Treatment of age-related frailty including administration of bone marrow-derived mesenchymal stem cells

Abstract

Disclosed herein are compositions and methods for the treatment of aging frailty with bone marrow-derived mesenchymal stem cells. The method of treatment includes administering a composition of bone marrow-derived mesenchymal stem cells to a subject in need thereof, and the efficacy of the method of treatment can be determined through the measurement of certain biomarkers and improved physical activity.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 261,092, filed September 10, 2021, which is incorporated by reference as if fully recited herein.

[0002] The present disclosure relates to methods and compositions for the treatment of aging frailty in a subject in need thereof. [Background technology]

[0003] Age-related frailty poses very serious problems to the overall health and well-being of an individual. Age-related frailty is a geriatric syndrome characterized by weakness, low physical activity, slow exercise performance, extreme fatigue, and unintentional weight loss. See Yao, X. et al., Clinics in Geriatric Medicine 27(1):79-87 (2011). Furthermore, there are many studies showing a direct correlation between age-related frailty and inflammation. See Hubbard, RE et al., Biogerontology 11(5):635-641 (2010).

[0004] Immunosenescence is characterized by a state of chronic low-grade systemic inflammation known as inflammaging. See Franceshi, C. et al., Annals of the New York Academy of Sciences 908:244-254 (2000). This state of high inflammation or chronic inflammation seen in aging and age-related frailty leads to immune dysregulation and complex remodeling of both innate and adaptive immunity. In immunosenescence, the T and B cell repertoire is skewed, with CD8+ / CD45ra re-expressing. + Effector memory T cells (TEMRA) and CD19 + Increase in late / exhausted memory B cells and CD8 + Naive T cells and switched memory B cells (CD27 +(See Blomberg, B.B. et al., Immunologic Research 57(1-3):354-360 (2013); Colonna-Romano, G. et al., Mechanisms of Ageing and Development 130(10):681-690 (2009); and Koch S. et al., Immunity & Ageing:5:6 (2008).

[0005] This change in the T and B cell repertoire leads to a refractory or less efficient immune state. This immune system deterioration leads to increased susceptibility to infectious diseases and reduced response to vaccination. Optimal B cell function is essential for the generation of effective antibody responses to vaccines and protection from infectious pathogens. It is well known that age-related increases in systemic inflammation (TNF-α, IL-6, IL-8, INFγ, and CRP) induce B cell dysfunction, leading to inadequate antibody responses and reduced vaccine efficacy.

[0006] Inflammation has attracted considerable attention because it presents a link between immune changes and several diseases and conditions common in older adults, such as age-related frailty. Circulating inflammatory mediators, such as cytokines and acute phase proteins, are markers of low-grade inflammation that are observed to increase with age. These pro-inflammatory cytokines (e.g., TNF-α, IL-6) impair the ability of B cells to produce protective antibodies against exogenous antigens and vaccines. This impaired B cell response is measured by a decrease in class switch recombination (CSR), the ability of immunoglobulins to switch isotype from IgM to a secondary isotype (IgG, IgA, or IgE). Immunoglobulin isotype switching is essential for proper immune responses, as effector functions differ for each isotype. A central player in CSR and somatic hypermutation (SHM) is the enzyme, activation-induced cytidine deaminase (AID), encoded by the Aicda gene. The essential function of AID in CSR and SHM is to initiate DNA cleavage by converting cytosines to uracils in immunoglobulin switch and variable regions.

[0007] E47, encoded by the Tcfe2a (E2A) gene, is a transcription factor belonging to the class I basic helix-loop-helix (bHLH) proteins, also known as E proteins. Absence of E47 expression results in the absence of expression of the B cell-specific transcription factors EBF1 (early B cell factor) and Pax-5 (paired box protein). Both E47 and Pax-5 are important transcription factors in early development of the B cell lineage and in the function of mature B cells. See Hagman J. et al., Immunity 27(1):8-10 (2007); Horcher M. et al., Immunity 14(6):779-790 (2001); Riley RL et al., Seminars in Immunology 17(5):330-336 (2005). The Pax-5 gene encodes the B cell lineage-specific activating protein (BSAP), which is expressed at all stages of B cell differentiation but not in terminally differentiated B cells. Pax-5 controls B cell commitment by suppressing inappropriate genes in the B cell lineage and activating B cell-specific genes to produce the B cell gatekeeper, Pax-5, which is expressed exclusively in the B lymphocyte lineage from the committed pro-B cell to the mature B cell stage. The B cell-specific transcription factor Pax-5 is not only crucial in early B cell development and B cell lineage commitment, but is also involved in CSR.

[0008] It has also been shown in humans that the amount of TNF-α produced (1) depends on the amount of inflammation in the system and (2) impairs the ability of the same B cells to be stimulated by mitogens or antigens. See Frasca, D. et al., Journal of Immunology 188(1):279-286 (2012). Thus, immune responses in subjects suffering from aging frailty are impaired for several reasons.

[0009] Age-related frailty can affect a subject's quality of life in many ways. For example, age-related frailty can cause a decrease in immune response after vaccination, thereby reducing the efficacy of the vaccine. In fact, vaccination against influenza is strongly recommended for individuals over 65 years of age to protect against infection. Commercially available vaccines against influenza provide protection and ensure long-lasting immune memory in children and adults, but are less effective in elderly and frail individuals. See Frasca D. et al., Current Opinion in Immunology 29:112-118 (2014) and Yao X. et al., Vaccine 29(31):5015-5021 (2011). Despite regular influenza vaccination, elderly individuals are at higher risk of influenza infection, leading to secondary complications, hospitalization, physical debilitation, and ultimately death. See Gross, P. et al., Annals of Internal Medicine 123(7):518-527 (1995); Simonsen L. et al., The Journal of Infectious Diseases 178(1):53-60 (1998); and Vu T. et al., Vaccine 20(13-14):1831-1836 (2002).

[0010] These increased risks may be due to the onset of age-related frailty in this population. Influenza vaccines also prevent other complications resulting from influenza infection (e.g., pneumonia) in most older individuals and reduce hospitalization rates to some extent. Nichol KL et al., The New England Journal of Medicine 331(12):778-784 (1994). However, within this population, the rate of hospitalization due to influenza-related illness remains very high. See Thompson, WW et al., JAMA 292(11):1333-1340 (2004).

[0011] Previously published results have reported that B cell specific responses to influenza vaccines in vitro (measured by AID) and in vivo serological responses (measured by HAI assay and ELISA) decrease with age and are significantly correlated. See Frasca, D. et al., Vaccine 28(51):8077-8084 (2010). It was also reported that the percentage of switched memory B cells and CpG-induced AID, both measured before vaccination (t0), decrease with age and are significantly correlated with in vivo responses. Thus, these markers appear to be predictive of in vivo responses.

[0012] Therefore, in view of the above problems, the development of therapeutic methods and compositions that can improve immune responses and immune system function in elderly people would be beneficial for the advancement of the field of geriatrics. Summary of the Invention [Problem to be solved by the invention]

[0013] An object of the present disclosure is to provide methods for the treatment or alleviation of age-related frailty in a subject in need thereof, comprising administering to a subject in need thereof a therapeutic amount of bone marrow-derived mesenchymal stem cells.

[0014] Another object of the present disclosure is to provide novel biomarkers for diagnosing and assessing the progression of age-related frailty in subjects in need thereof. These novel biomarkers can also be measured to determine the efficacy of the treatment methods described herein. [Means for solving the problem]

[0015] Some embodiments are directed to compositions comprising a therapeutically effective amount of bone marrow derived stem cells, particularly bone marrow derived mesenchymal stem cells (bMSCs), used to reduce symptoms of aging frailty, such as a decreased immune response or increased systemic inflammation. Other embodiments are directed to therapeutic methods of administering a therapeutically effective amount of a composition comprising bMSCs to a subject suffering from symptoms of aging frailty. The efficacy of these treatments can be assessed by measuring the concentration and expression of certain biomarkers, such as Tie2, VEGF, and TGF-β, in the subject after administration of a composition comprising bMSCs. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 shows a Phase 2b randomized, blinded, placebo-controlled clinical trial to evaluate the safety and efficacy of Lomecel-B injection in patients suffering from age-related frailty. [Figure 2A] Figure 1 shows change in 6MWT over time after treatment with Lomecel-B cells. All four Lomecel-B groups showed a trend or significant increase in walking distance, with the 200M dose showing the largest change from baseline. In contrast, the placebo group showed a trend toward a decrease up to 9 months after treatment. *=p<0.05, change from baseline. **=p<0.01, change from baseline. #=p<0.05, change from baseline for Lomecel-B group vs. change from baseline for placebo. ##=p<0.01, change from baseline for Lomecel-B group vs. change from baseline for placebo. [Figure 2B] FIG. 2D shows the dose-response effect for each treatment group in FIG. 2A, calculated using the MCP-Mod method. All five models were statistically significant, but the linear model yielded the lowest AIC value. [Diagram 3] FIG. 1 shows a modified therapeutic intent dose-response curve between the amount of Lomecel-B cells administered to a patient and the change in the patient's 6MWT score. [Figure 4A] FIG. 1 shows the change in PROMIS physical function score at 6 months after treatment, selected as a secondary endpoint, which correlated significantly with the change in 6MWT. [Figure 4B] FIG. 1 shows change in PROMIS Mobility at 6 months post-treatment, selected as a secondary endpoint, which correlated significantly with change in 6MWT. [Figure 4C] FIG. 1 shows PROMIS Upper Limb change at 6 months post-treatment, selected as a secondary endpoint, which correlated significantly with change in 6MWT. [Figure 5A] Figure 1 shows the change in soluble Tie2 (sTie2) after treatment with Lomecel-B cells. sTie2 was decreased in the Lomecel-B200M group and was significant at 3 months post-infusion. At 9 months post-infusion, the difference between the Lomecel-B200M group and placebo was highly significant. *=p<0.05, change from baseline. ##=p<0.01, change from baseline in Lomecel-B group vs. change from baseline in placebo. [Figure 5B] FIG. 5B shows the dose-response effect for each treatment group from FIG. 5A calculated using the MCP-Mod method, modeled with sigmoidal Emax (p=0.0175). [Figure 5C] FIG. 1 shows the inverse correlation between changes in sTie2 levels and changes in 6MWT. [Figure 6A] FIG. 1 shows the change in TGF-β concentration following administration of 200 million bone marrow-derived Lomecel-B cells compared to placebo. [Figure 6B] FIG. 1 shows the change in VEGF concentration following administration of 200 million bone marrow-derived Lomecel-B cells compared to placebo. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Mesenchymal stem cells are multipotent cells capable of migrating to sites of injury and are also immune privileged due to undetectable expression of major histocompatibility complex class II (MHC-II) molecules and low levels of expression of MHC-I molecules. See Le Blanc, K. et al., Lancet 371(9624):1579-1586 (2008) and Klyushnenkova E. et al., J. Biomed. Sci. 12(1):47-57 (2005). Thus, allogeneic mesenchymal stem cells hold great promise for therapeutic and regenerative medicine and have repeatedly been shown to have a high safety and efficacy profile in clinical trials for multiple disease processes. See Hare, JM et al., Journal of the American College of Cardiology 54(24):2277-2286 (2009); Hare, JM et al., Tex. Heart Inst. J. 36(2):145-147 (2009); and Lalu, MM et al., PloS One 7(10):e47559 (2012). Allogeneic mesenchymal stem cells have also been shown not to undergo malignant transformation following transplantation into patients. See Togel F. et al., American Journal of Physiology Renal Physiology 289(1):F31-F42 (2005).

[0018] Treatment with mesenchymal stem cells has been shown to reverse severe graft-versus-host disease, protect against ischemic acute renal failure, contribute to pancreatic islet and glomerular repair in diabetes, reverse fulminant liver failure, regenerate damaged lung tissue, alleviate sepsis, reverse remodeling after myocardial infarction, and improve cardiac function. Le Blanc K. et al., Lancet 371(9624):1579~1586(2008);Hare, JM et al., Journal of the American College of Cardiology 54(24):2277~2286(2009);Togel F. et al., American Journal of Physiology Renal Physiology 289(1):F31~F42(2005);Lee RH et al., PNAS 103(46):17438~17442(2006);Parekkadan, B. et al., PloS One 2(9):e941(2007);Ishizawa K. et al., FEES Letters 556(1-3):249~252(2004);Nemeth K. et al. Nature Medicine 15(1):42~49(2009);Iso See Y. et al., Biochem. Biophys. Res. Comm. 354(3):700-706 (2007); Schuleri KH et al., Eur. Hearth J. 30(22):2722-2732 (2009); and Heldman AW et al., JAMA 311(1):62-73 (2014). In addition, mesenchymal stem cells are also a potential source of numerous cell types for use in tissue engineering. (See Gong Z. et al., Methods in Mol. Bio. 698:279-294 (2011); Price, AP et al., Tissue Engineering Part A 16(8):2581-2591 (2010); and Togel F. et al., Organogenesis 7(2):96-100 (2011)).

[0019] Mesenchymal stem cells have immune-modulating capabilities: they control inflammation and cytokine production of lymphoid and bone marrow-derived immune cells without evidence of immunosuppressive toxicity, and are low immunogenic (see Bernardo ME et al., Cell Stem Cell 13(4):392-402 (2013)).

[0020] Mesenchymal stem cells also have the ability to differentiate into cells of mesodermal origin, as well as endodermal and ectodermal origin (see Le Blanc K. et al., Exp. Hematol. 31(10):890-896 (2003)). For example, mesenchymal stem cells cultured in vitro in airway growth medium differentiate to express lung-specific epithelial markers, such as surfactant protein C, Clara cell secretory protein, and thyroid transcription factor 1 (see Jiang Y. et al., Nature 418(6893):41-49 (2002) and Kotton DN et al., Development 128(24):5181-5188 (2001)).

[0021] However, despite being a safe therapeutic agent, it has been reported in the literature that mesenchymal stem cells exert an inhibitory effect on antibody production, as well as on the proliferation and maturation of B cells (see Uccelli, A. et al., Trends in Immunology 28(5):219-226 (2007)). Mesenchymal stem cells have also been reported to inhibit the generation and function of antigen-presenting cells (see Hoogduijn MJ et al., Int. Immunopharmacology 10(12):1496-1500 (2010)). Finally, mesenchymal stem cells have been reported to suppress the proliferation of CD4+ and CD8+ T cells (see Ghannam S. et al., Stem Cell Res. & Ther. 1:2 (2010)).

[0022] Surprisingly, despite reports that mesenchymal stem cells have suppressive effects on aspects of the immune system, the present inventors have discovered a method for enhancing a subject's immune response and the performance of a subject's immune system through the administration of therapeutic amounts of bone marrow derived mesenchymal stem cells.

[0023] Henceforth, it is an object of the present disclosure to provide methods for the treatment or alleviation of age-related frailty in a subject in need thereof, which methods comprise administering to a subject in need thereof a therapeutic amount of bone marrow derived mesenchymal stem cells.

[0024] The therapeutic methods described herein may also be used to treat subjects who have a weakened immune system due to reasons not related to aging, such as chemotherapy, AIDS / immune dysfunction, toxin exposure, Lyme disease, and organ dysfunction / failure. The therapeutic methods described herein may also be used to treat subjects with pre-weakened or compromised immune systems to prevent the subject from developing a weakened immune system.

[0025] In some embodiments, a method of treating or alleviating symptoms of age-related frailty in a subject in need thereof comprises administering to a subject in need thereof a therapeutic amount of LOMECEL-B™ brand isolated allogeneic human stem cells. (LOMECEL-B™ is the brand name of Longeveron, Inc.'s isolated allogeneic human stem cells.)

[0026] Further uses and information regarding the preparation of stem cells that may be suitable for use herein can be found in the following U.S. Patent Application Publication No. 2019003874; U.S. Patent Application Publication No. 20190290698; U.S. Patent Application Publication No. 20200129558, all of which are incorporated by reference herein.

[0027] Without being bound by any theory, bone marrow derived mesenchymal stem cells can treat or alleviate symptoms of age-related frailty in a subject by either improving endothelial cell function, promoting anti-inflammatory cytokine expression or cellular pathways, stimulating inherent regenerative or repair pathways in adjacent somatic or stem cells, improving immune system function, improving mitochondrial function in adjacent somatic or stem cells, promoting anti-fibrotic pathways, or a combination thereof.

[0028] In some embodiments, the method further comprises measuring the concentration of a biomarker or biomarkers in a subject suffering from a symptom of age-related frailty before and after administration of a therapeutically effective amount of a composition comprising bone marrow-derived MSCs.

[0029] Therapeutic amounts of bone marrow-derived MSCs may range from 25 million cells to 200 million cells, hi some embodiments, the therapeutic amount is 25 million cells, 50 million cells, 100 million cells, or 200 million cells.

[0030] A subject in need thereof may be any subject suffering from symptoms of age-related frailty, such as an elderly patient or a patient over the age of 55, or any subject suffering from symptoms of a weakened immune system not attributable to age-related frailty. In some embodiments, the patient is over 65 years old, and in other embodiments, the patient is over 75 years old.

[0031] In some embodiments, the method further comprises determining whether there is a change or improvement in the subject's functional mobility and / or exercise tolerance following administration of the stem cells. A change in the subject's functional mobility and / or exercise tolerance can be determined, for example, by examining whether there is an improvement in the subject's 6-minute walking distance test following administration of the bone marrow-derived MSCs. In these embodiments, "improvement" means that the subject is able to walk or move a greater distance after administration of a therapeutic amount of bone marrow-derived MSCs, as compared to the distance achieved before administration of the MSCs.

[0032] In other embodiments, the method further comprises determining whether there is a change or improvement in the subject's ability to perform activities of daily living. The PROMIS physical function score can be used to determine whether the subject's ability to perform activities of daily living has improved. In these embodiments, "improvement" means that the subject has a higher PROMIS physical function score after receiving a therapeutic amount of bone marrow-derived MSCs as compared to the score achieved before administration of the MSCs.

[0033] In some embodiments, the method further comprises determining whether there is a change or improvement in the subject's PROMIS mobility score. In these embodiments, "improvement" means that the subject has a higher PROMIS mobility score after being administered a therapeutic amount of bone marrow-derived MSCs as compared to the score achieved before administration of the MSCs.

[0034] In other embodiments, the method further comprises determining whether there is a change or improvement in the subject's grip strength. In these embodiments, "improvement" means that the subject is able to hold an object longer or more firmly after receiving a therapeutic amount of bone marrow-derived MSCs when compared to the time or pressure / force achieved prior to administration of the MSCs.

[0035] In some embodiments, the method further comprises determining whether a change or improvement occurs in the subject's gait or balance. The Tinetti Performance Oriented Mobility Assessment (POMA) test can be used to determine whether an improvement occurs in the subject's gait or balance. In these embodiments, "improvement" means that the subject achieves a higher Tinetti POMA score after administration of a therapeutic amount of bone marrow-derived MSCs, as compared to the score achieved before administration of the MSCs.

[0036] Another object of the present disclosure is to provide novel biomarkers for diagnosing and assessing the progression of age-related frailty in subjects in need thereof. These novel biomarkers can also be measured to determine the efficacy of the treatment methods described herein.

[0037] In some embodiments, the novel biomarkers include changes in concentration levels of pro-inflammatory cytokines in a subject in need thereof. These pro-inflammatory cytokines may be selected from TNF-α, TGF-β, IL-1β, IL-2, D-dimer, C-reactive protein (CRP), or combinations thereof. In a preferred embodiment, the concentration of pro-inflammatory cytokines is decreased in serum, plasma, or blood of a subject in need thereof suffering from age-related frailty symptoms after administering a therapeutic amount of bone marrow-derived MSCs to said subject. The decrease in pro-inflammatory cytokine concentration may be in the range of 0%-10%, 0.5%-10%, 1.0%-10%, 3%-10%, 5%-10%, 7%-10%, greater than 0% to less than or equal to 10%, 10%-50%, 20%-50%, 30%-50%, or greater than 50%. In preferred embodiments, the pro-inflammatory cytokine concentration is reduced to a stable concentration level that does not increase by more than 0%-10%, 0%-5%, or 0%-1%, once it reaches and is maintained at a concentration level different from the concentration level prior to administration of the bone marrow-derived MSCs to a subject in need thereof.

[0038] In some embodiments, the novel biomarker comprises a change in the concentration level of an anti-inflammatory cytokine in a subject in need thereof. These anti-inflammatory cytokines may be selected from IL-8, soluble IL-2 receptor alpha (sIL-2Rα), IL-4, IL-10, IL-12, TNF-α stimulated gene 6 (TSG-6), or a combination thereof. In a preferred embodiment, the concentration of the anti-inflammatory cytokine is increased in the serum, plasma, or blood of a subject in need thereof suffering from age-related frailty symptoms after administering a therapeutic amount of bone marrow-derived MSCs to said subject. The increase in the anti-inflammatory cytokine concentration may be in the range of 0%-10%, 0.5%-10%, 1.0%-10%, 3%-10%, 5%-10%, 7%-10%, greater than 0% to less than or equal to 10%, 10%-50%, 20%-50%, 30%-50%, or greater than 50%. In a preferred embodiment, the anti-inflammatory cytokine concentration is increased to a stable concentration level that reaches and is maintained at a concentration level different from the concentration level prior to administration of the bone marrow-derived MSCs to a subject in need thereof, and the concentration does not increase by more than 0%-10%, 0%-5%, or 0%-1%.

[0039] In another embodiment, the novel biomarker comprises changes in the concentration of soluble Tie2 (sTie2). Tie2 is a receptor tyrosine kinase for angiopoietin and is involved in anti-inflammatory, endothelial integrity, and angiogenic cellular pathways. In an inflammatory or cellular environment, Tie2 is proteolytically cleaved to form sTie2, which is typically detected in serum. sTie2 can inhibit the pro-vascular signaling of full-length membrane-bound Tie2. VEGF has also been shown to stimulate Tie2 signaling (see Singh et al., Cellular Signaling 2009).

[0040] Without being bound by any theory, administration of therapeutic amounts of bone marrow-derived MSCs can reduce the concentration of sTie2 and increase vascular stabilization via Tie2 signaling and VEGF / VEGFR signaling in subjects suffering from age-related frailty.

[0041] In some embodiments, the concentration of sTie2 is decreased in serum, plasma, or blood of a subject suffering from age-related frailty symptoms in need thereof after administering a therapeutic amount of bone marrow-derived MSCs to said subject. The decrease in sTie2 concentration may be in the range of 0%-10%, 0.5%-10%, 1.0%-10%, 3%-10%, 5%-10%, 7%-10%, more than 0% to less than 10%, 10%-50%, 20%-50%, 30%-50%, or more than 50%. In preferred embodiments, the concentration of sTie2 is decreased to a stable concentration level that does not increase by more than 0%-10%, 0%-5%, or 0%-1% once it has reached and maintained a concentration level different from the concentration level prior to administration of bone marrow-derived MSCs to a subject in need thereof.

[0042] In other embodiments, the novel biomarker comprises a change in the concentration level of VEGF in a subject in need thereof. In a preferred embodiment, the concentration of VEGF is increased in the serum, plasma, or blood of a subject in need thereof suffering from age-related frailty symptoms after administering a therapeutic amount of bone marrow-derived MSCs to said subject. The increase in VEGF concentration may be in the range of 0%-10%, 0.5%-10%, 1.0%-10%, 3%-10%, 5%-10%, 7%-10%, more than 0% to less than 10%, 10%-50%, 20%-50%, 30%-50%, or more than 50%. In a preferred embodiment, the VEGF concentration is increased to a stable concentration level, and when it reaches and is maintained at a concentration level different from the concentration level prior to administration of bone marrow-derived MSCs to a subject in need thereof, the concentration is not decreased by more than 0%-10%, 0%-5%, or 0%-1%. EXAMPLES

[0043] Example 1: A Phase 2b Randomized, Blinded, Placebo-Controlled Clinical Trial Evaluating the Safety and Efficacy of Lomecel-B Infusion in Aging Frail Patients Clinical Trials The Phase 2b study was a multicenter, randomized, double-blind, placebo-controlled, parallel-group study (Figure 1). The total duration for each subject following infusion of Lomecel-B cells was 12 months, with up to an additional 2 months for screening and baseline visits. The baseline demographics of participants enrolled in this study are seen in Table 1 below. [Table 1]

[0044] Lomecel-B and placebo: Lomecel-B is a preparation of allogeneic MSCs harvested from healthy young adult donors in compliance with Code of Federal Regulations 1271 and culture-expanded using current Good Manufacturing Practices (cGMP) under the Chemistry, Manufacturing, and Controls (CMC) section of an FDA-approved IND. Placebo consisted of the vehicle (PlasmaLyte-A with 1% human serum albumin) in which Lomecel-B MSCs were resuspended. Lomecel-B and placebo were prepared in identically labeled and identically appearing infusion bags and delivered by peripheral intravenous infusion in the outpatient setting.

[0045] Clinical evaluation: Clinical evaluations were performed at baseline, day 90, day 180, and day 270 of treatment.

[0046] Objectives and Evaluation Items: Primary objective: To determine whether Lomecel-B provides benefits in functional mobility and exercise tolerance compared with placebo in frail older patients.

[0047] Endpoint: Change in 6-minute walk distance at 180 days post-treatment compared to placebo.

[0048] Secondary objectives: (i) to assess the relationship between changes in physical performance and function-specific patient-reported outcomes (PROs), and changes in functional mobility and exercise tolerance, and (ii) to assess the relationship between changes in TNF-α and changes in functional mobility and exercise tolerance.

[0049] Evaluation item (i): PROMIS Physical Function Short Form 20a End point (ii): Serum TNF-α level Exploratory endpoints are listed in Table 2 below. [Table 2]

[0050] Statistical methods: Primary endpoint: The primary efficacy endpoint was the change from baseline in 6MWT at day 180. Each Lomecel-B arm was compared to placebo in pairwise comparisons using mixed-effects model for repeated measures (MMRM) procedures. Four pairwise comparisons used appropriate simple contrasts for the change from baseline at day 180 post-infusion (primary endpoint). Lomecel-B25 million formulations vs. placebo Lomecel-B 50 million formulations vs. placebo Lomecel-B 100 million formulations vs. placebo Lomecel-B 200 million formulations vs. placebo

[0051] As a secondary analysis of the primary efficacy endpoint, dose-response effects were analyzed using multiple comparisons and modeling approaches. To control for the overall type I error rate, statistical significance of each comparison was determined by the Hochberg method.

[0052] Secondary / Exploratory Endpoints: Statistical tests for the key secondary endpoint, PROMIS-Physical Function-Short Form 20a, and all other secondary / exploratory endpoints were performed without adjustment for multiple testing. Exploratory endpoints were analyzed using MMRM in a similar manner as the primary endpoint.

[0053] Biomarkers: Assays were performed to determine changes in concentrations of vascular endothelial growth factor (VEGF), TGF-β, TNF-α, and sTIE2.

[0054] Physical Function and Performance Assessment: Physical function and performance assessments were determined by examining changes in grip strength (dynamometer), Short Physical Performance Battery (SPPB) test, Forced Vital Capacity-1 second (FEV-1) test, Performance Oriented Mobility Assessment (POMA) test, 4-meter walking speed test (gait speed), and 6-minute walk distance test.

[0055] result: The major novel finding of this placebo-controlled study is that after rigorous testing and evaluation, the trial demonstrated a dose-response (DR) relationship between the amount of Lomecel-B cells administered and the change in 6MWT for each cohort (see Figure 3).

[0056] PROMIS Physical Function PRO scores were highly significantly correlated with the combined Lomecel-B group. Changes in PROMIS Physical Function and PROMIS Mobility scores for each cohort are seen in Tables 3 and 4 below. [Table 3] [Table 4]

[0057] DR was also found along with other biomarkers. Specifically, as the amount of administered Romecel-B cells increased, the levels of Tie2 and VEGF also increased (see FIG. 6B). Furthermore, as the amount of administered Romecel-B cells increased, the level of TGF-β decreased (see FIG. 6A).

[0058] Grip strength was shown to be increased in the dominant hand in the 100 million cell cohort when compared to both baseline measurements and the placebo cohort. Grip strength was also increased in the non-dominant hand in the 100 million cell cohort when compared to the placebo cohort. In addition to the resulting balance and gait scores, total balance and total gait were significantly increased in the 100 million cell cohort at day 270.

[0059] This trial was shown to be safe and effective, with no treatment-related adverse events of grade 3 or higher. The safety results of this trial are seen in Table 5 below. [Table 5]

[0060] Overall, this clinical trial demonstrated that administration of bone marrow-derived Romecel-B cells to subjects suffering from symptoms of age-related frailty can alleviate those symptoms and improve the subjects' quality of life.

[0061] Example 2: A randomized, double-blind, placebo-controlled study to evaluate the safety and efficacy of Lomecel-B injection in elderly frail patients Patient demographics Subjects met the inclusion criteria of being 70-85 years of age, cognitively unimpaired (Mini-Mental Scale exam score ≥ 24), and mild to moderate frailty as assessed by the Canadian Health and Ageing Survey (CSHA) Clinical Frailty Scale (CFS) (scores of 5 or 6, respectively). (See Juma S, Taabazuing MM, Montero-Odasso M. Clinical Frailty Scale in an Acute Medicine Unit: a Simple Tool That Predicts Length of Stay. Canadian geriatrics journal: CGJ 2016;19:34-9; Ritt M, Ritt JI, Sieber CC, Gassmann KG. Comparing the predictive accuracy of frailty, comorbidity, and disability for mortality: a 1-year follow-up in patients hospitalized in geriatric wards. Clinical interventions in aging 2017,12:293-304.) In addition, each subject underwent screening with a 6MWT over a distance between 200 and 400 m (see Cesari M, Bernabei R, Vellas B, et al. Challenges in the Development of Drugs for Sarcopenia and Frailty-Report from the International Conference on Frailty and Sarcopenia Research (ICFSR) Task Force. J Frailty Aging 2022;11:135-42) and had baseline serum TNF-α ≥ 2.5 pg / ml.Oversight was provided by a single Institutional Review Board (Western IRB; Puyallup, WA), an independent pharmacovigilance group (ProPharma Group; Washington, DC), a Data and Safety Monitoring Board (DSM21B) appointed by the National Institute on Aging (NIA) of the National Institutes of Health (NIH), and an independent clinical monitoring site (Syneos / Joule Inc.; Edison, NJ). IQVIA (Durham, NC) was the CRO for this trial.

[0062] Subjects received placebo or 2.5 × 10 7 Cells (“25M”), 5.0×10 7 Cells (“50M”), 1.0×10 8 cells ("100M"), or 2.0 x 10 8 Subjects were administered a dose of 1000 M cells ("200M"). Subjects were initially randomly assigned 1:1:1:1 to placebo, or 25 M, 50 M, or 100 M Lomecel-B, respectively, using a block size of 4. The addition of the Lomecel-B 200M group was introduced after 92 patients had been enrolled. Therefore, to balance this new group with the other groups within each study site, the randomization method was changed from center randomization to center-stratified randomization. To maintain blinding, treatment assignments were designed such that the "200M" group was the most likely to be randomized, but all other groups could also be randomized.

[0063] Lomecel-B cells and placebo: Lomecel-B and placebo were manufactured in accordance with the Chemistry, Manufacturing, and Controls (CMC) section of the Investigational New Drug (IND) application. Allogeneic MSCs for Lomecel-B were obtained from healthy young adult donors in compliance with Code of Federal Regulations Title 1271 and culture expanded to high homogeneity using current Good Manufacturing Practice (cGMP). Release criteria included: cell viability ≥ 70%; endotoxin ≤ 5 EU / mL; mycoplasma negative; United States Pharmacopeia. <71> Sterility test negative / no proliferation; CD73, CD90, and CD105 positive >= 95% by flow cytometry; CD45, CD11b, and CD19 positive <= 2%; CD34 positive <= 5%. Each dose was stored frozen until needed for infusion.

[0064] The placebo was PlasmaLyte-A containing 1% human serum albumin, which was the excipient used in the final formulation of Lomecel-B. Lomecel-B and placebo were delivered via peripheral venous infusion at approximately 2 mL / min (total volume of 80 mL administered over approximately 40 minutes) in the outpatient setting. To maintain blinding, Lomecel-B and placebo were prepared in indistinguishably labeled, identical appearing infusion bags.

[0065] Patient-Reported Outcomes (PROs): The Patient-Reported Outcomes (PRO) Measurement Information System (PROMIS) is a set of validated PROs developed by the NIH to assess physical, mental, and social health (see Cella D, Yount S, Rothrock N, et al. The Patient-Reported Outcomes Measurement Information System (PROMIS): progress of an NIH Roadmap cooperative group during its first two years. Medical care 2007;45:S3-S11). The adult PROMIS Physical Function-Short Form 20a (SF20) was used to assess patient-reported global physical function and was used as a secondary outcome measure because it has been shown to have good test-retest reliability and a minimal clinically meaningful change of 2 points (approximately 0.20 SD). PROMIS Mobility and PROMIS Upper Extremity were used as prespecified exploratory outcomes to assess mobility and upper body function, respectively.

[0066] Biomarkers To minimize circadian rhythm variation, blood draws were performed between 9:00 and 11:00 am (see Born J, Lange T, Hansen K, Molle M, Fehm HL. Effects of sleep and circadian rhythm on human circulating immune cells. Journal of Immunology 1997;158:4454-64). Serum and plasma samples were centrifuged on-site immediately after collection, aliquoted, flash frozen, and stored frozen until use. Biomarker analysis was performed in a double-blind fashion. To the best extent possible, samples from each patient were processed in parallel at all time points to minimize inter-experiment variation. A central laboratory performed Q 2(an IQVIA company; Durham NC) performed blood and urine safety analyses and sensitive electrochemiluminescence multiplex immunoassays of serum samples using the Meso QuickPlex system and V-Plex pro-inflammatory panels K151A9H and K15049D (Meso-Scale Discovery (MSD): Rockville, MD). Longeveron ran the V-Plex Angiogenesis Panel (K15190D) using the Meso QuickPlex system.

[0067] Safety assessment Safety evaluations included assessment of frequency, severity, and blinded relationship of adverse events (AEs) and serious AEs (SAEs) to testing products. AEs were coded by primary system organ class (SOC) and preferred term (PT) according to the Medical Dictionary for Regulatory Activities (MedDRA) 23.0. Treatment-emergent (TE-)AEs and TE-SAEs were summarized by number and percentage (n and %) of subjects in each SOC and PT. When multiple AEs were reported with the same preferred term, the AE with the strongest relationship was included in the summary by relationship and the AE with the most severe grade was included in the summary by severity table.

[0068] Statistical analysis: Unblinded statistical analyses were performed by an independent group of statisticians (Pharma Data Associates, LLC: Piscataway, NJ). Sample size was calculated based on the primary endpoint of change from baseline in 6MWT (see Oliva AA et al., Results and Insights from a Phase 1 Clinical Trial of Lomecel-B for Alzheimer's Disease. Alzheimer's & Dementia 2022: Accepted). Using a one-sided test α = 0.025 and an effect size of 0.75 (calculated as the difference in change from baseline in 6MWT between intervention group vs placebo divided by a common standard deviation of 75m), the power of 30 subjects per group was approximately 80% for a difference between treatment groups of 56m. This distance is shorter than the changes seen in previous Phase 1 / 2 trials (up to 76.6 m) (see Golpanian S, DiFede DL, Khan A, et al. Allogeneic Human Mesenchymal Stem Cell Infusions for Aging Frailty. J Gerontol A Biol Sci Med Sci 2017;72:1505-12; Tompkins BA, DiFede DL, Khan A, et al. Allogeneic Mesenchymal Stem Cells Ameliorate Aging Frailty: A Phase II Randomized, Double-Blind, Placebo-Controlled Clinical Trial. J Gerontol A Biol Sci Med Sci 2017;72:1513-22).

[0069] Efficacy endpoint analyses were performed on the modified intention-to-treat (MITT) population, defined as all randomized subjects who received an infusion and completed at least one post-baseline assessment for the primary efficacy endpoint. A repeated measures mixed-effects model (MMRM) was used to compare each Lomecel-B arm pairwise with placebo, and least squares means (LSMs) were calculated for the comparison of changes in Lomecel-B arms with changes in placebo. Correlations between repeated measures were modeled using an unstructured variance-covariance matrix. Dose-response effects were calculated using the multiple comparison procedure modeling (MCP-Mod) method, a hybrid approach combining hypothesis testing and modeling to analyze phase 2 dose-ranging studies to find the appropriate dose(s) for confirmatory phase 3 trials (see Bretz F, Pinheiro JC, Branson M. Combining multiple comparisons and modeling techniques in dose-response studies. Biometrics 2005;61:738-48; Menon SM, Zink RC. Modern Approaches to Clinical Trials Using SAS: Classical, Adaptive, and Bayesian Methods: SAS Institute;2105). Candidate models included linear, quadratic, exponential, Emax, and sigmoidal Emax dose-response models. In addition to adjusted p-values, Akaike's Information Criterion (AIC) was used to evaluate the most parsimonious and predictive models. A smaller AIC means a better model. Model means of dose-response curves were plotted with 95% confidence intervals. The safety population for evaluating safety was defined as all subjects who received an infusion.

[0070] To account for multiple testing of different dose groups versus placebo, the step-up Hochberg method was used for the primary analysis of the primary endpoint.Secondary analysis of the primary endpoint was the dose-response effect by the MCP-Mod method.

[0071] Simple linear regression and correlations were calculated for absolute values ​​and changes from baseline between 6MWT and patient-reported outcome questionnaires.

[0072] result: Between August 2017 and February 2020, 365 patients were screened, and 155 met all inclusion / exclusion criteria and were randomized. Reasons for screening dropout were TNF-α < 2.5 pg / mL (n = 57; 26.9%), hepatitis B virus positivity (n = 28; 13.2%), 6MWT distance out of range (n = 19; 9.0%), HbA1c > 8.0% (n = 19; 9.0%), and various other reasons (each < 9%). Seven subjects were excluded from the analysis due to withdrawal before infusion. The remaining 148 subjects received a single infusion of study product (Lomecel-B or placebo) and were included in the safety population analysis. Of these, 137 completed the trial (95.8%), 5 withdrew of their own choice (3.4%), 4 were lost to follow-up (2.7%), and 2 died during the study (1.4%). Of these, 143 had at least one follow-up visit and constituted the mITT population for efficacy analyses. Table 6 shows balanced characteristics among the five groups. Participants' mean age ranged from 74.3 to 76.8 years, 20.6% to 53.3% were female, and the mean CFS score was 5.1, with overall mild frailty. [Table 6]

[0073] Baseline 6MWT distance was comparable across all groups, approximately 300 meters across all groups (see Table 6). The first component of the primary endpoint was the change in 6MWT for each individual dose of Lomecel-B compared to placebo at 6 months post-infusion (Figure 2A, see Table 7). A formal dose-response analysis, the prespecified second component of the primary endpoint, demonstrated a statistically significant relationship of increasing Lomecel-B dose to incremental change in 6MWT distance (Figure 2B). All five candidate dose-response models tested were significant (p<0.05 each), and the dose-response was best modeled by a linear curve with a significant p-value (p=0.0321) and the smallest AIC within the dose range tested. [Table 7]

[0074] The highest dose of Lomecel-B (2x10 8 The difference in 6MWT between the 50M and 200M doses of Lomecel-B and placebo was 41.3 m (95% CI: -2.4 to 84.9 M p = 0.0635) at 6 months. When analyzing the difference at 9 months, the changes in the 50M and 200M doses of Lomecel-B reached significance, reflecting a continuous time-dependent separation between the active treatment group and placebo (see Figure 2A). At 9 months after infusion, the difference in change between the Lomecel-B 200M group and placebo was 63.4 m (95% CI [17.1, 109.6] m; p = 0.0077). In addition, the change in 6MWT from baseline is also shown (see Figure 2A). The change from baseline in 6MWT was significant at 6 and 9 months.

[0075] Patient-reported outcomes (PROs) PROMIS Physical Function SF20 was used as a secondary outcome measure to assess changes in patient-perceived global physical function. Similarly, PROMIS Mobility and PROMIS Upper Limb were used as pre-specified exploratory outcomes tested in advance to assess mobility and upper body function, respectively. Changes in 6MWT and PROMIS Physical Function SF20 were correlated. The Pearson correlation coefficient at 6 months after injection was 0.3124 (p=0.0002, Figure 4A). Similarly, the correlation coefficient between 6MWT and PROMIS Mobility at 6 months after injection was 0.3046 (p=0.0003, Figure 4B), and the correlation coefficient between 6MWT and PROMIS Upper Limb was 0.2318 (p=0.0070, Figure 4C).

[0076] sTie2 as a biomarker of activity The predefined goal of this clinical trial was to identify biomarkers that may predict functional outcomes of Lomecel-B. Among the various potential biomarkers investigated in this study, soluble Tie2 (sTie2) was identified as a potential biomarker that met these criteria. sTie2 was decreased in the Lomecel-B200M group (see Figure 5A), which was significantly different from baseline at 3 months post-infusion (-484.1 pg / mL; 95% CI [-925.33, -42.90] pg / mL; p=0.0318). At 9 months post-infusion, the Lomecel-B200M group differed from placebo by -936.9 pg / mL (95% CI [-1640.3, -233.4] pg / mL; p = 0.0095), as did the Lomecel-B50M group (-601.2 pg / mL; 95% CI [-1137.2, -65.2] pg / mL; p = 0.0283), and the Lomecel-B100M group (-755.4 pg / mL; 95% CI [-1294.1, -216.8] pg / mL; p = 0.0064). Dose-response analysis showed optimal modeling to a sigmoidal Emax (p = 0.0175) that appeared to plateau at the Lomecel-B50M dose (see Figure 5B). Furthermore, changes in sTie2 correlated with changes in 6MWT (r=-0.1850; p=0.0397) (see Figure 5C).

[0077] Safety and Clinical Events No safety concerns were raised regarding this study by either the NIA appointed DSMB or the pharmacovigilance officer. Overall, the proportion of subjects with TE-SAEs was comparable across the different study arms (see Table 8). There were two deaths during the study. A pulmonary embolism occurred 296 days after infusion in the Lomecel-B100M group, and a complication of cerebral arteriosclerosis / coronary artery disease / aspiration pneumonia occurred 167 days after infusion in the placebo group. There were no SAEs attributable to the study product. Two infusions were temporarily suspended (both in the Lomecel-B25M group) but continued to completion, and both subjects completed follow-up visits. All AEs occurring during the infusion were deemed not product related by the investigator and DSMB. There were no statistically significant differences in the rates of falls, fractures, hospitalizations, and admissions to medical facilities in either the Lomecel-B group versus placebo. [Table 8]

[0078] One of the major novel findings of this study is that a single infusion of Lomecel-B led to a dose-dependent increase in walking distance in older adults with mild to moderate frailty when compared with placebo. The increases seen in the highest dose group were just above the clinical threshold for significance (see Shoemaker MJ, Curtis AB, Vangsnes E, Dickinson MG. Clinically meaningful change estimates for the six-minute walk test and daily activity in individuals with chronic heart failure. Cardiopulmonary physical therapy journal 2013;24:21-9; Kwok BC, Pua YH, Mamun K, Wong WP. The minimal clinically important difference of six-minute walk in Asian older adults. BMC geriatrics 2013;13:23; Perera S, Mody SH, Woodman RC, Studenski SA. Meaningful change and responsiveness in common physical performance measures in older adults. Journal of the American Geriatrics Society 2006;54:743-9) and correlated with improvements in patient self-reported outcomes. Additionally, serum levels of sTie2, a factor associated with vascular dysfunction, were improved (decreased) in a dose-dependent manner with Lomecel-B. These findings are consistent with the possibility that Lomecel-B may be able to treat frailty by improving patients' quality of life, improving mobility, and reducing dependency on others.

[0079] These findings are also consistent with previous studies in frail patients in which a single infusion of allogeneic MSCs suggested improvements in walking distance (see Golpanian S et al., 2017; Tompkins et al., 2017). A further predefined objective of this trial was to establish whether Lomecel-B exhibited a dose-response effect. Indeed, a clear and significant dose-response relationship for increases in 6MWT was evident at 6 months after treatment, supporting evidence of bioactivity. Furthermore, at 9 months after a single infusion, improvements were sustained and reached significance versus placebo, which had begun to decline from baseline. The larger differences between treatment and placebo groups observed at 9 months may indicate ongoing and sustained bioactivity, suggesting that future trials should monitor responses to treatment for more than 6 months.

[0080] Our findings are consistent with previous studies in frail patients in which a single infusion of allogeneic MSCs suggested improvements in walking distance (see Golpanian S et al., 2017; Tompkins et al., 2017). A further predefined objective of this trial was to establish whether Lomecel-B exhibited a dose-response effect. Indeed, a clear and significant dose-response relationship for increases in 6MWT was evident at 6 months after treatment, supporting evidence of bioactivity. Furthermore, at 9 months after a single infusion, improvements were sustained and reached significance versus placebo, which had begun to decline from baseline. The larger differences between treatment and placebo groups observed at 9 months may indicate ongoing and sustained bioactivity, suggesting that future studies should monitor responses to treatment for more than 6 months.

[0081] PROMIS measures provide insight into the impact of frailty on function and mobility and are essential for patient-centered clinical decisions. As part of this study, we sought to determine the extent of patients' perception of improved physical function. Indeed, the PROMIS Physical Function SF20, an assessment of global physical function, showed improvements that correlated significantly with increases in 6MWT distance (see Figure 4A). Furthermore, PROMIS Mobility showed an even more significant correlation (see Figure 4B). These results indicate that the positive effects of Lomecel-B were not limited to objective physical measures but generalized to individual older adults' perceptions of improved function.

[0082] Another goal of this study was to identify biomarkers that may correlate with biological activity. Among a set of potential biomarkers, we found that sTIE2 levels were more closely associated with Lomecel-B infusion in a dose-response manner. TIE-2 is a receptor tyrosine kinase present on microvascular endothelium and endothelial progenitor cells and is activated through binding of angiopoietins, Ang1, and Ang2 (see Sack KD, Kellum JA, Parikh SM. The Angiopoietin-Tie2 Pathway in Critical Illness. Crit Care Clin 2020;36:201-16). Cleavage of the extracellular domain of Tie2 (sTie2) mediated by matrix metalloproteinase (MMP) 14 (see Idowu TO, Etzrodt V, Seeliger B, et al. Identification of specific Tie2 cleavage sites and therapeutic modulation in experimental sepsis. eLife 2020;9) can be detected in serum and increased levels indicate endothelial dysfunction. In the context of the mechanism of action of Lomecel-B, the reduction in sTie2 levels supports a pro-vascular activity in frailty and is consistent with the suggestive pro-vascular activity of Lomecel-B for Alzheimer's disease. 25Prevention of Tie2 cleavage is a biologically plausible effect of Lomecel-B, since MSCs are known to secrete high levels of tissue inhibitors of MMPs (TIMPs) (see Lozito TP, Jackson WM, Nesti LJ, Tuan RS. Human mesenchymal stem cells generate a distinct pericellular zone of MMP activities via binding of MMPs and secretion of high levels of TIMPs. Matrix Biol 2014;34:132-43). Future studies, including assessment of other vascular biomarkers and determination of endothelial function, are needed to confirm these findings.

[0083] In summary, the results of the study demonstrate that age-related frailty may be responsive to infusion of Lomecel-B, which leads to clinically meaningful dose-dependent improvements in the 6MWT that correlate with physical function PROs, and also reveals a potential dose-dependent effect on the mechanistically relevant biomarker, sTie2. These data support the advancement of Lomecel-B as a potential treatment for the unmet medical need of frailty. Furthermore, this trial clearly demonstrates a dose-dependent relationship for the cell-based therapy and provides evidence of bioactivity.

Claims

1. A pharmaceutical composition for treating and alleviating symptoms of age-related frailty in a subject with symptoms of age-related frailty, comprising a therapeutically effective amount of bone marrow-derived mesenchymal stem cells (MSCs).

2. A pharmaceutical composition for treating age-related frailty or inhibiting the progression of age-related frailty in a subject with symptoms of age-related frailty, comprising a therapeutically effective amount of allogeneic mesenchymal stem cells (MSCs).

3. The pharmaceutical composition of claim 1 or 2, wherein the treatment includes determining whether a change occurs in the concentration of a biomarker in the subject by comparing the concentration of the biomarker before and after administration of the pharmaceutical composition containing the therapeutically effective amount of MSCs.

4. The pharmaceutical composition of claim 3, wherein the treatment includes determining whether an improvement occurs in the subject's functional mobility and / or exercise tolerance by comparing the subject's functional mobility and / or exercise tolerance before and after administration of the pharmaceutical composition containing the therapeutically effective amount of MSCs.

5. The pharmaceutical composition of claim 3, wherein the treatment includes determining whether an improvement occurs in the subject's ability to perform activities of daily living by comparing the subject's ability to perform activities of daily living before and after administration of the pharmaceutical composition containing the therapeutically effective amount of MSCs.

6. 6. The pharmaceutical composition of claim 5, wherein the improvement in the subject's ability to perform activities of daily living is determined by examining the subject's PROMIS physical function score before and after administration of the pharmaceutical composition comprising the therapeutically effective amount of MSCs.

7. The pharmaceutical composition of claim 3, wherein the treatment includes determining whether there is an improvement in the subject's PROMIS mobility score by comparing the subject's PROMIS mobility score before and after administration of the pharmaceutical composition containing the therapeutically effective amount of MSCs.

8. The pharmaceutical composition of claim 3, wherein the treatment includes determining whether an improvement occurs in the subject's grip strength by comparing the subject's grip strength before and after administration of the pharmaceutical composition containing the therapeutically effective amount of MSCs.

9. The pharmaceutical composition of claim 3, wherein the treatment includes determining whether an improvement occurs in the subject's gait or balance by comparing the subject's gait or balance before and after administration of the pharmaceutical composition containing the therapeutically effective amount of MSCs.

10. The pharmaceutical composition of claim 3 , wherein the biomarker comprises a pro-inflammatory cytokine.

11. 11. The pharmaceutical composition of claim 10, wherein the pro-inflammatory cytokine is selected from the group consisting of TNF-α, TGF-β, IL-1β, IL-2, D-dimer, C-reactive protein (CRP), and combinations thereof.

12. The pharmaceutical composition described in claim 3, wherein administration of the pharmaceutical composition to a subject having symptoms of age-related frailty reduces the concentration of pro-inflammatory cytokines in the subject before administration by 0.5% to 10%, 5% to 10%, 10% to 50%, or more than 50%.

13. The pharmaceutical composition of claim 3 , wherein the biomarker comprises an anti-inflammatory cytokine.

14. 14. The pharmaceutical composition of claim 13, wherein the anti-inflammatory cytokine is selected from the group consisting of IL-8, soluble IL-2 receptor alpha (sIL-2Rα), IL-4, IL-10, IL-12, TNF-α stimulated gene 6 (TSG-6), and combinations thereof.

15. The pharmaceutical composition described in claim 3, wherein administration of the pharmaceutical composition to a subject having symptoms of age-related frailty increases the concentration of anti-inflammatory cytokines in the subject by 0.5% to 10%, 5% to 10%, 10% to 50%, or more than 50% compared to before administration.

16. The pharmaceutical composition of claim 3 , wherein the biomarker comprises soluble Tie2 (sTie2).

17. The pharmaceutical composition described in claim 3, wherein administration of the pharmaceutical composition to a subject having symptoms of age-related frailty reduces the concentration of sTie2 in the subject before administration by 0.5% to 10%, 5% to 10%, 10% to 50%, or more than 50%.

18. The pharmaceutical composition of claim 3 , wherein the biomarker comprises Tie2.

19. The pharmaceutical composition described in claim 3, wherein administration of the pharmaceutical composition to a subject having symptoms of age-related frailty increases the Tie2 concentration in the subject by 0.5% to 10%, 5% to 10%, 10% to 50%, or more than 50% compared to the concentration before administration.

20. The pharmaceutical composition of claim 3 , wherein the biomarker comprises VEGF.

21. The pharmaceutical composition described in claim 3, wherein administration of the pharmaceutical composition to a subject having symptoms of age-related frailty increases the concentration of VEGF in the subject before administration by 0.5% to 10%, 5% to 10%, 10% to 50%, or more than 50%.

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