubated with CD63 capture beads (Abeam #ab239686, clone TEA3 / 18) according to manufacturer’s instructions in PBS at RT overnight, followed by staining of EVs for PE / Cy7 anti-human CD63 (BioLegend #353010, clone H5C6) and FITC anti-human CD73 (BioLegend 344016, clone AD2).

[0107] EVs were further characterized by Western blot for the presence and absence of discrete proteins according to recommendations MISEV2023 (Welsh et al., 2024). EVs were analyzed by Western blot to confirm protein marker expression. EV and MSC lysates were prepared in 4x Laemmli buffer containing lOOx protease inhibitors under non-reducing conditions and heated at 70°C for 10 min. Samples of equal amount of total protein (15ug) were separated by 4-20% Mini-PROTEAN® TGX™ Precast Protein Gels (Invitrogen, 4561093) using Invitrogen lOx Tris / Glycine / SDS running buffer (1610732) and transferred to PVDF membranes (BioRad, 1620177). Membranes were blocked with 3% BSA in TBS-T, incubated with primary antibodies against positive EV markers (CD9, CD81, ALIX, TSG101) and negative markers (GRP94, Calnexin, TOM20) at 1:1000 dilution, followed by HRP-conjugated secondary antibody (Goat anti Rabbit IgG HRP Conjugate, B oster Biological Tech, BA1054-0.5) 1:5000. Primary Antibodies used were from Cell Signaling: CD9 (D8O1A), CD81 (D3N2D) (56039S), TSG101 (E6V1X), Alix (E6P9B) 80S, Calnexin (2433S), Grp94 (2104S), and Tom20 (D8T4N). Bands were visualized by SuperSignal West Pico PLUS Chemiluminescent Substrate, ThermoFisher, 34577).RNA isolation and bulk RNA sequencing

[0108] For sequencing of monocytes, freshly thawed cells were cultured at a concentration of 106cells per well of a 6-well plate in 2 mL for 24 hrs. Monocytes were either cultured with 50% MSC-CM, 50 ng / mL GM-CSF plus 100 ng / mL IFNy, 50 ng / mL M-CSF plus 10 ng / mL IL- 10, or left untreated.

[0109] RNA was isolated using the RNeasy kit (Qiagen, 74104) according to manufacturer’s instructions. Isolated RNA was quantified using the NanoDrop8000 spectrometer. Samples were processed by poly-A-enrichment-based library preparation for sequencing in a NovaSeq PE150 Flowcell performed by NovoGene (Sacramento, CA, USA).

[0110] The aligned FPKM reads were loaded into R. Differentially expressed genes were identified using the DESeq2 package. Gene ontology analysis was performed using the clusterProfiler package. The full codes are deposited on GitHub.cDNA generation and quantification via qPCR

[0111] cDNA was generated using High-capacity cDNA Reverse Transcription Kit (Applied Biosystems by ThermoFisher, 4368814) according to the manufacturer’s instructions. The reverse transcription was performed in BioRad T100 Thermal Cycler. The generated cDNA was used for quantification PCR procedure, with TaqMan Universal PCR Master Mix (Applied Biosystems by ThermoFisher ,4304437) and qPCR primers, in Mic PCR machine (Bio Molecular Systems). The cycling conditions were as follow: 50°C for 2 min followed by 95 °C for 10 min, 40 cycles of 95 °C for 15 sec and 60°C for 60 sec, with a final hold 72°C for 60 sec. Primers were obtained from ThermoFisher (IL10: Hs00961622 ml IL10, TNF: Hs00174128 ml TNF, IL1B: Hs01555410 ml IL1B). GAPDH (Applied Biosystems, 4332649) was used as housekeeping gene to calculate fold change in gene expression.Cellular interaction analysis

[0112] A publicly available scRNA seq dataset (GSE253355) (Bandyopadhyay et al., 2024)of the human bone marrow niche enriched for MSCs was used to analyze cellular interaction networks. CellChat package was employed to analyze cellular interactions using the CellChatDB database. Singe cell RNA-seq data was mapped onto a protein-protein interaction network. Significant cell-cell interactions were identified using the default settings, incorporating population size adjustments and calculating mean gene expression using the “trimean” method. The full code is deposited on GitHub.CBO-labeled EV uptake assay

[0113] Freshly thawed MSCs were stained with CBO (Biotium, 30022) for 20 mins at 37°C in PBS by adding 5 pL of CBO per mL PBS at a cell density of 106cells / m . Cells were washed twice with access RPMI / 10%FBS. MSCs were cultured for 3 days in RPMV10%FBS at 37°C at a seeding cell density of 4.5 x 105cells per well of a 6-well plate to generate CBO-labeled EVs. EVs were isolated as described above. Freshly thawed monocytes were rested for Ihr at 37°C in RPMI / 10%FBS. EVs were added to 300 x 103monocytes and the monocytes were incubated with EVs for 6, 24, and 48hrs. At the indicates time points, the monocytes were harvested and washed twice with PBS. For detection using imaging flow cytometry, the cells were stained with APC anti-CD14 (BioLegend, 398706) and acquired using the Cytek Amnis FlowSight Imaging Flow Cytometer (Cytek Bioscience). For detection via conventional flow cytometry, the cells were stained with the following conjugated antibodies and stain (Biolegend) APC / Cy7 anti-CD14 (M5E2), APC anti-MERTK (590H11G1E3), PE / Cy7 anti-CD163 (GHI / 61), and BV785 anti-CD80 (2D10) and DAPI (422801) and acquired using the NovoSight flow cytometer.Phagocytosis assay

[0114] Monocytes were primed with MSC-derived EVs for 2 days in 96-well plates at a density of 8 x 104cells per well. As controls, monocytes were differentiated to Ml or M2 macrophages as described above, or monocytes were incubated with EVs isolated from cRPMI. 3 x 104microspheres (Poly sciences, 19519-500) were incubated with IgG-FITC (MilliporeSigma, F9636) at a concentration of 5 mg / mL overnight at RT at 300 rpm shaking. Beads were washed 3x with access PBS to remove unbound IgG-FITC. A small aliquot was checked for labeling efficiency (>90%) and beads were added to monocytes at a multiplicity of infection (MOI) of 2.5 (2x105 beads added per well to 8 x 104seeded monocytes). Plates were centrifuged at 1000 g for 5 mins to synchronize phagocytosis and then incubated at 37 °C for 1 hr. Cells were detached on ice with 5 mM EDTA for 15 mins, washed and stained with CD45-APC (BioLegend, 304012) for 15 mins followed by acquisition with the FlowSight Imaging Flow Cytometer.Potency matrix

[0115] To generate a potency factor matrix, the average values of IDO1, M-CSF, CD63, and CCL2 for 8 donors were determined. The coefficient of variation was calculated by dividing the standard deviation for each factor through the mean. The potency matrix was generated by first calculating the quartiles for each factor and then assigning 0 to 3 points for each factor depending on the quartile placement for each factor. For the weighted potency matrix, the scores for IDO1 and CD63 were multiplied by 2 and the scores for CCL2 were divided by 2.Statistical analysis

[0116] Statistical analyses, as indicated, were performed using GraphPad Prism statistical analysis software package (version 10.6.1).Data and code availability

[0117] Software and source code created for this study are publicly available and can be obtained at GitHub. Bulk and single cell RNA-seq data can be downloaded from the NCBI Gene Expression Omnibus (GEO) database with the accession numbers GSE000001 and GSE000002. Public single cell RNA-seq data can be downloaded from the NCBI GEO database with the accession number GSE253355.Example 3: MSCs possess multiple immunosuppressive properties

[0118] In vitro proliferation assays are a standard method for assessing the T cell inhibitory function by cells of interest. Previous studies demonstrated that inter-donor variability as well as differences in culture conditions can influence the suppressive potential of MSCs. To initially evaluate the variation in T cell suppression activity between donors, MSCs derived from two different donors (donors identified as 063 and 043) were co-cultured at different cell densities with CFSE-labeled PBMCs for 4 days (Fig. 6, panels A+B). MSCs dose-dependently suppressed activated CD4+and CD8+T cell proliferation, with a clear difference in the potency between MSC isolated from the two different donors. The concentration of MSCs required to inhibit T cell proliferation by 50% (i.e., IC50) was 2.6-fold lower for CD4+T cells and 2-fold lower for CD8+T cells with donor 063 MSCs compared to043 (Fig. 6, panels A+B). Suppression was independent of other cells in the PBMCs as demonstrated by a strong inhibition of activated purified T cell proliferation, suggesting that MSCs directly suppress T cell proliferation (data not shown).

[0119] Previous work suggests that MSCs induce CD4+T cell differentiation to Tregs. In line with this, co-culture of MSCs with T cells was demonstrated to induced a higher percentage of FoxP3+CD25+CD4+T cells compared to T cells cultured alone (Fig. 6. panel C). It is unclear if MSCs stimulate Treg differentiation via a cell contact-dependent mechanism, a cell contact-independent mechanism, or both. To test this, conditioned media (CM) were generated and added at two different concentrations (50% and 12.5%) to T cells plated in complete (c)RPMI medium. Both CM concentrations induced higher percentages of FoxP3+CD25+CD4+ Tregs as well as increased interleukin- 10 (IL- 10) secretion compared to the control (cRPMI) condition (Fig. 6, panels D, E). These results demonstrate that MSCs secrete substances that induce FoxP3+Treg formation.

[0120] MSCs have been reported to drive monocyte polarization towards antiinflammatory M2 macrophages. To validate this, isolated CD14+monocytes were cultured with MSCs and used established Ml and M2 macrophages polarization conditions as control. Co-culture with MSCs for 3 days selectively polarized purified monocytes to antiinflammatory CD163+M2 macrophages (Fig. 6, panel F) with comparatively fewer pro-inflammatory CD80+Ml macrophages present (Fig. 6, panel G). Additionally, co-culture induced IL-10 expression (Fig. 6, panel H). IL-10 was not expressed by MSCs alone in the conditions used here. To test if M2 polarization is cell contact-dependent, MSC-CM was used as described above and found to be sufficient to recapitulate the co-culture phenotype (Fig. 6, panels 1-1). Further analysis of cytokine gene expression confirmed that MSC-CM selectively polarized monocytes to anti-inflammatory M2 macrophages without induction of pro-inflammatory Ml macrophages (Fig. 6, panel K).

[0121] Overall, these results confirmed that the deceased donor vertebral bone marrow-MSCs used in this study potently suppress T cell proliferation, induce Treg differentiation, and promote M2 macrophage polarization (i.e. early skewing of monocytes towards a M2 macrophage phenotype). Validating these important immunomodulatory functions establishes the therapeutic potential of this abundant source of MSCs for treating inflammatory diseases. The next steps in development for clinical testing, as described below,involved developing a matrix of potency assays based on each of these functions to monitor the quality of GMP-compliant manufactured cells.Example 4: Development of a robust T cell suppression surrogate assay

[0122] T cell suppression is a well described MSC immunomodulatory function. Attempts have been made to adapt this assay to a standard which is compatible with regulatory requirements for comparing clinical lots to ensure consistent potency. However, the assay is difficult to standardize, primarily due to the variability in responses between stimulated PBMCs isolated from different donors. An alternative would be to measure a surrogate function of the MSCs which strongly correlates with T cell suppression activity. The first described and best characterized mediator of T cell suppression in vitro is the IFNy-inducible cytoplasmic protein IDO1. IDO1 is the first, rate-limiting enzyme metabolizing tryptophan (Trp) in the kynurenine (Kyn) pathway. T cells require Trp for proliferation and previous work showed that macrophages and cancer cells deplete Trp in the tumor microenvironment via cell-associated IDO1 activity to inhibit T cell proliferation. A positive correlation between IDO1 expression by primed MSCs isolated from different donors and activated T cell suppression has been noted; therefore, levels of IDO1 expression by IFNy-primed MSCs could be a useful surrogate assay for activated T cell suppression activity.

[0123] As a first step to developing a robust surrogate assay, IDO1 expression was confirmed to be the dominate mediator of T cell suppression in the in vitro assay conditions used. An IFNy neutralizing antibody (alFNy) was used to block IDO1 expression, reducing IDO1 expression from -60% to <10% at the highest antibody concentration (Fig. 7, panels D-E). The degree of T cell suppression was correspondingly reduced by increasing concentrations of alFNy (Fig. 7, panels C-D). Neutralizing IFNy without MSC co-culture had no effect on the proliferation of T cells (data not shown). Additionally, an IDO1 inhibitor as well as resupplementing the medium with Trp was sufficient to rescue T cell proliferation (Fig. 7, panel E). To further demonstrate the dependence of T cell suppression on IFNy-induced IDO1 expression, the inability of murine IFNy to cross-react with human IFNy receptor was used. Intracellular IDO1 expression was undetectable and murine T cell proliferation was unaffected when human MSCs were co-cultured with mouse splenocytes (Fig. 7, panel F). However,adding recombinant human IFNy to the co-culture induced IDO1 expression in MSCs and significantly suppressed mouse T cell proliferation (Fig. 7, panel F).

[0124] The above data confirmed that MSC IDO1 expression is obligately dependent on IFNy priming in a dose-dependent manner and, therefore, suggested that measurement of IDO1 expression could be a facile surrogate assay for T cell suppression activity. To test this, MSCs derived from 10 different donors were assayed for IFNy-induced IDO1 expression and activated T cell suppression activity. A strong negative correlation (r = -0.9; P<0.001) was observed between IDO1 expression (range: 12% to 91%) and CD4 as well as CD8 T cell proliferation (Fig. ID and Fig. 7, panel G). Overall, these results demonstrate that assaying for IDO1 expression is a valid surrogate for MSC-mediated suppression of activated T cell proliferation and, as such, is a suitable assay for measurement of potency.Example 5: MSCs drive M2 macrophage polarization through secretion of macrophage-colony stimulating factor (M-CSF)

[0125] To understand how MSCs drive M2 polarization, publicly available scRNA seq data of the human bone marrow niche, including stromal cells, was used. The authors identified five different MSC populations in the bone marrow. CellChat package was used to systematically interrogate interactions between stromal cells and immune cell populations. One of the strongest predicted interactions between MSCs and monocytes is CSF1 (the gene encoding M-CSF) signaling (Fig. 8. panel A). CSF1 is expressed by multiple MSC subsets, including Adipo-MSCs, Fibro-MSCs and THY 1+ MSCs, while the cognate receptor (CSF1R) is expressed at high levels by monocytes (Fig. 8, panel B). The expression of M-CSF was verified in the supernatants of cultured MSCs obtained from three different donors (Fig. 8, panel C). To test if MSC-secreted M-CSF induces M2 polarization, an M-CSF neutralizing antibody was used to inactivate receptor binding. Neutralization of M-CSF significantly reduced MSC-induced upregulation of the M2 macrophage-specific marker CD163 (Fig. 2A). Additionally, M-CSF neutralization reduced live CD45+ CD163+ cell numbers and IL-10 expression (Fig. 2B-2C). In line with this observation, blocking CSF1R signaling using a CSF1R inhibitor also significantly reduced the live CD45+ CD163+ cell numbers and IL-10 expression (Fig. 2D-E). To test the suitability of measuring M-CSF as a surrogate for M2 macrophage polarization, MSCs isolated from 10 different donors, or CM derived from each,were separately added to monocyte / macrophage cultures. A significant correlation was observed (PcO.OOl and P<0.05 when either MSC or CM, respectively, was used) between M-CSF levels in the supernatant and IL- 10 released by monocytes / macrophages (Fig. 2G-2H). These data suggest that measuring M-CSF release is an additional surrogate assay for MSC immunomodulatory potency.Example 6: CCL2 secretion by MSCs facilitates macrophage polarization through recruitment of monocytes

[0126] Neutralizing M-CSF significantly reduced M2 polarization but did not completely abolish it, suggesting that other factors support polarization. Bulk RNA-seq of monocytes cultured for 24 hrs with MSC-CM was employed to identify candidate factors driving M2 polarization (data not shown). Controls were untreated monocytes (Ctrl) and monocytes polarized to either Ml or M2 macrophages by the addition of recombinant cytokines. Ml macrophages demonstrated the most extreme differences in gene expression profile compared to the three other groups (data not shown). To further understand the similarities and differences between Ctrl, MSC-CM and M2 samples, Ml macrophages were excluded (data not shown). This analysis revealed multiple gene clusters that were regulated by MSC-CM. For instance, genes in group (i) that were decreased in M2 macrophages compared to Ctrl and MSC-CM include HLA-DMA, HLA-DMB, HLA-DPB, and HLA-DRA, which are involved in antigen presentation (data not shown). Genes in group (ii) that were increased in MSC-CM-induced macrophages and M2 macrophages controls compared to the monocyte control include CD163, IL10, and MERTK. Each of the proteins expressed from these transcripts are implicated in anti-inflammatory processes.

[0127] To further understand how MSC-CM affects monocytes / macrophages polarization to M2 macrophages, genes upregulated in CM-treated monocytes compared to the control were compared and evaluated using gene ontology (GO) analysis (Fig. 3A). Pathways enriched in CM-treated monocytes included “leukocyte migration” and “cell chemotaxis” (Fig 3A+ 9A). MSCs express relatively high levels of CCL2, a potent chemokine necessary for monocyte recruitment (Fig. 3C). To test if MSC-secreted CCL2 drives monocyte migration, transwell experiment was performed, whereby MSCs or CM were added to the bottom well and migration of monocytes added to the top well was tracked. Both, MSCs and CM effectivelystimulated monocyte migration from the upper chamber to the lower chamber, which was abolished by CCL2 neutralization (Fig. 3D and 9, panel B).

[0128] To determine if migration towards MSCs is required for effective M2 macrophage polarization, the migration experiment was repeated followed by culturing migrated (lower chamber) and non-migrated (upper chamber) monocytes for an additional 3 days. Monocytes that migrated towards MSCs or CM followed by 3-day culture expressed high levels of CD 163 and MerTK but relatively low levels of CD80, confirming M2 polarization (Fig. 3E and 9, panels C-D). However, non-migrated monocytes or monocytes migrated via establishing a recombinant CCL2 gradient did not upregulate CD 163 or MerTK expression. These results indicate that CCL2 secreted by MSCs recruit monocytes which are then converted to M2 macrophages via localized high concentrations of MSC-derived molecules.Example 7: MSC potency is mediated through secretion of EVs.

[0129] Interestingly, GO pathways analysis indicated that cell-free MSC-CM enriched phagocytosis in monocytes / macrophages (Fig. 9, panel A). Upregulated genes in the phagocytosis pathway included MERTK and GAS6, which mediate efferocytosis and uptake of extracellular vesicles (EVs) (Fig. 10, panel A). Increased MerTK protein levels were confirmed in monocytes / macrophages co-cultured with MSCs or cultured with CM (Fig. 10B). Next, EVs from CM were isolated using columns with a molecular cut-off of 300 kDa. followed by characterization and quantification of the EVs using nanoparticle tracking analysis (NTA), CD63 ELIS As, and CD63 capture beads (data not shown). The EVs were positive for the markers CD9, ALIX, TSG101 and CD81; while negative for GRP94 and Calnexin (data not shown). The average EV size was ~80 nm, with the majority of particles ranging between 30 and 150 nm in size, which are classically defined as exosomes 3 (data not shown). On average, ~5.2 x 1011EVs / mL were present in CM and there was a clear correlation (r=0.86; P<0.001) between CD63 concentration measured by ELISA and EV numbers determined with NTA (data not shown).

[0130] To test if MSC EVs are taken up by monocytes, MSCs were labeled with the cytoplasmic membrane dye CellBrite Orange (CBO), the supernatant was harvested, and EVs were isolated. Uptake of labeled EVs was assessed by both imaging and conventionalflow cytometric analysis of monocytes (Fig. 4A and 10, panel C). After 6hrs, <10% of monocytes had a detectable CBO-signal (Fig. 10, panel C). However, after 24hrs and 48hrs the percentage of CBO-positive monocytes increased to 87% and 98%, respectively. Interestingly, EV uptake correlated with an early increase in MerTK expression followed by CD 163 upregulation (Fig. 10, panel C). As expected, CD80 expression remained low.

[0131] Using four different EV preparations and four different monocyte donors, strong M2 macrophage polarization of monocytes by MSC-derived EVs was demonstrated, as shown by increased expression of CD163, MerTK and IL-10 after a 3-day culture (Fig. 10, panel D-E). There was also a clear concentration-dependent relationship between EV numbers added to monocytes and CD163, MerTK and IL-10 induction (Fig. 10, panel F). Additionally, when comparing CBO-positive and CBO-negative monocytes from the same sample, CBO uptake was clearly correlated (r=0.86; P<0.001) with CD163 expression (data not shown). As MerTK expression by monocytes was rapidly upregulated by EVs and previous research suggests a role of MerTK in the uptake of EVs, blocking MerTK would reduce CBO-labeled EV uptake. Both a MerTK neutralizing antibody or a MerTK inhibitor clearly reduced the uptake of CBO-labeled EVs (Fig. 4C). A recent publication indicated that M-CSF is expressed on EVs secreted by cancer cells. Indeed, M-CSF was detected in both EVs and the flow-through obtained during column purification and, moreover, was highly enriched in purified EV preparations (Fig. 10, panel G). Of note, EV isolation resulted in a 15-fold concentration of EVs (start volume vs. final EV volume). This process likely contributed to the high levels of M-CSF measured on EVs compared to the FT or compared to CM (Fig. 8, panel C).

[0132] To demonstrate the direct involvement of MerTK-mediated uptake of EVs, leading to M2 polarization by EV-associated M-CSF, both pathways were inhibited. Blocking MerTK activity on monocytes / macrophages using either a neutralizing antibody or small molecule inhibitor reduced CD163 expression (Fig. 5J). Similarly, blocking either EV M-CSF activity or monocyte / macrophage CSF1R activity also reduced CD163 expression (Fig. 10, panel H).

[0133] Finally, expression of genes increased in the “phagocytosis” pathway included those involved in the conventional phagocytosis pathway, such as THBS1, CD93, FCGR1A, and VAV3 (Fig. 3A). Phagocytic clearing of autoreactive T cells has also been implemented in the anti-inflammatory function of M2 macrophages in diseases such as GvHD.Therefore, priming monocytes with EVs was tested to determine whether it increased the phagocytic potential of monocytes / macrophages. M2 macrophages and monocytes primed with MSC-derived EVs phagocytized IgG-opsonized microbeads more effectively compared to Ml macrophages or untreated monocytes (data not shown).

[0134] Overall, these data suggest that MSCs induce M2 macrophage polarization through EVs displaying M-CSF which activates CSF1R either preceding or following endocytosis through the MerTK7GAS6 pathway with the net result of increased antiinflammatory activity.Example 8: MSC EVs drive Tree polarization

[0135] Considering that MSC-CM induces Treg polarization (Fig. 6, panel D), EVs may also contribute to Treg induction. To test this, CD4+T cells were incubated with different concentrations of EVs (Fig. 4F). Indeed, there was an EV concentration-dependent increase in FoxP3 expression, with approximately twice as many CD4+cells expressing FoxP3 when incubated with 2 x 1011EVs compared to without EVs (Fig. 6A). Similarly, IL-10 levels in the culture supernatant increased proportionally to EV concentration (Fig. 11, panel A). To understand if EVs released by MSCs promoted de novo Treg differentiation or preferentially supported Treg proliferation, the replication index of FoxP3+and FoxP3“ CD4+cells was compared (Fig. 11, panel B). Both CD4+T cell populations replicated to the same extent in the presence or absence of EVs, suggesting that EVs did not preferentially stimulate Treg proliferation.

[0136] Using imaging flow cytometry showed that EVs labeled with CBO were taken up by CD4+and CD8+T cells (Fig. 11, panel C). To identify specific components that induce Treg formation, EVs were first assayed for TGFpi, a well described induction factor for Treg differentiation. Relatively high levels of TGFpi were detected on EVs (Fig. 11, panel D). Interestingly, the TGFpi concentration on EVs varied between EVs derived from MSC isolated from different donors (Fig. 11, panel D). To confirm that EV-associated TGFpi was functional and directly induced Treg formation, its activity was blocked using two different TGFpRl pathway inhibitors, resulting in significantly reduced EV-mediated Treg induction (Fig. 11, panel E). Additionally, a neutralizing antibody was used as a complementary method to confirm EV-mediated Treg induction via associated TGFpi (Fig. 11, panel F). Of note,FoxP3 expression in the control sample varied significantly based on the specific donor T cells used, which is a well-documented phenomenon. It was confirmed that TFGpi associated with EVs was necessary for Treg induction and not other potential sources by reisolating EVs to remove free antibody before testing (data not shown).

[0137] The Latency- Associated-Peptide (LAP) noncovalently binds TGFpi, thereby preventing activity 37659098. Neutralizing LAP function could increase active TGFpi concentrations. Indeed, a 50% increase in Treg abundance was observed when EVs were incubated for 3hrs with an anti-LAP antibody before addition of EVs to T cells (data not shown). Additionally, neutralization of LAP with an anti-LAP antibody was sufficient to detect active TFGpi without the need of activation using HC1 (data not shown). Collectively, these data indicate that functional TGFpi associated with MSC-derived EVs is able to promote de novo Treg induction in vitro.Example 9: A quantitative potency assay matrix for qualifying MSCs used in clinical testing

[0138] Heterogeneity of potency between manufactured lots has severely impeded commercial development of some MSC therapies. To ensure inter-batch consistency of cellular therapies used in clinical trials, the FDA requires potency assays as release criteria. The present study has identified four potency factors (IDO1, M-CSF, EVs, and CCL2) and demonstrated correlations between the quantities of each and the corresponding in vitro immunosuppressive activity.

[0139] To demonstrate the utility of this assay matrix for assessing MSC potency, MSCs recovered from vertebral body bone marrow obtained from different 8 different donors, were assayed for M-CSF, CD63 (EV concentration) and CCL2 expression as well as percentage of IDO1+cells following IFNy stimulation (Fig. 5A). For each potency factor, the range between the highest value and lowest value varied with a high coefficient of variation (CV) for IDO1 (47.3%) and CCL2 (60.5%) and smaller CVs for M-CSF (25.8%) and CD63 (20.5%). To evaluate overall potency, scores were assigned to each donor depending on the detected levels of each potency factor (Fig. 12, panels A-B). For each factor, 0 to 3 points were assigned depending on quartile levels. For instance, donor 320 received a total score of 6 based on the following calculation: IDO1 levels were in the second lowest quartile (1 point), M-CSF levels were in the highest quartile (3 points). CD63 levels were in the lowest quartile (0 points)and CCL2 levels were in the second highest quartile (2 points). Overall analysis of the potency matrix for each of the 8 MSC isolates suggests a high potency of donor 017, followed by donors 257 and 074.

[0140] An additional consideration is that not all four potency factors are equal in their impact on immune cell function. For example, activated T cell suppression by IDO1 activity as well as M2 polarization induced by EVs both potently affected immune suppression activity; whereas, CCL2 by itself elicited chemoattraction of monocytes, but, in isolation, did not alter immune cell function. Therefore, each factor was assigned a weighted score to reflect its potential to directly modulate the immune system. Thus, 2X multipliers were added to scores for IDO1, M-CSF and CD63 to account for their relative importance for immune suppression. Conversely, a multiplier of 0.5X was used for CCL2 to account for its relatively lesser importance. The resulting weighted matrix shifts the ranking slightly. MSCs from donor 017 is still predicted to have the highest potency, followed by the MSCs from donor 063 and 257.

[0141] These data demonstrate the utility of the developed matrix of surrogate potency assays for screening donor-derived MSCs. By analogy, the matrix is fully expected to similarly provide meaningful comparisons between manufactured lots produced from a single donor MSC to detect changes in the final product. The utility of the potency matrix will be validated before incorporating these assays into testing of GMP manufactured lots. Future planned clinical trials in GvHD patients will evaluate the correspondence between in vitro potency and clinical efficacy.Example 10

[0142] In this study, multiple immunomodulatory mechanisms employed by MSCs that are in clinical development for GvHD and other inflammatory diseases were characterized. In so doing the influence on T cell and monocyte functions and phenotypes were ascertained through secreted factors, metabolic regulation (i.e. Trp depletion), and EV signaling. These data demonstrate that MSCs suppress effector T cell proliferation, promote Treg induction, and drive monocyte polarization toward an anti-inflammatory M2 macrophage phenotype. The broad applicability of these findings was enhanced by testing immunomodulatory function of a total of 10 MSC isolates from donors age 14-48 (both male and female) using immune cellsisolated from a total of 22 male and female donors (ages 18-84). These findings provide further insights into the multiple mechanisms by which MSCs regulate immune cell populations. Moreover, the variability observed in individual potency factor expression between MSCs obtained from different donors may explain the heterogeneity observed in clinical responses to MSC therapies. The development of a robust matrix of orthogonal immunomodulatory potency assays allows for multifactorial assessment of in vitro activity to better maintain quality control during manufacturing; thus, potentially limiting variations in clinical responses.

[0143] The demonstration that IFNy- stimulated MSCs isolated from deceased donor vertebral body bone marrow suppress T cell proliferation validates previous findings that these cells are no different than iliac crest-derived MSCs obtained from healthy, living volunteer donors. These results also corroborate previous reports establishing the importance of IDO1 in MSC-mediated immune regulation and highlight its central role in MSC potency. Importantly, a strong correlation was observed between levels of IDO1 expressed by MSCs isolated from different donors and the degree of suppression of T cell proliferation, supporting its use as an indicator of functional potency.

[0144] In addition to suppression of T cell activity, it was demonstrated that MSCs orchestrate monocyte polarization toward an M2 phenotype through a coordinated process involving chemoattraction, surface receptor signaling, and EV-mediated communication. Chemoattraction towards MSCs via a CCL2 gradient enhances the polarizing effect of secreted M-CSF, which is reinforced through enhanced MerTK signaling induced by EV uptake. These results build on prior work implicating M-CSF in macrophage polarization and further establish EVs as essential vectors of MSC-mediated immune regulation. MSC-derived EVs are shown to carry bioactive M-CSF and that blocking M-CSF or MerTK signaling impairs the acquisition of an M2 phenotype.

[0145] The combinatorial effects of MSC-secreted factors provide a coordinated mechanism for affecting immunomodulation within the inflammatory milieu of diseased tissues. Alternatively, given the predominant entrapment of intravenously infused MSCs in lungs, the cells could promote systemic effects. A mechanism based on lung-entrapped MSCs promoting circulating macrophage efferocytosis has been proposed to explain therapeutic effects on distal tissues. These findings suggest that MSC-secreted CCL2 could promote recruitment of circulating monocytes to the lungs where they would be polarized by M-CSFand subsequently migrate to lesions in distal tissues. It is also possible that soluble molecules and EVs secreted by MSCs circulate to regulate immune cells in other tissues.

[0146] In particular, these findings highlight a critical immunoregulatory role of MSC-derived EVs in promoting the induction of Tregs. These EVs were found to carry bioactive TGF 1, a well-established cytokine that drives FoxP3 expression and Treg lineage commitment. The EV-mediated Treg induction was dependent on TGF 1, as blocking its signaling abrogated Treg formation, and enhancing its activation via LAP neutralization further amplified the response. These data underscore the capacity of MSC-derived EVs

[0147] Together, these examples demonstrate that MSCs engage multiple, complementary pathways to modulate the immune microenvironment, explaining observations of potent immunomodulation in diverse diseased tissues. From a translational perspective, this work underscores the importance of using a matrix of functionally validated surrogate potency factors that modulate distinct pathways directly relevant to the proposed mechanism of action for treating a specific disease indication. These markers not only reflect the biological activity of MSCs but also serve as potency metrics for manufactured lots that are required by regulatory agencies to ensure quality and consistency of products used in the clinic.

Claims

WHAT TS CLAIMED TS:

1. A method of developing a potency assay matrix for use in monitoring the quality of a manufactured cell, the method comprising:(i) testing the manufactured cell for activity in one or more screening factors; and (ii) assigning a value to the activity for each screening factor;wherein the value for each screening factor is combined to form the potency assay matrix.

2. The method of claim 1, wherein the one or more screening factors comprise at least one of: immune cell migration, immune cell chemotaxis, phagocytosis, leukocyte mediated immunity, activated lymphocyte suppression, macrophage polarization, regulatory T cell stimulation, or any combination thereof.

3. The method of claim 2, wherein the one or more screening factors comprise at least one of: activated lymphocyte suppression, macrophage polarization, regulatory T cell stimulation, or any combination thereof.

4. The method of claim 2, wherein the one or more screening factors comprise at least one of: leukocyte migration, leukocyte chemotaxis, myeloid leukocyte migration, granulocyte migration, neutrophil chemotaxis, neutrophil migration, mononuclear cell migration, lymphocyte migration, or any combination thereof.

5. The method of claim 1 , wherein the manufactured cell is a stem cell or a stromal cell.

6. The method of claim 5, wherein the manufactured cell is a Mesenchymal Stem / Stromal Cell (MSC).

7. A method of insuring inter-batch consistency across a group of manufactured cells in a cell batch, the method comprising:(i) administering the potency assay matrix of claim 1; and(ii) removing from the cell batch any manufactured cell that does not reach a suitable activity value in at least one screening factor.

8. A method of determining the quality of a stored cell, the method comprising: (i) measuring the expression level of an at least one gene involved in an at least one activity pathway in a fresh cell; and(ii) measuring the expression level of the at least one gene in the stored cell;wherein no significance in expression levels between the fresh and stored cell is indicative of a good quality of the stored cell, and a significant difference in expression level is indicative of a poor quality of the stored cell.

9. The method of claim 8, wherein the stored cell is cryopreserved.

10. The method of claim 8, wherein the stored cell is a stem cell or a stromal cell.

11. The method of claim 10, wherein the stored cell is an MSC.

12. The method of claim 8, wherein the at least one activity pathway comprises at least one of: immune cell migration, immune cell chemotaxis, phagocytosis, leukocyte mediated immunity, activated lymphocyte suppression, macrophage polarization, regulatory T cell stimulation, or any combination thereof.

13. The method of claim 8, wherein the at least one gene comprises at least one of: VAV1, IDO-1, TREM1, S1PR1, MYO1G, ITGA7, EDNRB, CCL18, ADAM8, CCL2, CSF1, CD63, or any combination thereof.

14. The method of claim 13, wherein the at least one gene comprises 1, 2, 3, or 4 of: IDO1, CSF1, CD63, CCL2, or any combination thereof.

15. The method of claim 8, wherein when the stored cell is of poor quality, the expression level of the at least one gene is significantly reduced in the stored cell compared to the fresh cell.

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