Candidates For Cell Therapy Research Articles

Proteomic analysis of human Wharton's jelly mesenchymal stem/stromal cells and human amniotic epithelial stem cells: a comparison of therapeutic potential.

Perinatal stem cells have prominent applications in cell therapy and regenerative medicine. Among them, human Wharton's jelly mesenchymal stem/stromal cells (hWJMSCs) and human amniotic epithelial stem cells (hAESCs) have been widely used. However, the distinction in the therapeutic potential of hWJMSCs and hAESCs is poorly understood. In this study, we reported the phenotypic differences between these two distinct cell types and provided the first systematic comparison of their therapeutic potential in terms of immunomodulation, extracellular matrix (ECM) remodelling, angiogenesis and antioxidative stress using proteomics. The results revealed that the two cell types presented different protein expression profiles and were both promising candidates for cell therapy. Both types of cells demonstrated angiogenic and antifibrotic potential, whereas hAESCs presented superior immunological tolerance and antioxidant properties, which were supported by a series of relevant in vitro assays. Our study provides clues for the selection of appropriate cell types for diverse indications in cell therapy, which contributes to the advancement of their clinical translation and application.

Human Umbilical Cord-Mesenchymal Stem Cells Promote Extracellular Matrix Remodeling in Microglia.

Human mesenchymal stem cells modulate the immune response and are good candidates for cell therapy in neuroinflammatory brain disorders affecting both adult and premature infants. Recent evidence indicates that through their secretome, mesenchymal stem cells direct microglia, brain-resident immune cells, toward pro-regenerative functions, but the mechanisms underlying microglial phenotypic transition are still under investigation. Using an in vitro coculture approach combined with transcriptomic analysis, we identified the extracellular matrix as the most relevant pathway altered by the human mesenchymal stem cell secretome in the response of microglia to inflammatory cytokines. We confirmed extracellular matrix remodeling in microglia exposed to the mesenchymal stem cell secretome via immunofluorescence analysis of the matrix component fibronectin and the extracellular crosslinking enzyme transglutaminase-2. Furthermore, an analysis of hallmark microglial functions revealed that changes in the extracellular matrix enhance ruffle formation by microglia and cell motility. These findings point to extracellular matrix changes, associated plasma membrane remodeling, and enhanced microglial migration as novel mechanisms by which mesenchymal stem cells contribute to the pro-regenerative microglial transition.

EMF treatment delays mesenchymal stem cells senescence during long-term in vitro expansion by modulating autophagy.

Bone marrow mesenchymal stem cells (BMSCs) are widely used in tissue engineering and regenerative medicine as seed cells. Due to low amount in bone marrow, BMSCs must be expanded and cultured in vitro before application. However, the senescence of stem cell caused by long-term in vitro culture greatly limits its efficacy of transplantation. In this study, we propose an approach based on electromagnetic fields (EMF) treatment to rejuvenate aged BMSCs due to long-term in vitro culture. Aged BMSCs were treated with sinusoidal EMF (50Hz, 0.4mT), and stem cell senescence, cell proliferation, cell differentiation, cell stemness and autophagy level were detected. Additionally, aged BMSCs-laden hydrogels were transplanted into the rat critical-sized calvarial defect with or without EMF treatment. The bone formation was evaluated 8weeks after surgery. Our results indicated that the BMSCs age significantly after long-term in vitro passaging. The self-renew, multiple differentiation capacity, senescence phenotypes and stemness of aged BMSCs are partly reversed by EMF treatment with a frequency of 50Hz and strength of 0.4mT. Moreover, declined autophagy level is observed in BMSCs during long-term in vitro passaging and BMSCs senescence is closely associated with autophagy regulation. Additionally, the mechanistic investigation reveals that EMF treatment rejuvenate senescent BMSCs by enhancing autophagy. Furthermore, EMF treatment significantly promote the therapeutic effect of long-term passaged BMSCs on bone formation in vivo. Overall, our study identifies a practical approach for the rejuvenation of old BMSCs and may provide a promising candidate in tissue engineering and stem cell therapy.

Feeder cell training shapes the phenotype and function of in vitro expanded natural killer cells.

Natural killer (NK) cells are candidates for adoptive cell therapy, and the protocols for their activation and expansion profoundly influence their function and fate. The complexity of NK cell origin and feeder cell cues impacts the heterogeneity of expanded NK (eNK) cells. To explore this, we compared the phenotype and function of peripheral blood-derived NK (PB-NK) and umbilical cord blood-derived NK (UCB-NK) cells activated by common feeder cell lines, including K562, PLH, and 221.AEH. After first encounter, most PB-NK cells showed degranulation independently of cytokines production. Meanwhile, most UCB-NK cells did both. We observed that each feeder cell line uniquely influenced the activation, expansion, and ultimate fate of PB eNK and UCB eNK cells, determining whether they became cytokine producers or killer cells. In addition, they also affected the functional performance of NK cell subsets after expansion, that is, expanded conventional NK (ecNK) and expanded FcRγ- NK (eg-NK)cells. Hence, the regulation of eNK cell function largely depends on the NK cell source and the chosen expansion system. These results underscore the significance of selecting feeder cells for NK cell expansion from various sources, notably for customized adoptive cell therapies to yield cytokine-producing or cytotoxic eNK cells.

Modulation of human induced neural stem cell-derived dopaminergic neurons by DREADD reveals therapeutic effects on a mouse model of Parkinson’s disease

BackgroundStem cell-based therapy is a promising strategy for treating Parkinson’s disease (PD) characterized by the loss of dopaminergic neurons. Recently, induced neural stem cell-derived dopaminergic precursor cells (iNSC-DAPs) have been emerged as a promising candidate for PD cell therapy because of a lower tumor-formation ability. Designer receptors exclusively activated by designer drugs (DREADDs) are useful tools for examining functional synaptic connections with host neurons.MethodsDREADD knock-in human iNSCs to express excitatory hM3Dq and inhibitory hM4Di receptors were engineered by CRISPR. The knock-in iNSCs were differentiated into midbrain dopaminergic precursor cells (DAPs) and transplanted into PD mice. The various behavior test such as the Apomorphine-induced rotation test, Cylinder test, Rotarod test, and Open field test were assessed at 4, 8, or 12 weeks post-transplantation with or without the administration of CNO. Electrophysiology were performed to assess the integrated condition and modulatory function to host neurons.ResultsDREADD expressing iNSCs were constructed with normal neural stem cells characteristics, proliferation ability, and differentiation potential into dopaminergic neuorns. DAPs derived from DREADD expressing iNSC showed matched function upon administration of clozapine N-oxide (CNO) in vitro. The results of electrophysiology and behavioral tests of transplanted PD mouse models revealed that the grafts established synaptic connections with downstream host neurons and exhibited excitatory or inhibitory modulation in response to CNO in vivo.ConclusioniNSC-DAPs are a promising candidate for cell replacement therapy for Parkinson’s disease. Remote DREADD-dependent activation of iNSC-DAP neurons significantly enhanced the beneficial effects on transplanted mice with Parkinson’s disease.

Effects of Human Neural Stem Cells Overexpressing Neuroligin and Neurexin in a Spinal Cord Injury Model.

Recent studies have highlighted the therapeutic potential of stem cells for various diseases. However, unlike other tissues, brain tissue has a specific structure, consisting of synapses. These synapses not only transmit but also process and refine information. Therefore, synaptic regeneration plays a key role in therapy of neurodegenerative disorders. Neurexins (NRXNs) and neuroligins (NLGNs) are synaptic cell adhesion molecules that connect pre- and postsynaptic neurons at synapses, mediate trans-synaptic signaling, and shape neural network properties by specifying synaptic functions. In this study, we investigated the synaptic regeneration effect of human neural stem cells (NSCs) overexpressing NRXNs (F3.NRXN) and NLGNs (F3.NLGN) in a spinal cord injury model. Overexpression of NRXNs and NLGNs in the neural stem cells upregulated the expression of synaptophysin, PSD95, VAMP2, and synapsin, which are synaptic markers. The BMS scores indicated that the transplantation of F3.NRXN and F3.NLGN enhanced the recovery of locomotor function in adult rodents following spinal cord injury. Transplanted F3.NRXN and F3.NLGN differentiated into neurons and formed a synapse with the host cells in the spinal cord injury mouse model. In addition, F3.NRXN and F3.NLGN cells restored growth factors (GFs) and neurotrophic factors (NFs) and induced the proliferation of host cells. This study suggested that NSCs overexpressing NRXNs and NLGNs could be candidates for cell therapy in spinal cord injuries by facilitating synaptic regeneration.

IL-10-modulated dendritic cells from birch pollen- and hazelnut-allergic patients facilitate Treg-mediated allergen-specific and cross-reactive tolerance.

Approximately 70% of individuals allergic to birch pollen (Bet v 1.01 [Bet]) develop a secondary food allergy (e.g., hazelnut: Cor a 1.04 [Cor]), due to allergen cross-reactivity. However, standard immunotherapy for type I allergies often does not improve the food allergy sufficiently. We analyzed the allergen-specific and cross-reactive suppressive capacity of primary human regulatory T cells (Treg) induced by autologous IL-10-modulated dendritic cells (IL-10 DC) invitro and invivo. CD4+ T cells of patients with birch pollen and associated hazelnut allergies were differentiated into Bet-specific or non-specific induced Treg (iTreg). After Bet- or Cor-specific restimulation the phenotype, proliferation, and suppressive capacity of iTreg subsets were analyzed. iTreg function was further investigated in humanized mouse models of airway and intestinal allergy, generated by engraftment of peripheral blood mononuclear cells from allergic donors into immunodeficient animals. After IL-10 DC priming and allergen-specific restimulation (Bet or Cor), non-specific control iTreg remained anergic, whereas Bet-specific iTreg proliferated extensively and exhibited a regulatory phenotype (enhanced expression of CTLA-4, PD-1, TNFR2, IL-10). Accordingly, activated Bet-specific iTreg displayed a high capacity to suppress Bet- and Cor-induced responder Th2 cell responses invitro, indicating induction of both allergen-specific (birch) and cross-reactive tolerance (hazelnut). Invivo, the beneficial effect of Bet-specific iTreg was verified in humanized mouse models of allergic airway and intestinal inflammation, resulting in reduced allergen-induced clinical symptoms, and immune responses. Human IL-10 DC-induced iTreg facilitate allergen-specific and cross-reactive tolerance. Therefore, they are potential candidates for regulatory cell therapy in allergic and autoimmune diseases.

Plasticity Comparison of Two Stem Cell Sources with Different Hox Gene Expression Profiles in Response to Cobalt Chloride Treatment during Chondrogenic Differentiation.

The limited self-repair capacity of articular cartilage is a challenge for healing injuries. While mesenchymal stem/stromal cells (MSCs) are a promising approach for tissue regeneration, the criteria for selecting a suitable cell source remain undefined. To propose a molecular criterion, dental pulp stem cells (DPSCs) with a Hox-negative expression pattern and bone marrow mesenchymal stromal cells (BMSCs), which actively express Hox genes, were differentiated towards chondrocytes in 3D pellets, employing a two-step protocol. The MSCs' response to preconditioning by cobalt chloride (CoCl2), a hypoxia-mimicking agent, was explored in an assessment of the chondrogenic differentiation's efficiency using morphological, histochemical, immunohistochemical, and biochemical experiments. The preconditioned DPSC pellets exhibited significantly elevated levels of collagen II and glycosaminoglycans (GAGs) and reduced levels of the hypertrophic marker collagen X. No significant effect on GAGs production was observed in the preconditioned BMSC pellets, but collagen II and collagen X levels were elevated. While preconditioning did not modify the ALP specific activity in either cell type, it was notably lower in the DPSCs differentiated pellets compared to their BMSCs counterparts. These results could be interpreted as demonstrating the higher plasticity of DPSCs compared to BMSCs, suggesting the contribution of their unique molecular characteristics, including their negative Hox expression pattern, to promote a chondrogenic differentiation potential. Consequently, DPSCs could be considered compelling candidates for future cartilage cell therapy.

Manufacturing mesenchymal stromal cells in a microcarrier-microbioreactor platform can enhance cell yield and quality attributes: case study for acute respiratory distress syndrome

Mesenchymal stem and stromal cells (MSCs) hold potential to treat a broad range of clinical indications, but clinical translation has been limited to date due in part to challenges with batch-to-batch reproducibility of potential critical quality attributes (pCQAs) that can predict potency/efficacy. Here, we designed and implemented a microcarrier-microbioreactor approach to cell therapy manufacturing, specific to anchorage-dependent cells such as MSCs. We sought to assess whether increased control of the biochemical and biophysical environment had the potential to create product with consistent presentation and elevated expression of pCQAs relative to established manufacturing approaches in tissue culture polystyrene (TCPS) flasks. First, we evaluated total cell yield harvested from dissolvable, gelatin microcarriers within a microbioreactor cassette (Mobius Breez) or a flask control with matched initial cell seeding density and culture duration. Next, we identified 24 genes implicated in a therapeutic role for a specific motivating indication, acute respiratory distress syndrome (ARDS); expression of these genes served as our pCQAs for initial in vitro evaluation of product potency. We evaluated mRNA expression for three distinct donors to assess inter-donor repeatability, as well as for one donor in three distinct batches to assess within-donor, inter-batch variability. Finally, we assessed gene expression at the protein level for a subset of the panel to confirm successful translation. Our results indicated that MSCs expanded with this microcarrier-microbioreactor approach exhibited reasonable donor-to-donor repeatability and reliable batch-to-batch reproducibility of pCQAs. Interestingly, the baseline conditions of this microcarrier-microbioreactor approach also significantly improved expression of several key pCQAs at the gene and protein expression levels and reduced total media consumption relative to TCPS culture. This proof-of-concept study illustrates key benefits of this approach to therapeutic cell process development for MSCs and other anchorage-dependent cells that are candidates for cell therapies.

Expansion and characterization of immune suppressive CD56(bright)Perforin(-) regulatory-like natural killer cells in chronic graft-versus-host disease

Background: Chronic graft-versus-host disease (cGvHD) is a major cause of morbidity and mortality after Hematopoietic Stem Cell Transplantation (HSCT). Previously, in large patient cohorts, we identified increased numbers of CD56brightPerforin− regulatory-like NK cells (NKreg-like) associated with cGvHD suppression. Thus, we hypothesized that NKreg-like cells may be a potential candidate for cGvHD cell therapy. Aim: To expand NKreg-like cells while maintaining regulatory phenotype and function. Methods: Total NK cells were first expanded with IL-2, which was then combined with rapamycin, Transforming Growth Factor Beta 1 (TGF-β1), NECA (Adenosine A2A receptor (A2AR) agonist), metformin, or dexamethasone, to prevent change in cell phenotype/function. The functional characteristics were evaluated via T cell suppression assays and the phenotype was measured using flow cytometry. The optimal expansion protocol was compared in terms of function and metabolism for three NK expansion media, and cells from cord vs. peripheral blood. Further, expanded NKreg-like cell gene expression was characterized using bulk RNA sequencing. Finally, NKreg-like cells were differentiated from CD34+ hematopoietic stem and progenitor cells (HSPCs) and compared in terms of proliferation and function. Results: The expansion of total NK cells found the addition of TGF-β1 and/or NECA with the pulsing of rapamycin in IL-2 containing media to prevent NKreg-like differentiation (up to 200-fold expansion). Expanded NKreg-like cells maintained a phenotype, transcriptome, and T cell suppression similar to freshly isolated NKreg-like cells. NKreg-like expansion was greatest in the Immunocult media (up to 300-fold), and NKreg-like cells from peripheral blood demonstrated significantly greater proliferation than cells isolated from cord blood (65-fold). The metabolic profile of NKreg-like and cytolytic NK cells appeared similar at baseline, though rapamycin induced a shift to oxidative over glycolytic metabolism. Further, we demonstrated that suppressive NKreg-like cells may alternatively be expanded from CD34+ cells isolated from cord blood, reaching an average 340-fold expansion. Conclusions: In conclusion, our studies have optimized two alternative expansion approaches for deriving functional NKreg-like cells. Additionally, evaluating the transcriptomic and metabolic characteristics provides useful information regarding NKreg-like cell function and differentiation. With further optimization and in vivo validation, we may work towards preparing these cells as a therapy for cGvHD.

The Effect of Exosomes Isolated from Poly (I:C) Treated Human Wharton's Jelly Mesenchymal Stem Cells on CD4+CD25+Foxp3+ Regulatory T Cells.

Mesenchymal stem cells (MSCs) are a potential cell therapy candidate for autoimmune and inflammatory diseases due to their multilineage capacity and immune modulating function. MSCs exert immunomodulatory effects on target cells through the secretion of exosomes. Inflammatory conditions such as Toll-like receptors (TLRs) engagement can change the biological functions and immunomodulatory activities of MSCs and the contents of exosomes derived from MSCs are changed. Regulatory T-cells (Treg) are crucial for maintaining immune cell homeostasis and self-tolerance. Our study aimed to investigate the impact of isolated exosomes from hWJ-MSCs that were treated with Poly (I:C) on regulatory CD4 CD25 Foxp3 T-cells. MSCs were harvested from human umbilical cord Wharton's Jelly by explant method. Stem cells were treated by Polyinosinic-polycytidylic acid sodium salt (Poly (I:C)) for 48 hours. Exosomes were extracted from supernatant of cells and Scanning electron microscopy (SEM) and Dynamic light scattering (DLS) were performed for them. Peripheral blood mononuclear cells (PBMCs) isolated from the healthy donors were stimulated with PHA (Phytohemagglutinin) and co-cultured with Poly (I:C) treated hWJ-MSCs derived exosome and untreated hWJ-MSCs derived exosome or without hWJ-MSCs-derived exosome for 6 days. Then, frequency of CD4+CD25+ Foxp3+ regulatory T cells was measured by flow cytometry. Our results showed that exosomes isolated from Poly (I:C) treated hWJ-MSCs significantly increased frequency of CD4+CD25+ Foxp3+ regulatory T cells compared to the untreated hWJ-MSCs derived exosome group and control group. Stimulation by TLR3 improved the anti-inflammatory features of exosomes that were derived from hWJ-MSCs by increasing the frequency of Treg cells.

Flame spray pyrolyzed carbon-encapsulated Au/Fe3O4 nanoaggregates enabled efficient photothermal therapy and magnetic hyperthermia of esophageal cancer cells.

Multifunctional magneto-plasmonic nanoparticles with magnetic hyperthermia and photothermal therapy could kill cancer cells efficiently. Herein, carbon-encapsulated Au/Fe3O4 (Au/Fe3O4@C) was fabricated using an enclosed flame spray pyrolysis. The nanostructures, including an Fe3O4 core (51.9-55.2nm) with a decreasing carbon shell thickness and an Au core (4.68-8.75nm) coated with 2-4 graphite layers, were tailored by tuning the C2H4 content in the reacting gas mixture. Saturation magnetization (33.7-48.2emu/g) and optical absorption were determined. The carbon shell facilitated the dispersion of Au/Fe3O4 and restrained their laser-induced and magnetic field-induced coalescence and growth. Au/Fe3O4@C exhibited excellent magnetic resonance imaging capability (91.4mM-1 s-1) and photothermal performance (65.4°C for 0.8mg/mL Au/Fe3O4@C at a power density of 1.0W/cm2 after 300s near-IR laser irradiation (808nm)). Moreover, the combined application of photothermal and magnetic-heating properties reduced the required intensity of both laser and magnetic field compared to the intensity of separate situations. Our work provides a unique, intriguing approach to preparing multicomponent core/shell nanoaggregates that are promising candidates for esophageal cancer cell therapy.

Human amniotic epithelial stem cell is a cell therapy candidate for preventing acute graft-versus-host disease.

Graft-versus-host disease (GVHD), an immunological disorder that arises from donor T cell activation through recognition of host alloantigens, is the major limitation in the application of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Traditional immunosuppressive agents can relieve GVHD, but they induce serious side effects. It is highly required to explore alternative therapeutic strategy. Human amniotic epithelial stem cells (hAESCs) were recently considered as an ideal source for cell therapy with special immune regulatory property. In this study, we evaluated the therapeutic role of hAESCs in the treatment of GVHD, based on our previous developed cGMP-grade hAESCs product. Humanized mouse model of acute GVHD (aGVHD) was established by injection of huPBMCs via the tail vein. For prevention or treatment of aGVHD, hAESCs were injected to the mice on day -1 or on day 7 post-PBMC infusion, respectively. We showed that hAESCs infusion significantly alleviated the disease phenotype, increased the survival rate of aGVHD mice, and ameliorated pathological injuries in aGVHD target organs. We demonstrated that hAESCs directly induced CD4+ T cell polarization, in which Th1 and Th17 subsets were downregulated, and Treg subset was elevated. Correspondingly, the levels of a series of pro-inflammatory cytokines were reduced while the levels of the anti-inflammatory cytokines were upregulated in the presence of hAESCs. We found that hAESCs regulated CD4+ subset polarization in a paracrine mode, in which TGFβ and PGE2 were selectively secreted to mediate Treg elevation and Th1/Th17 inhibition, respectively. In addition, transplanted hAESCs preserved the graft-versus-leukemia (GVL) effect by inhibiting leukemia cell growth. More intriguingly, hAESCs infusion in HSCT patients displayed potential anti-GVHD effect with no safety concerns and confirmed the immunoregulatory mechanisms in the preclinical study. We conclude that hAESCs infusion is a promising therapeutic strategy for post-HSCT GVHD without compromising the GVL effect. The clinical trial was registered at www.clinicaltrials.gov as #NCT03764228.

Evaluation of Human Platelet Lysate as an Alternative to Fetal Bovine Serum for Potential Clinical Applications of Stem Cells from Human Exfoliated Deciduous Teeth

In recent years, there has been a surge in demand for and research focus on cell therapy, driven by the tissue-regenerative and disease-treating potentials of stem cells. Among the candidates, dental pulp stem cells (DPSCs) or human exfoliated deciduous teeth (SHED) have garnered significant attention due to their easy accessibility (non-invasive), multi-lineage differentiation capability (especially neurogenesis), and low immunogenicity. Utilizing these stem cells for clinical purposes requires careful culture techniques such as excluding animal-derived supplements. Human platelet lysate (hPL) has emerged as a safer alternative to fetal bovine serum (FBS) for cell culture. In our study, we assessed the impact of hPL as a growth factor supplement for culture medium, also conducting a characterization of SHED cultured in hPL-supplemented medium (hPL-SHED). The results showed that hPL has effects in enhancing cell proliferation and migration and increasing cell survivability in oxidative stress conditions induced by H2O2. The morphology of hPL-SHED exhibited reduced size and elongation, with a differentiation capacity comparable to or even exceeding that of SHED cultured in a medium supplemented with fetal bovine serum (FBS-SHED). Moreover, no evidence of chromosome abnormalities or tumor formation was detected. In conclusion, hPL-SHED emerges as a promising candidate for cell therapy, exhibiting considerable potential for clinical investigation.

The impact of high nicotine concentrations on the viability and cardiac differentiation of mesenchymal stromal cells: a barrier to regenerative therapy for smokers.

Background: Current treatment methods are not successful in restoring the lost cardiomyocytes after injury. Stem cell-based strategies have attracted much attention in this regard. Smoking, as a strong cardiovascular risk factor, not only affects the cardiac cells adversely but also deteriorates the function of stem cells. Since mesenchymal stem cells (MSCs) are one of the popular candidates in cardiovascular disease (CVD) clinical trials, we investigated the impact of nicotine on the regenerative properties (viability and cardiac differentiation) of these cells. Methods: MSCs were isolated from rat bone marrow and characterized based on morphology, differentiation capability, and the expression of specific mesenchymal markers. The MTT assay was used to assess the viability of MSCs after being exposed to different concentrations of nicotine. Based on MTT findings and according to the concentration of nicotine in smokers' blood, the growth curve and population doubling time were investigated for eight consecutive days. Cells were treated with 5-azacytidine (an inducer of cardiac differentiation), and then the expressions of cardiac-specific markers were calculated by qPCR. Results: MSCs were spindle-shaped, capable of differentiating into adipocyte and osteocyte, and expressed CD73 and CD90. The viability of MSCs was reduced upon exposure to nicotine in a concentration- and time-dependent manner. The growth curve showed that nicotine reduced the proliferation of MSCs, and treated cells needed more time to double. In addition, the expressions of GATA4 and troponin were downregulated in nicotine-treated cells on day 3. However, these two cardiac markers were overexpressed on day 7. Conclusion: Nicotine decreased normal growth and reduced the expression of cardiac markers in MSCs. This aspect is of eminent importance to smokers with cardiovascular disease who are candidates for stem cell therapy.

Abstract 55: CD70-specific CAR-T cell therapy for the treatment of glioblastoma

Abstract Despite the remarkable success of chimeric antigen receptor (CAR)-T cell treatment for patients with hematologic malignancies, this method has yet failed to confer meaningful survival benefits to patients suffering from glioblastoma (GB), the most lethal type of brain tumor. This highlights the need to develop novel CAR-T cell approaches for this disease. CD70 is a member of the tumor necrosis factor receptor (TNFR) superfamily. Although absent on normal brain tissue, it is ectopically expressed in a substantial fraction of GB patients, indicating its suitability as a CAR-T cell therapy candidate. In this study, we generated CD70-targeting CAR-T cells and tested their cytotoxicity in vitro and in vivo. First, we detected CD70 in a panel of primary GB cell lines and to investigate its role, it was overexpressed in human and murine GB cells. In a syngeneic glioma model, C57BL/6J mice injected with CD70-overexpressing GL261 cells developed significantly larger tumors compared to the control counterparts. Additionally, RNA-sequencing revealed that CD70-overexpression in the same cells led to higher expression levels of immunosuppressive marker CD200 and lower levels of tumor inhibition genes Prkg2 and Sh3bgrl2, suggesting a tumor-promoting role in GB. For our immunotherapeutic intervention, we designed CD70-targeting CAR-T cell constructs featuring different co-stimulatory domains (CD27, CD28 or 4-1BB) and successfully transduced primary T-Cells from healthy donors. In an in vitro co-culture, all CAR-T cells recognized and eliminated primary cancer cells in a target-dependent and donor-independent manner, while secreting high levels of Granzyme B. The killing capacity of these cells was further highlighted in a 3D system in which in vitro-generated cortical organoids were treated with CAR-T cells after being infiltrated by CD70-expressing or control cells. Immunofluorescence (IF) staining and enzyme-linked immunosorbent assay (ELISA) revealed increased levels of Granzyme B and IFN-γ in all treated organoids previously infiltrated by CD70+ tumor cells. Importantly, generated CAR-T cells showed high specificity and efficiency in killing CD70+ tumors in vivo in the brains of immunodeficient mice. Namely, 80% of NSG mice orthotopically implanted with CD70+ GB cells and subsequently treated by CARs but not mock-transduced T-Cells showed complete tumor remission by the end of the experimental endpoint, determined by bioluminescence imaging (BLI). IF analysis of these brains showed high levels of apoptotic marker cleaved caspase-3, enhanced effector cell presence and significantly lower tumor cell occupancy compared to control-treated animals. In conclusion, we provide evidence in favor of utilization of CAR-T cells against CD70-expressing gliomas. Based on these findings, a phase-I clinical trial to assess the safety and efficacy of autologous CD70-specific CAR-T cells for relapsed CD70+ GB is being planned. Citation Format: Alexandros Kourtesakis, Hiu Nam Hannah Chow, Dennis Alexander Agardy, Eileen Bailey, Sandra Horschitz, Ammar Jabali, Rainer Will, Denise Reibold, Sonja Pusch, Christoph Schifflers, Manuel Fischer, Ling Hai, Dirk C. Hoffmann, Yu-Chan Chih, Robin Wagener, Leon Kaulen, Philipp Koch, Michael Breckwoldt, Michael Schmitt, Wolfgang Wick, Tim Sauer, Tobias Kessler. CD70-specific CAR-T cell therapy for the treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 55.

Abstract 3604: NKX101, an allogeneic off-the-shelf NKG2D CAR-NK cell therapy, has potent in vitro cytotoxicity against patient-derived AML leukemic stem cells and non-leukemic stem cell blasts

Abstract Introduction: Acute myeloid leukemia (AML) is a malignancy of immature myeloid cells characterized by rapid proliferation of abnormal myeloblasts. Treatment options for patients with AML are limited, especially for relapsed/refractory (r/r) disease. Leukemic cells with stem cell features, known as leukemic stem cells (LSCs), have been implicated as the origin of relapse in minimal residual disease positive (MRD+) AML (Khaldoyanidi et al. 2022, Kamel et al. 2022). NKX101 is an allogeneic, off-the-shelf, healthy donor-derived chimeric antigen receptor (CAR) NK cell therapy candidate engineered to express an NKG2D CAR and membrane bound IL-15. In a clinical trial, NKX101 has shown promise for the treatment of r/r AML, including generating complete responses (CR) with MRD negativity (MRD−). In this study, we utilize a flow cytometry-based approach to (i) evaluate the cell surface expression of NKG2D-Ls on normal cells vs. primary AML blasts, (ii) assess the expression pattern of NKG2D-Ls on AML blast subsets, and (iii) determine NKX101 cytotoxic killing of LSC and non-LSC AML blasts. Methods: NKX101 cells were generated from peripheral blood leukopaks from healthy donors. Cryopreserved bone marrow mononuclear cells (BMMCs) were obtained from AML patients (n=20) and healthy donors (n=10) in accordance with approved IRB protocols. AML somatic mutation status was determined using an Illumina TruSight® Myeloid Sequencing Panel. NKX101 cytotoxicity against patient-derived AML blasts was assessed using a flow cytometry-based assay that measured specific cytotoxicity and AML blast/LSC markers. NKG2D-L expression was determined using flow cytometry with antibodies specific to individual NKG2D-Ls. Results: Utilizing patient-derived primary samples, we demonstrate that NKG2D-Ls are expressed on AML blast cells and expressed at higher levels on AML blast cells compared to healthy normal hematopoietic cells (p = 0.002). Furthermore, we show that NKG2D-Ls are expressed at similar levels on LSCs compared to non-LSC AML blast populations. Target cell specific flow cytometry-based cytotoxicity assays revealed that NKX101 potently kills patient-derived AML blasts in a dose-dependent manner. Moreover, NKX101 had similar cytotoxic potency against both LSC and non-LSC AML blasts. Conclusions: In summary, we show that NKG2D-Ls are expressed on the surface of both LSC and non-LSC AML blasts and at levels above those observed in normal hematopoietic cells. Additionally, the ability of NKX101 to potently kill AML LSC blasts suggests that NKG2D-L targeting may be a viable mechanism for eliminating LSCs in the blood and bone marrow of patients with r/r AML. Taken together, these data support NKG2D-Ls as promising therapeutic targets for AML and support further investigation of NKX101 CAR-NK therapy for the treatment of r/r AML. Citation Format: Kyle Hansen, Cynthia Cho, Nishi Kothari, David Shook, James Trager. NKX101, an allogeneic off-the-shelf NKG2D CAR-NK cell therapy, has potent in vitro cytotoxicity against patient-derived AML leukemic stem cells and non-leukemic stem cell blasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3604.

Abstract 3603: NKX101, an allogeneic off-the-shelf CAR NK cell therapy targeting NKG2D-Ls, has potent anti-leukemic activity alone or in combination with Ara-C

Abstract Introduction: NKG2D-Ligands (NKG2D-Ls) are a family of immunomodulatory proteins that are upregulated on damaged or transformed cells but have limited expression on normal tissues. Natural killer (NK) cells target and eliminate NKG2D-L+ cells through NKG2D receptor binding and subsequent activation. NKX101 is an allogeneic, off-the-shelf healthy donor-derived chimeric antigen receptor (CAR) NK cell therapy candidate engineered to express an NKG2D-L targeting CAR and membrane-bound IL-15 for extended persistence. In a clinical trial, NKX101 treatment combined with modified lymphodepletion incorporating cytarabine (Ara-C) has shown promise for the treatment of relapsed/refractory (r/r) AML, including generating complete responses (CR) with MRD negativity (MRD−). To support the rationale of NKX101 combined with modified lymphodepletion, we developed assays to (i) investigate the cytotoxic activity of NKX101 against individual NKG2D-Ls, (ii) determine the effect of Ara-C pre-treatment on NKG2D-L expression on AML cell lines, and (iii) evaluate the combined anti-leukemic activity of NKX101 and Ara-C. Methods: NKX101 cells were generated from peripheral blood leukopaks from healthy donors. CHO-K1 cells were engineered to stably express individual NKG2D-Ls or transduced with empty vector controls and were combined with NKX101 cells for cytotoxicity experiments. AML cell lines were treated with increasing levels of Ara-C and evaluated for NKG2D-L expression by flow cytometry. To measure NKX101 cytotoxicity against AML cell lines, NucRed™-labeled target cells were pre-treated with Ara-C using IC50 and IC90 concentrations and co-cultured with NKX101. Specific cell killing of target cells was measured using an Incucyte® instrument for five days. Assessment of the contribution of each agent was determined using a dose-response matrix of increasing concentrations of NKX101 and Ara-C to generate specific cytotoxicity values. Combination effects were analyzed using the SynergyFinder Plus web application. Results: Expression of a single NKG2D-L was sufficient to trigger NKX101 cytotoxicity against target cell lines, further validating NKG2D-Ls as therapeutic targets. Flow cytometry analysis revealed that pre-treatment with Ara-C upregulated NKG2D-Ls on AML cell lines in a dose-dependent manner. NKX101 showed enhanced anti-leukemic activity against AML cells pre-treated with Ara-C compared to untreated controls, demonstrating that the combination can enhance NKX101 potency. Assessment of the combination of NKX101 and Ara-C using a dose-response matrix revealed that the two modalities combine to potently kill AML cells in a synergistic manner, supporting continued investigation of NKX101 CAR NK cell therapy after lymphodepletion incorporating Ara-C for the treatment of r/r AML. Citation Format: Cynthia Cho, Nikki Kimura, Jessica Sood, Damie Juat, Max Zhang, Carmel Chan, Tao Geng, Kyle Hansen, James Trager. NKX101, an allogeneic off-the-shelf CAR NK cell therapy targeting NKG2D-Ls, has potent anti-leukemic activity alone or in combination with Ara-C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3603.

Roles of PD-L1 in human adipose-derived mesenchymal stem cells under inflammatory microenvironment.

Mesenchymal stem cells (MSCs) display unique homing and immunosuppression features which make them promising candidates for cell therapy in inflammatory disorders. It is known that C-X-C chemokine receptor type 4 (CXCR4, also known as CD184) is a critical receptor implicated in MSCs migration, and the protein programmed death ligand-1 (PD-L1) is involved in MSC's immunosuppression. However, it remains unclear how the molecular mechanisms regulate PD-L1 expression for migration and immunosuppression of MSCs under the inflammatory microenvironment. In this article, we used the human adipose-derived mesenchymal stem cells (hADMSCs) treated with lipopolysaccharide (LPS) as an in vitro inflammatory model to explore the roles of PD-L1 on the migration and immunosuppression of MSC. Our results demonstrate that in hADMSCs, LPS significantly increased PD-L1 expression, which mediated the migration of the LPS-treated hADMSCs via CXCR4. In addition, we found that the increased PD-L1 expression in the LPS-treated hADMSCs inhibited B cell proliferation and immunoglobulin G secretion through nuclear factor-κB. Our study suggests that the PD-L1 plays critical roles in the homing and immunosuppression of MSCs which are a promising cell therapy to treat inflammatory diseases.

High-Specificity CRISPR-Mediated Genome Engineering in Anti-BCMA Allogeneic CAR T Cells Suppresses Allograft Rejection in Preclinical Models.

Allogeneic chimeric antigen receptor (CAR) T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR T cells. Key considerations in the development of allogeneic CAR T cell therapies include prevention of graft-vs-host disease (GvHD) and suppression of allograft rejection. Here, we describe preclinical data supporting the ongoing first-in-human clinical study, the CaMMouflage trial (NCT05722418), evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is a hypoimmunogenic, allogeneic anti-B-cell maturation antigen (BCMA) CAR T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR T cells were engineered to prevent endogenous HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition, T-cell receptor (TCR) expression was disrupted at the TCR alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell cocultures derived from patients with multiple myeloma. In addition, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer cell-mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M-HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma. See related Spotlight by Caimi and Melenhorst, p. 385.