Leiden, The Netherlands, April 3, 2024: Pharming Group N.V. (“Pharming”) (Euronext Amsterdam: PHARM/Nasdaq: PHAR) announces that Pharming’s management will participate in the following investor conferences in the month of April:
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Pharming Group to participate in April investor conferences
Genetic algorithm
We used the GA to optimize the conductance of each current so that the AP model reproduced the experimental values in previous studies24,29. We executed the GA optimization using a program implemented in C# with reference to the method of Bot et al.55, and its type was a real-coded GA. We evaluated the degree of adaptation of each model in the population using the score calculated using Eq.(1). We calculated the AP for each model 60s after the initial state. We performed numerical integration to compute APs using the forward Euler method with a time step of 0.01ms. The initial values of ion concentration inside and outside the cell, and temperature were equivalent to the conditions of the experiments. We fixed the intracellular potassium and sarcoplasmic reticulum calcium concentrations to accelerate convergence. We estimated the cell volume from the cell surface area data24,56. We used the same value as that in the Paci model for the ratio of the sarcoplasmic reticulum volume to the cytoplasmic volume23. We used a model population with random values assigned to each conductance as the starting generation. The upper and lower scaling limits were 0.010.0 for GNa, GCaL, and Gf in the ventricular-type model and 0.05.0 for all others. We describe the details of the GA optimization of AP models in the Supplementary Methods.
To estimate magnetic signals from iPS-CMs, we simulated the 2D electrical activity of the cell population. We set the intracellular ion concentrations using adult mouse cardiac AP model values57. We determined the extracellular ion concentrations from the composition of the culture medium and set the temperature to room temperature (RT: 24C). The temperature coefficients (Q10) used in the AP models referred to values from the published literature58. We computed the solution to the partial differential Eq.(2) using the CrankNicolson method, with the spatial step set to x=y=60m and the time step set to t=0.01ms. We determined the averaged cellular resistivity to reproduce the conduction velocity measured in neonatal rat cardiac cell sheets40 (Supplementary Methods and Supplementary Fig. S7). The list of parameters used in the simulation is summarized in Supplementary Table S3. We calculated the magnetic field using BiotSavart's law from the currents flowing in the cells at each time point. We estimated the observed waveforms using integration in the area of each pickup coil. We assumed that the cancellation component of the magnetic field caused by the extracellular return current was negligibly small because the volume of the medium was sufficiently large relative to the spreading of cultured cells. We also checked how much the observed waveforms were affected when the cell position was shifted from directly under the sensor. As a result, we confirmed that a displacement of2mm in the x-axis or y-axis directions had almost no effect on the measurements (Supplementary Fig. S8).
The procedure for dataset preparation is as follows: A peak region (250ms) was cut from the magnetic signals estimated using simulation and subjected to random stretching and scaling. Non-peak regions between peak regions were linearly interpolated to make data of 120s each. For the background noise, four data of the x component of the magnetic field with no current applied (for artificial signal experiments) or eight data of the y component with no cell sample placed (for cell experiments) were used in equal proportions within each dataset. Random time shifts were performed in the superposition of these magnetic signals and noise data. Even when the cycle length and amplitude were fixed, this shift brought diversity to the dataset. Finally, datasets (n=160 for artificial signal experiments or 640 for cell experiments) were generated, including three data types in a 1:1:2 ratio: positive peak direction, negative peak direction, and background noise only. Representative waveforms are shown in Supplementary Fig. S9.
The window used in the spectral calculation with the FSST33 was the Kaiser window, with a size of 512 points. The sidelobe attenuation was 13.6dB. The real and imaginary parts of the spectral were input as separate features. The input values were pre-standardized by subtracting the mean and dividing by the standard deviation. Training was iterated for up to 60 epochs (one epoch means one round of data). The network was validated using the validation data for each epoch. If the validation loss exceeded the previous minimum value more than ten times, it was decided that there was no further improvement and training was stopped. The initial learning rate was set to 0.001 and the learning rate was dropped by a factor of 0.1 every 20 epochs. The training data were divided into segments of 10s lengths and the mini-batch size (a subset of the training data used in one step to evaluate the gradient of the loss function and update the weights) was set to 16.
We calculated the AUROC36 to evaluate network classification performance. The AUROC is the area under the curve plotted with the false positive rate on the horizontal axis and the true positive rate on the vertical axis. The AUROC is 1.0 when separation performance is best and 0.5 when classification is performed randomly. In this study, we defined each data point as positive if it was peak (P) or negative if it was non-peak (N).
From the output label data, we plotted a histogram of the lengths of segments labeled as class P (Fig.4f). Using this histogram as a reference, we estimated the appropriate distribution of peak region lengths, set a lower limit, and identified segments longer than this threshold as peak regions (Fig.1c). We used the average count number for the analysis of samples measured multiple times. We obtained the average waveform by superimposing magnetic signals of 175ms before and after the center position of each peak region and averaging their amplitudes. Then, we repeated the adaptive correlation filter59 ten times to correct for positional fluctuations.
A vector-type SQUID magnetometer12,15 was applied to measure magnetic fields. The vector-type SQUID magnetometer had an axial-type first-order gradiometric pickup coil with a diameter of 15.5mm and two planar-type first-order gradiometric squared pickup coils of 915.5 mm2 and 1115.5 mm2. The baseline length of each gradiometric pickup coil was 50mm. The three gradiometric pickup coils were oriented perpendicular to each other and assembled on a cylindrical bobbin. Three Ketchen-type low-temperature SQUIDs were individually coupled to each pickup coil and simultaneously detected the three independent components of the magnetic field: Bx, By, and Bz. The SQUID readouts were connected to double-integrator type flux-locked loop (FLL) circuits for output linearization and dynamic range improvement. The total noise level, including environmental noise, was 1020 fT/Hz at 10Hz. The SQUID magnetometer was installed in a glass-fiber reinforced plastic (GFRP) cryostat with an MSB. The MSB comprised two 1mm thick mu-metal layers with double front doors. The shielding factor of the MSB was more than 40dB at 10Hz. The GFRP cryostat consisted of a cylindrical main body that stored 6-L liquid helium and a narrow GFRP tube that dropped from the bottom of the main body. The main body was installed in the ceiling of the MSB and only the GFRP tube penetrated the MSB through a hole in its top. The SQUID magnetometer was installed at the bottom end of the GFRP tube and placed at the center of the MSB.
The cell sample was placed on a height-adjustable stage made of non-magnetic materials and adjusted to 3mm from the bottom edge of the pickup coil. Measurements were taken at room temperature, and the FLL readout signals were digitally recorded at the sampling rate of 1kHz with HPF at 3Hz, LPF at 100Hz, and notch filters at 60Hz.
We kept the resistance fixed and varied the output voltage of the function generator to adjust the current that generated magnetic signals. With no filtering, we increased the voltage until the peak of the magnetic signal could be identified by visual inspection and recorded the peak amplitude at that point. Based on that value, we adjusted the voltage to generate the desired magnetic signals. We enhanced the artificial signal (2.74) so that the signal-to-noise ratio was equivalent to that in the cell sample experiment. To confirm the validity of this procedure, we compared the amplitude spectrum densities of the background noise between the artificial signal experiment and the cell sample experiment (Supplementary Fig. S4). Although differences in amplitude existed, the spectral distribution had the same trend within the range of 3.540Hz used to train the LSTM networks.
We implemented the scaled template technique following previous research22. We slid the template (the event waveform of interest to detect) along the time series data and scaled it to fit the data at each position. Then, we divided the template scaling factor by the standard error of the time series data, which was the detection criterion, and we considered the event waveform of interest to be detected when this criterion exceeded a threshold value. The template was a peak waveform of 250ms in length cut from the magnetic signal estimated using numerical simulation, which we also used as the training data in deep learning. To compare the two methods without bias, we set the threshold so that the number of detected peaks from background noise was equal to that of deep learning.
The mouse iPS cell line iPS-MEF-Ng-20D-17 (Expressing GFP by Nanog promoter)44, established by the Center for iPS Cell Research and Application, Kyoto University, was provided by the RIKEN BRC through the National BioResource Project of the Ministry of Education, Culture, Sports, Science, and Technology, Japan. For the culture method, we referred to previous studies44,60,61. To maintain the undifferentiated state of iPS cells, MEFs (EmbryoMax Primary Mouse Embryonic Fibroblasts, PMEF-NL, Neo Resistant, Strain FVB; purchased from Sigma-Aldrich, St Louis, MO, USA), in which cell proliferation was arrested by mitomycin C (Nacalai Tesque, Kyoto, Japan) treatment, were cocultured as feeder cells. The maintenance medium was composed of Dulbecco's modified Eagle's medium (Sigma-Aldrich) with 15% fetal bovine serum (Equitech-Bio Inc., Kerrville, TX, USA), 50 U/ml penicillin, 50g/ml streptomycin (Sigma-Aldrich), 2mM L-glutamine (Sigma-Aldrich), nonessential amino acids (100) (Sigma-Aldrich), 0.1mM 2-mercaptoethanol (FUJIFILM Wako Chemicals, Osaka, Japan), and 0.1% human leukemia inhibitory factor (FUJIFILM Wako Chemicals). The medium was refreshed daily and iPS cells were passaged every two days. Colonies were detached with 0.25% trypsin/1mM EDTA (FUJIFILM Wako Chemicals), dispersed in cell suspension, counted, and 1.0106 cells were seeded into MEFs on 60mm plates.
Based on previous studies37,62, cardiomyocyte differentiation was induced by forming EB. The differentiation medium was Iscove's modified Dulbecco's medium (Sigma-Aldrich) containing 20% fetal bovine serum, 50 U/ml penicillin, 50g/ml streptomycin, 2mM L-glutamine, nonessential amino acids (100), and 0.1mM 2-mercaptoethanol. Mouse iPS cells were suspended at 1.5104 cells/ml in the differentiation medium and seeded 0.2ml into each well of a 96-well U-shaped-bottom microplate (Nunclon Sphera; Thermo Fisher Scientific, Waltham, MA, USA). The plates had a cell-nonadherent surface treatment, which allowed uniform and stable EBs to form. For further differentiation, the culture was switched from floating to adherent on day 5. Plastic dishes of 100mm diameter and MEA (Alpha MED Scientific, Osaka, Japan) were used for magnetic measurement, and glass bottom dishes (AGC Techno Glass, Shizuoka, Japan) were used for fluorescence microscopy. These dishes were coated with 0.1 w/v% gelatin solution (FUJIFILM Wako Chemicals) and one EB was transplanted at the center of each dish. Beating areas began to appear on day 7. Magnetic measurement was performed during days 1921 when the area of differentiated cells was extensive and synchronized beating was observed. Fluorescence microscopy was also performed at this time. To ensure that one peak corresponded to the electrical activity of the entire cell population, samples with a single beating area larger than 3mm square were selected for measurement. To bring the cells closer to the sensor, the cylinder of the MEA was excised to a height of 1mm. For comparison with iPS-CMs, MEFs were also cultured in cloning rings with an inner diameter of 5mm. In the experiment to detect the drug's chronotropic effects from magnetic signals, the medium was replaced with a medium supplemented with isoproterenol at a final concentration of 10M, and magnetic signals were measured from iPS-CMs after incubation for 30min.
Cardiomyocytes were immunostained on day 19 of differentiation, and the expression of cardiomyocyte marker cardiac troponin T and connexin 43 that forms gap junctions was confirmed. iPS-CMs were fixed in 4% paraformaldehyde for 20min at 4C, followed by blocking with 5% goat serum (Nichirei, Tokyo, Japan) and 0.1% Triton-X diluted in Dulbecco's phosphate buffered saline (DPBS) for 20min at RT. The cells were washed three times for 5min with DPBS and incubated with a primary antibody diluted in DPBS containing 1% goat serum for 1h at RT and then overnight at 4C. The primary antibodies were rabbit polyclonal IgG anti cardiac troponin T antibody (1.4g/mL; Proteintech, Rosemont, IL, USA) and rabbit polyclonal IgG anti connexin 43 antibody (10g/mL; Thermo Fisher Scientific). The cells were washed three times for 5min with DPBS with shaking and further incubated with the secondary antibody Alexa fluor 546 goat anti rabbit IgG (Invitrogen, Carlsbad, CA, USA; 1:1000 dilution in DPBS/0.05% Triton X-100) for 30min at RT. The cells incubated with connexin 43 antibody were also treated with Alexa fluor 488 Phalloidin (Thermo Fisher Scientific; 1:50 dilution in DPBS/0.05% Triton X-100) and stained for actin filaments. The cells were washed three times with tris buffered saline for 5min and once with DPBS for 5min, and immersed in 4',6-diamidino-2-phenylindole (DAPI)-added anti-fading agent (Nacalai Tesque). Observation and imaging were performed with an IX71 fluorescence microscope (Olympus, Tokyo, Japan).
We measured FPs using MEA38. We selected the electrode near the center of the beating area and recorded the potential difference between it and a reference electrode not in contact with the cells. To avoid noise when measuring simultaneously with magnetic signals, we output the electrical signals from the probe externally through an IC clip and did not use the attached connector. When measuring FP only, we used it. We performed the measurement at room temperature and recorded data at the sampling rate of 1kHz with HPF at 0.16Hz, LPF at 160Hz, and notch filters at 60Hz.
Deep learning network training and data classification were performed in MATLAB (Mathworks Inc., Natick, MA, USA). The GA for parameter optimization of the AP model and the numerical simulation of the electrical activity of cardiomyocytes were performed using our programs implemented in C#.
Data are presented as meanstandard error of the mean (SEM). Comparisons between two groups were analyzed using the unpaired t-test unless otherwise indicated. For comparisons of three or more groups, when equal variances could be assumed, one-way ANOVA was used, followed by Tukey's test as a post hoc test. When equal variances could not be accepted, the BrownForsythe correction was performed, followed by the GamesHowell test as a post hoc test. Differences between data were considered statistically significant at p<0.05.
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Detection of biomagnetic signals from induced pluripotent stem cell-derived cardiomyocytes using deep learning with ... - Nature.com
Human subjects
PD patients were recruited from the outpatient clinic for Movement Disorders of the Department of Neurology of the Leiden University Medical Center (Leiden, the Netherlands) and nearby university and regional hospitals. All participants fulfilled the U.K. Parkinsons Disease Society Brain Bank criteria for idiopathic PD. The study was approved by the medical ethics committee of the Leiden University Medical Center (P12.194/NV/ib), and written informed consent was obtained from all PD patients.
Fibroblasts were isolated at Leiden University Medical Center from skin biopsies derived from the ventral side of the upper leg and cultured under highly standardized conditions as previously described in [14]. Peripheral whole blood was collected from PD patients at Leiden University Medical Center and PBMCs were isolated at the Department of Molecular Genetics at the Erasmus Medical Center in Rotterdam. Control iPSC were obtained from the Eramsus MC iPS Core facility.
Peripheral whole blood from 24 age-matched healthy controls (age >55 years) was obtained from Sanquin Rotterdam (NVT0585.00 Mantel, NVT0585.01 Annex).
Bioinformatic analysis was performed using the Parkinsons Progression Markers Initiative (PPMI) database.
To generate erythroblasts, PD patients peripheral blood mononuclear cells (PBMC) were extracted from 10ml of freshly extracted blood with the use of Lympholyte-H (Cedarline) and Leucosep polypropylene tubes (227290, Greiner) according to manufacturers indications. Briefly, blood was diluted in PBS at a 1:2 ratio and loaded on a 15mL Lympholyte Leucosep tube. Blood was centrifuged at 800g for 25min with no brakes at 4C. Upon removal of the plasma, the PBMC enriched cell fraction was collected, washed several times with sterile PBS and upon PBMCs were cultured in StemSpan SFEM medium (Stemcell Technologies) containing 2mM Ultraglutamine (Lonza), 1% Nonessential aminoacids (NEAA), 1% penicillin/streptomycin, 50ng/ml Stem Cell Factor, 2U/ml Erythropoietin, 1uM Dexamethasone (Sigma), 10ng/ml Interleukin-3 (R&D Systems), 10ng/ml Interleukin-6 (R&D Systems), 40ng/ml IGF-1 (R&D Systems) and 50ug/ml Ascorbic Acid (Merck) for 69 days refreshing half of the medium every other day starting from day 2. Erythroblasts were isolated when reaching 6070% of the total cell population by gradient centrifugation at 1000g for 20minutes at room temperature over Percoll (GE Healthcare). Isolated erythroblasts were frozen in FBS containing 10% DMSO at 80C. Metabolic analysis was performed within 2 days after thawing.
PD patients fibroblasts used in this study were prepared and isolated at Leiden University Medical Center from skin biopsies derived from the ventral side of the upper leg and cultured under highly standardized conditions as previously described in [14]. The study was approved by the medical ethics committee of the Leiden University Medical Center, and written informed consent was obtained from all PD patients.
Fibroblasts were reprogrammed to pluripotent stem cells using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (A16517, Thermo Fisher) according to the manufacturers protocol.
Human iPSC lines were generated as previously described [18]. Briefly, to generate embryoid bodies with neuroepithelial outgrowths (EBs), iPSC colonies were dissociated with 2mg/mL collagenase IV and transferred to non-adherent plates in hESC medium.
(Dulbeccos modified Eagles medium (DMEM)/F12 (Thermo Fisher Scientific), 20% knockout serum (Thermo Fisher Scientific), 1% minimum essential medium/non-essential amino acid (NEAA, Sigma-Aldrich, St Louis, MO, USA), 7nlml1 -mercaptoethanol (Sigma-Aldrich), 1% L-glutamine (Thermo Fisher Scientific) and 1% penicillin/streptomycin (P/S, Thermo Fisher Scientific) supplemented with 10M SB-431542 (Ascent Scientific), 1M dorsomorphin (Tocris), 3M CHIR 99021 (Axon Medchem) and 0.5M Purmorphamine (Alexis) on a shaker in an incubator at 37C/5% CO2. On the second day, medium was replaced with N2B27 medium [DMEM-F12/neurobasal 50:50 (Thermo Fisher Scientific), 1% P/S, 1:100 B27 supplement lacking vitamin A (Thermo Fisher Scientific) and 1:200 N2 supplement (Thermo Fisher Scientific)] containing 10M SB-431542, 1M dorsomorphin, 3M CHIR99021 and 0,5M Purmorphamine. On day 4, N2B27 medium was replaced and supplemented with 3M CHIR99021, 0.5M Purmorphamine, and 150M Ascorbic Acid (Sigma).
On day 6, EBs were slightly triturated and plated on Matrigel-coated (Matrigel - 354277, Corning) plates at a density 1015 EB per well containing smNPC expansion medium (N2B27 medium containing 3M CHIR 99021, 200M Ascorbic Acid, 0.5M Purmorphamine) and expanded for 5 passages before final differentiation. The medium was refreshed every other day.
smNPC were dissociated with Accutase at RT, diluted, and seeded on Poly-D-lysine Matrigel-coated cover glasses in a 12-well plate at the concentration of 5104 cells per well in the Patterning medium [N2B27 medium containing 1ng/mL GDNF (Peprotech), 2ng/ml BDNF (Pepotech), 200M Ascorbic Acid and 0.5M Smoothened Agonist (SAG Pepotech)]. The medium was refreshed every 2 days.
At day 8, the medium wash switched to the Maturation medium containing N2B27 medium, 2ng/ml GDNF, 2ng/ml BDNF, 1ng/mL TGF-b3 (Peprotech), 200M ascorbic Acid and 5ng/ml of ActivinA for the first feeding and 2ng/ml ActivinA for the following feedings. Medium change occurred every third day.
Erythroblasts and iPSCs were washed and resuspended in FC buffer (HBSS w/o calcium and magnesium + 0.5% BSA) and incubated with PE Mouse Anti-Human CD44 antibody (1:25, BD, 550989), FITC Mouse Anti-Human CD71 antibody (1:50, BD, 555536), or 7-AAD (Thermo Fisher, A1310) for 30min at 4C. Mitochondria were stained with Mitotracker Green FM (100nm, Cell Signaling, 9074) and active mitochondria with TMRM (100nm, Thermo Fisher, T668) for 30min at 37C. Cells were detected by flow cytometry using a LSRFortessa Cell Analyzer (BD, USA). Flowjo software (BD, USA) was used for data analysis.
Oxygen consumption rates (OCR) and extracellular acidification rate (ECAR) were measured using a XF-24 Extracellular Flux Analyzer (Agilent Technologies), as previously described [14]. Erythroblasts were seeded at a density of 2105 cells/well on Cell-Tak (Corning, 354240) coated Seahorse plates in unbuffered XF DMEM medium (Agilent Technologies) supplemented with 1mM sodium pyruvate, 2mM glutamine and 10mM glucose or galactose. Immediately after seeding, cells were centrifuged at 200g for 1minute to attach evenly to the bottom of the well and the plate was equilibrated for 30minutes at 37C in the absence of CO2. iPSCs derived from fibroblasts of PD patients and healthy controls were seeded at a density of 8103 cells/well on Seahorse plates and differentiated to dopaminergic neurons over a period of 3 weeks according to the described methodology. On an experimental day, the medium was changed to an unbuffered XF DMEM medium supplemented with 1mM sodium pyruvate, 2mM glutamine and 10mM glucose or galactose. Cells were incubated for 1h at 37C in the absence of CO2, before the Seahorse assay. For each assay, medium and reagent acidity were adjusted to pH 7.4 on the day of the assay, according to the manufacturers procedure. Optimal cell densities were determined experimentally to ensure a proportional response to FCCP (oxidative phosphorylation uncoupler).
After 3 measurements to detect the oxygen consumption ratio baseline, cells were then challenged with sequential injections of mitochondrial toxins: 1M oligomycin (Adenosine triphosphate ATP - synthase inhibitor), 1M FCCP, and 1M antimycin (complex III inhibitor). A minimum number of 5 replicates were performed for each cell line; data represent the mean of the different replicates. Basal respiration (measured as the average OCR rates at the baseline), maximal mitochondrial respiration (maximal respiration), reserve capacity (difference between maximal respiration and basal respiration), and respiration dedicated to ATP production (difference between basal respiration and oligomycin-dependent respiration) were used to investigate mitochondrial bioenergetics. Basal glycolysis, measured as extracellular acidification rate (ECAR) maximal glycolysis and reserve glycolytic capacity (difference between maximal glycolysis and basal glycolysis) were taken into account to investigate glycolytic properties.
Reprogrammed iPSCs cells cultured in an 8-chamber slide were fixed with 4% PFA for 15min at room temperature. After incubation in ice-cold methanol for 10min cells were permeabilized in 0.1% Triton in PBS for 10min and blocked using 1% BSA in PBS/0.05% Tween-20 for 30min. Next, cells were incubated with primary antibodies diluted in blocking buffer overnight at 4C - Mouse Anti-Human TRA1-81 (1:75, Abcam, AB16289#20), Rabbit Anti-Human OCT4 (1:250, Abcam, AB19857#8), Goat Anti-Human NCAM (1:100, R&D, AF2408), Goat-Anti Human SOX17 (1:100, R&D, AF1924) or Mouse Anti-Human Beta-Tubulin (1:1000, Merck, T8660) primary Chicken Anti-Human MAP2 (1:2000, Abcam, AB5392), Mouse Anti-Human TH (1:200, Millipore, MAB318). After washing with PBS cells were incubated with respective secondary Goat Anti-Mouse Alexa Fluor 546 (1:500, Invitrogen, A21045), Goat Anti-Rabbit Alexa Fluor 488 (1:500, Invitrogen, A11008#8a), Donkey Anti-Goat Alexa Fluor 488 (1:500, Invitrogen, A11055) or Goat Anti-Mouse Dylight 594 (1:500, Jackson, 115-515-166#7) antibodies diluted in blocking buffer for 1h at room temperature. Nuclei were stained with Hoechst 33342 (1:1000 in PBS, Thermo Fisher) for 10min. Cells were next washed with PBS, mounted with ProLong Diamond Antifade Mountant (P36965, Thermo Fisher), and imaged using a Leica Stellaris5 confocal microscope.
Blood transcriptome data from the Parkinson Progressive Markers Initiative (PPMI) cohort (PPMI website: https://ida.loni.usc.edu/pages/access/geneticData.jsp#441) were obtained. The libraries were prepared using the NEB/Kapa (NEBKAP) based library prep, with second-strand synthesis. RNA sequencing was performed at Hudson Alphas Genomic Services Lab on an Illumina NovaSeq6000, generating 100 million 125bp paired reads per sample. The Salmon files were imported into R using Tximport. To identify differentially expressed genes between PD groups and controls, the DESeq2 package was used. Normalized counts were subjected to Rlog transformation to improve distances/clustering for the principal component analysis (PCA). The cohort of subjects was divided into subgroups based on the delta-UPDRS-III (MDS-Unified Parkinsons Disease Rating Scale, UPDRS-III at last visit - UPDRS-III at first visit) of PD subjects: those with a delta-UPDRS-III less than 0 (defined as mild) and those with a delta-UPDRS-III greater than 0 (defined as severe), as well as controls (CTRL). A threshold of significance at FDR<0.05 was applied.
Gene Set Enrichment Analysis (GSEA) was conducted on an unfiltered, ranked list of genes. The analysis involved various terms from the Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome Pathway Databases, Hallmark Gene Set Collection, and WikiPathways (GSEA website: http://www.gsea-msigdb.org/gsea/msigdb/collections.jsp). Pathway information was obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG) available at the Molecular Signatures Database (http://www.broadinstitute.org/gsea/msigdb/index.jsp) or from the Hallmark Gene Set Collection (http://www.gsea-msigdb.org/gsea/msigdb/collections.jsp). Gene set enrichment with FDR<0.1 was considered significant. Genes in each PD group were ranked based on the level of differential expression using a signal-to-noise metric and a weighted enrichment statistic.
Transcriptomic analysis was performed using R Studio version 4.2.3. The experiments were conducted with a minimum of three independent biological replicates. GraphPad Prism version 9 (GraphPad Software, La Jolla California USA) was used for all statistical analyses and graphical representations. P values were denoted as *P<0.05, **P<0.01, ***P<0.001, and were considered significant. In the absence of indications, comparisons should be considered non-significant. Comparisons between two groups were analyzed using unpaired two-tailed Students t-tests, and comparisons between more than two groups were analyzed using either one-way ANOVA followed by Dunnetts (comparison of PD means vs. healthy subjects) or Tukeys (comparison of all the means) posthoc test for multiple comparisons.
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Orthogonal analysis of mitochondrial function in Parkinson's disease patients | Cell Death & Disease - Nature.com
Age-related macular degeneration (AMD), a common eye disorder leading to permanent loss of central vision, is the leading cause of blindness in developed countries. Current treatments for AMD are often ineffective, particularly for dry AMD, the most common form of the disorder. Cell therapy technologies offer potential new treatments for AMD, however, the efficacy of such treatments depends on the ability to deliver therapeutic cells to the target region and to produce enough RPE cells. On the back of the success of Induced pluripotent stem cell (iPSC) technology, a novel stem-cell therapy based upon iPSC-derived RPE cell transplantation was conceptualized as a promising new treatment for AMD and other retinal diseases. Bio-Techne aims to offer a range of animal-free cell culture products and GMP proteins for clinical manufacturing, which may boost the success of cell therapies. iPSC-based cell therapy is seen as a promising solution for AMD patients unresponsive to traditional treatments.
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Targeting Age-Related Macular Degeneration With Stem Cell Therapy - BioProcess Online
Neurosciences
April 1, 2024
ROCHESTER, Minn. A Mayo Clinic study shows stem cells derived from patients' own fat are safe and may improve sensation and movement after traumatic spinal cord injuries. The findings from the phase 1 clinical trial appear in Nature Communications. The results of this early research offer insights on the potential of cell therapy for people living with spinal cord injuries and paralysis for whom options to improve function are extremely limited.
In the study of 10 adults, the research team noted seven participants demonstrated improvements based on the American Spinal Injury Association (ASIA) Impairment Scale. Improvements included increased sensation when tested with pinprick and light touch, increased strength in muscle motor groups, and recovery of voluntary anal contraction, which aids in bowel function. The scale has five levels, ranging from complete loss of function to normal function. The seven participants who improved each moved up at least one level on the ASIA scale. Three patients in the study had no response, meaning they did not improve but did not get worse.
"This study documents the safety and potential benefit of stem cells and regenerative medicine," says Mohamad Bydon, M.D., a Mayo Clinic neurosurgeon and first author of the study. "Spinal cord injury is a complex condition. Future research may show whether stem cells in combination with other therapies could be part of a new paradigm of treatment to improve outcomes for patients."
No serious adverse events were reported after stem cell treatment. The most commonly reported side effects were headache and musculoskeletal pain that resolved with over-the-counter treatment.
In addition to evaluating safety, this phase 1 clinical trial had a secondary outcome of assessing changes in motor and sensory function. The authors note that motor and sensory results are to be interpreted with caution given limits of phase 1 trials. Additional research is underway among a larger group of participants to further assess risks and benefits.
The full data on the 10 patients follows a 2019 case report that highlighted the experience of the first study participant who demonstrated significant improvement in motor and sensory function.
Watch: Dr. Mohamad Bydon discusses improvements in research study
Journalists: Broadcast-quality sound bites are available in the downloads at the end of the post. Please courtesy: "Mayo Clinic News Network." Name super/CG: Mohamad Bydon, M.D./Neurosurgery/Mayo Clinic.
In the multidisciplinary clinical trial, participants had spinal cord injuries from motor vehicle accidents, falls and other causes. Six had neck injuries; four had back injuries. Participants ranged in age from 18 to 65.
Participants' stem cells were collected by taking a small amount of fat from a 1- to 2-inch incision in the abdomen or thigh. Over four weeks, the cells were expanded in the laboratory to 100 million cells and then injected into the patients' lumbar spine in the lower back. Over two years, each study participant was evaluated at Mayo Clinic 10 times.
Although it is understood that stem cells move toward areas of inflammation in this case the location of the spinal cord injury the cells' mechanism of interacting with the spinal cord is not fully understood, Dr. Bydon says. As part of the study, researchers analyzed changes in participants' MRIs and cerebrospinal fluid as well as in responses to pain, pressure and other sensation. The investigators are looking for clues to identify injury processes at a cellular level and avenues for potential regeneration and healing.
The spinal cord has limited ability to repair its cells or make new ones. Patients typically experience most of their recovery in the first six to 12 months after injuries occur. Improvement generally stops 12 to 24 months after injury. In the study, one patient with a cervical spine injury of the neck received stem cells 22 months after injury and improved one level on the ASIA scale after treatment.
Two of three patients with complete injuries of the thoracic spine meaning they had no feeling or movement below their injury between the base of the neck and mid-back moved up two ASIA levels after treatment. Each regained some sensation and some control of movement below the level of injury. Based on researchers' understanding of traumatic thoracic spinal cord injury, only 5% of people with a complete injury would be expected to regain any feeling or movement.
"In spinal cord injury, even a mild improvement can make a significant difference in that patient's quality of life," Dr. Bydon says.
Stem cells are used mainly in research in the U.S., and fat-derived stem cell treatment for spinal cord injury is considered experimental by the Food and Drug Administration.
Between 250,000 and 500,000 people worldwide suffer a spinal cord injury each year, according to theWorld Health Organization.
An important next step is assessing the effectiveness of stem cell therapies and subsets of patients who would most benefit, Dr. Bydon says. Research is continuing with a larger, controlled trial that randomly assigns patients to receive either the stem cell treatment or a placebo without stem cells.
"For years, treatment of spinal cord injury has been limited to supportive care, more specifically stabilization surgery and physical therapy," Dr. Bydon says. "Many historical textbooks state that this condition does not improve. In recent years, we have seen findings from the medical and scientific community that challenge prior assumptions. This research is a step forward toward the ultimate goal of improving treatments for patients."
Dr. Bydon is the Charles B. and Ann L. Johnson Professor of Neurosurgery. This research was made possible with support from Leonard A. Lauder, C and A Johnson Family Foundation, The Park Foundation, Sanger Family Foundation, Eileen R.B. and Steve D. Scheel, Schultz Family Foundation, and other generous Mayo Clinic benefactors. The research is funded in part by a Mayo Clinic Transform the Practice grant.
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Study documents safety, improvements from stem cell therapy after spinal cord injury - Mayo Clinic
Dr Mohamad Bydon, a Mayo Clinic neurosurgeon and first author of the study, said the results indicate that severe spinal cord injuries that were once thought hopeless, could be treatable in the future.
For years, treatment of spinal cord injury has been limited to supportive care, more specifically stabilisation surgery and physical therapy, said Dr Bydon.
Many historical textbooks state that this condition does not improve. In recent years, we have seen findings from the medical and scientific community that challenge prior assumptions.
This research is a step forward toward the ultimate goal of improving treatments for patients.
In Britain, an estimated 50,000 people are living with a spinal cord injury with about 2,500 new injuries each year.
Although operations to realign the spine and remove bone fragments or fuse bones can be effective, many people are left without movement below the site of the injury and show little improvement over time.
In the study, six people with neck injuries and four with back injuries, aged between 18 and 65 were assessed using the American Spinal Injury Association (ASIA) Impairment Scale, which has five levels, ranging from complete loss of function to normal function.
After treatment, seven participants who improved each moved up at least one level on the ASIA scale, with two patients moving up two levels. Three patients showed no improvement.
The spinal cord has limited ability to repair its cells or make new ones. Patients typically experience most of their recovery in the first six to 12 months after injuries occur. Improvement generally stops 12 to 24 months after injury.
However, during the trial two patients with cervical spine injuries of the neck received stem cells 22 months after their injuries and improved one level on the ASIA scale after treatment.
Some also regained movement and improved bowel function.
In spinal cord injury, even a mild improvement can make a significant difference in that patients quality of life, added Dr Bydon.
Spinal cord injury is a complex condition. Future research may show whether stem cells in combination with other therapies could be part of a new paradigm of treatment to improve outcomes for patients.
This study documents the safety and potential benefit of stem cells and regenerative medicine.
Fat tissue was used because it is abundant and easy to get hold of in the body and has the most mesenchymal stem cells.
Despite the success of the treatment, scientists are still unsure how the stem cells are boosting regeneration. In animal trials, it has been shown that they flock towards areas of inflammation, helping to regulate the immune response.
As part of the study, researchers took lumbar punctures from the patients to gather their cerebrospinal fluid before and after treatment to see if they could pick up any changes
After treatment, they found an increased level of a protein called Vascular endothelial growth factor, in seven patients. The protein promotes the growth of new blood vessels and forms part of the mechanism that restores the blood supply to cells and tissues.
The phase one trial, which was primarily looking at the safety of the treatment, reported no serious adverse events with only mild side effects such as headache and musculoskeletal pain that resolved with over-the-counter treatment. Further trials are expected to follow.
The study was published in the journal Nature Communications.
Original post:
Paralysed patients could regain movement and sensation, stem cell treatment trial finds - The Telegraph
Summary: A phase 1 clinical trial has revealed that stem cells derived from patients own fat may safely enhance sensation and movement in individuals with traumatic spinal cord injuries. In the study, seven out of ten adults showed measurable improvements on the ASIA Impairment Scale, experiencing increased sensation, muscle strength, and improved bowel function without serious side effects.
The findings challenge the longstanding belief that spinal cord injuries are irreparable, offering new hope for treatments. With the spinal cords limited repair capability, this research signifies a crucial step towards innovative therapies, emphasizing the need for further studies to unlock the full potential of stem cell treatments.
Key Facts:
Source: Mayo Clinic
AMayo Clinicstudy shows stem cells derived from patients own fat are safe and may improve sensation and movement after traumaticspinal cord injuries.
The findings from the phase 1 clinical trial appear inNature Communications.
The results of this early research offer insights on the potential of cell therapy for people living with spinal cord injuries and paralysis for whom options to improve function are extremely limited.
In the study of 10 adults, the research team noted seven participants demonstrated improvements based on the American Spinal Injury Association (ASIA) Impairment Scale. Improvements included increased sensation when tested with pinprick and light touch, increased strength in muscle motor groups, and recovery of voluntary anal contraction, which aids in bowel function.
The scale has five levels, ranging from complete loss of function to normal function. The seven participants who improved each moved up at least one level on the ASIA scale. Three patients in the study had no response, meaning they did not improve but did not get worse.
This study documents the safety and potential benefit of stem cells and regenerative medicine, saysMohamad Bydon, M.D., a Mayo Clinic neurosurgeon and first author of the study.
Spinal cord injury is a complex condition. Future research may show whether stem cells in combination with other therapies could be part of a new paradigm of treatment to improve outcomes for patients.
No serious adverse events were reported after stem cell treatment. The most commonly reported side effects were headache and musculoskeletal pain that resolved with over-the-counter treatment.
In addition to evaluating safety, this phase 1 clinical trial had a secondary outcome of assessing changes in motor and sensory function. The authors note that motor and sensory results are to be interpreted with caution given limits of phase 1 trials. Additional research is underway among a larger group of participants to further assess risks and benefits.
The full data on the 10 patients follows a 2019case reportthat highlighted the experience of the first study participant who demonstrated significant improvement in motor and sensory function.
Stem cells mechanism of action not fully understood
In the multidisciplinary clinical trial, participants had spinal cord injuries from motor vehicle accidents, falls and other causes. Six had neck injuries; four had back injuries. Participants ranged in age from 18 to 65.
Participants stem cells were collected by taking a small amount of fat from a 1- to 2-inch incision in the abdomen or thigh. Over four weeks, the cells were expanded in the laboratory to 100 million cells and then injected into the patients lumbar spine in the lower back. Over two years, each study participant was evaluated at Mayo Clinic 10 times.
Although it is understood that stem cells move toward areas of inflammation in this case the location of the spinal cord injury the cells mechanism of interacting with the spinal cord is not fully understood, Dr. Bydon says.
As part of the study, researchers analyzed changes in participants MRIs and cerebrospinal fluid as well as in responses to pain, pressure and other sensation. The investigators are looking for clues to identify injury processes at a cellular level and avenues for potential regeneration and healing.
The spinal cord has limited ability to repair its cells or make new ones. Patients typically experience most of their recovery in the first six to 12 months after injuries occur. Improvement generally stops 12 to 24 months after injury.
One unexpected outcome of the trial was that two patients with cervical spine injuries of the neck received stem cells 22 months after their injuries and improved one level on the ASIA scale after treatment.
Two of three patients with complete injuries of the thoracic spine meaning they had no feeling or movement below their injury between the base of the neck and mid-back moved up two ASIA levels after treatment.
Each regained some sensation and some control of movement below the level of injury. Based on researchers understanding of traumatic thoracic spinal cord injury, only 5% of people with a complete injury would be expected to regain any feeling or movement.
In spinal cord injury, even a mild improvement can make a significant difference in that patients quality of life, Dr. Bydon says.
Stem cells are used mainly in research in the U.S., and fat-derived stem cell treatment for spinal cord injury is considered experimental by the Food and Drug Administration.
Between 250,000 and 500,000 people worldwide suffer a spinal cord injury each year, according to theWorld Health Organization.
An important next step is assessing the effectiveness of stem cell therapies and subsets of patients who would most benefit, Dr. Bydon says. Research is continuing with a larger, controlled trial that randomly assigns patients to receive either the stem cell treatment or a placebo without stem cells.
For years, treatment of spinal cord injury has been limited to supportive care, more specifically stabilization surgery and physical therapy, Dr. Bydon says.
Many historical textbooks state that this condition does not improve. In recent years, we have seen findings from the medical and scientific community that challenge prior assumptions. This research is a step forward toward the ultimate goal of improving treatments for patients.
Dr. Bydon is the Charles B. and Ann L. Johnson Professor of Neurosurgery. This research was made possible with support from Leonard A. Lauder, C and A Johnson Family Foundation, The Park Foundation, Sanger Family Foundation, Eileen R.B. and Steve D. Scheel, Schultz Family Foundation, and other generous Mayo Clinic benefactors. The research is funded in part by a Mayo Clinic Transform the Practice grant.
Review thestudyfor a complete list of authors and funding.
Author: Megan Luihn Source: Mayo Clinic Contact: Megan Luihn Mayo Clinic Image: The image is credited to Neuroscience News
Original Research: Open access. Intrathecal delivery of adipose-derived mesenchymal stem cells in traumatic spinal cord injury: Phase I trial byMohamad Bydon et al. Nature Communications
Abstract
Intrathecal delivery of adipose-derived mesenchymal stem cells in traumatic spinal cord injury: Phase I trial
Intrathecal delivery of autologous culture-expanded adipose tissue-derived mesenchymal stem cells (AD-MSC) could be utilized to treat traumatic spinal cord injury (SCI).
This Phase I trial (ClinicalTrials.gov: NCT03308565) included 10 patients with American Spinal Injury Association Impairment Scale (AIS) grade A or B at the time of injury.
The studys primary outcome was the safety profile, as captured by the nature and frequency of adverse events.
Secondary outcomes included changes in sensory and motor scores, imaging, cerebrospinal fluid markers, and somatosensory evoked potentials. The manufacturing and delivery of the regimen were successful for all patients.
The most commonly reported adverse events were headache and musculoskeletal pain, observed in 8 patients. No serious AEs were observed. At final follow-up, seven patients demonstrated improvement in AIS grade from the time of injection.
In conclusion, the study met the primary endpoint, demonstrating that AD-MSC harvesting and administration were well-tolerated in patients with traumatic SCI.
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Stem Cells Spark Hope in Spinal Cord Recovery - Neuroscience News
Katy Rezvani, MD, PhD
Professor
Department of Stem Cell Transplantation
Division of Cancer Medicine
Section Chief
Cellular Therapy
Director
Translational Research
Department of Stem Cell Transplantation-Research
Division of Internal Medicine
Sally Cooper Murray Endowed Chair in Cancer Research
The University of Texas MD Anderson Cancer Center
Houston, TX
The landscape of cancer therapy has been witnessing a paradigm shift with the advent of immunotherapy treatments, especially for patients with hematologic malignancies. Immunotherapies such as immune checkpoint inhibitors and autologous chimeric antigen receptor (CAR) T-cell therapies have led to considerable improvements in survival yet still come with efficacy limitations, manufacturing challenges, financial toxicity, and significant safety risks. Switching to an allogeneic approach could help overcome such limitations and allow for treatment of more patients with fewer donors.
Natural killer (NK)cell therapies are increasingly being explored as an alternative and promising approach to immunotherapy. Katy Rezvani, MD, PhD, said NK-cell therapies are of interest as a possible faster, cheaper, safer alternative to CAR T-cell therapy.
These cells have a lot of promise[and they give] me a lot of hope that CAR NK cells could add to the armamentarium of what we have available for cancer immunotherapy, said Rezvani, a professor of medicine in the Department of Stem Cell Transplantation at The University of Texas MD Anderson Cancer Center in Houston, in an interview with Targeted Therapies in Oncology.
NK cells are part of the innate immune system and can target cancer cells that downregulate HLA class I molecules.1 These cytotoxic lymphocytes are tasked with the surveillance of stressed cells,2-4 making them our first line of defense to virally infected cells and abnormal cells, Rezvani explained.
NK cells are of interest for adoptive cell therapies because they do not require full HLA matching, reducing the risk of graft-vs-host disease (GVHD) as well as lengthy manufacturing times for unique products.1-5 This allows NK-cell therapies to be a more off-the-shelf, readily accessible treatment for more patients compared with first-generation CAR T-cell therapy.
CAR T-cell treatments are also associated with other significant safety concerns, such as cytokine release syndrome (CRS) and immune effector cellassociated neurotoxicity syndrome (ICANS) toxicity, which are reduced with NK-cell therapies.4,5
NK-cell therapies are viewed as a possibility for overcoming the manufacturing times of CAR T-cell therapies and the large price tag associated with the treatment as well as the safety risks.5 On the other hand, NK cells have a limited life span after transfusion.1,4 They can be modified with genetic engineering to allow for greater efficacy.4,5
Sources for NK-cell production include cell lines, peripheral blood cells, umbilical cord blood, and induced pluripotent stem cells (iPSCs)an emerging origin source.4,6 Current research focuses most on cord bloodderived NK cells, which require donation and expansion. iPSC-derived NK cells do not require collection of cells from a donor.6 Clinical trials are beginning to show promise for CAR-NK-cell therapies, based on the benefits seen with CAR T-cell therapies.
Although still in the early stages of discovery and study, NK-cell therapies are beginning to show promise in clinical trials for their safety and convenience, as well as for efficacy on par with current CAR T-cell therapies.1 At the recent 65th American Society of Hematology (ASH) Annual Meeting and Exposition, findings were presented from early-stage studies of CAR NK-cell therapies showing their potential.
Rezvani, the recipient of the E. Donnall Thomas Lecture and Prize, presented the latest research on this cellular therapy. Rezvani highlighted studies of an anti-CD19 CAR NK agent.2 Following the success and approval of autologous anti-CD19 CAR T-cell products, a CAR NK-cell therapy derived from cord blood was created that was directed against CD19 for patients with CD19-positive malignancies; CAR NK cells did not require HLA matching with the recipient.1,2
In the phase 1/2 trial (NCT03056339), 11 patients with relapsed or refractory CD19-positive hematologic malignancies5 with chronic lymphocytic lymphoma, 2 with diffuse large B-cell lymphoma, and 4 with follicular lymphoma, 3 of which were transformedreceived the therapy in a single treatment. Objective responses were seen in 8 of 11 patients (73%) within a month of treatment, and all but 1 were complete responses (CRs). CAR NK cells were also detectable for up to a year after infusion.
No cases of CRS, neurotoxicity, tumor lysis syndrome, hemophagocytic lymphohistiocytosis, or GVHD were observed in any of the patients. Observed grade 3/4 adverse events (AEs) were predominantly hematologic.
Further results of the study in 37 patients with relapsed/refractory CD19-positive B-cell malignancies showed an objective response rate (ORR) of 48.6% at day 30 and day 100. CRs were seen in 29.7% of patients by day 100 and 37.8% by 1 year. Responses were seen at a median of 30 days and were durable for 9 of the 10 patients who had a CR.2,7
The responses that we observed were pretty similar to what you would get with [autologous CD19 CAR] T cells, Rezvani noted. Neither neurotoxicity nor GVHD was reported in any of the patients, but 1 case of CRS was observed.7
Rezvani stressed that donor selection was vital in this study and for future studies of cord bloodderived NK cells. Optimal cord blood was determined to be units that were frozen within 24 hours of collection and had a nucleated red blood cell count of less than 80 million.6 [We found that] the most important determinants of whos going to respond or not was the quality of the cord blood that was used for the manufacturing of CAR NK cells, Rezvani said. The impact that we ended up seeing was huge.
The rate of overall survival (OS) at 1 year was 94% in patients who received cord blood from optimal donors and 48% from those who received cells from suboptimal donors. The progression-free survival rates at 1 year were 69% and 5% for patients who received cord blood from optimal and suboptimal donors, respectively.2
Rezvani added that optimal cord blood could maintain long-term cytotoxicity and had greater in vitro proliferation compared with suboptimal cord blood. Further, optimal cords had greater polyfunctionality vs suboptimal cords, which were characterized by a signature associated with hypoxia and exhaustion.7
A phase 1 study (NCT04623944) presented at ASH showed promise for another CAR NK-cell therapy, NKX101, in patients with acute myeloid leukemia (AML). The agent is composed of NK cells derived from healthy donors that were engineered to express a natural killer group 2D (NKG2D)directed CAR and IL-15.8
The first cohort included 6 patients with relapsed/refractory AML who received 3 doses of NKX101 per treatment cycle; 83% had poor-risk factors. Early responses were seen, with 67% of patients achieving a CR or CR with incomplete hematologic recovery (CRi). Two patients achieved minimal residual disease negativity after only 1 treatment cycle. Three patients were continuing treatment.
All 6 patients reported treatment-emergent AEs of grade 3 or higher, with myelosuppression and infection being most common. No cases of CRS, ICANS, or GVHD of any grade were reported in the cohort. One case of a grade 5 AE was observed but was considered not related to treatment.
A phase 1/2 study presented at ASH showed first-in-human data for the CD123 NK-cell engager SAR443579 in patients with relapsed/refractory AML, B-cell acute lymphoblastic leukemia, or high-risk myelodysplasia (NCT05086315). SAR443579 is a trifunctional anti-CD123 NK-cell engager that targets the CD123 antigen as well as engaging NKp46 and CD16a.9
In the dose-escalation portion of the study, CRs/CRis were reported in 33% of patients with AML treated with up to a maximum dose of 1000 g/kg/infusion. Treatment was ongoing in 3 patients who achieved a CR.
Grade 3 or higher treatment-emergent AEs were reported in 60.5% of patients across dose levels and grade 5 events were seen in 11.6%, although all were considered not related to treatment with SAR443579. The most common events observed were infusion-related reactions (67.4%) and constipation (25.6%). CRS was reported in 2 patients but no cases of ICANS were observed. In June 2023, the agent received an FDA fast track designation for the treatment of patients with hematologic malignancies.10
During the ASH meeting, Yago L. Nieto, MD, PhD, presented findings from a phase 1/2 study of AFM13 (Acimtamig), a tetravalent bispecific antibody construct with CD30 and CD16a, in combination with cord bloodderived, cytokine-induced, memory-like expanded NK cells in patients with relapsed/refractory CD30-positive lymphomas (NCT04074746).11
A total of 42 heavily pretreated patients who were double refractory to brentuximab vedotin (Adcetris) and checkpoint inhibitors, most with Hodgkin lymphoma (88%), received NK cells 15 days before treatment with AFM13. The ORR was 93% and the CR rate was 67%. At the recommended phase 2 dose level (108 NK/kg), the ORR was 94% and the CR rate was 72%. Responses were reported in 97% of patients with classical Hodgkin lymphoma (n =32) and CRs in 78%.
The median OS in patients who received 2 cycles of treatment was 85% at 6 and 12 months. In patients who received 4 cycles, the median OS was 87% at 6 months and 85% at 12 months. In patients with Hodgkin lymphoma, the median OS was 92% at 6 and 12 months for those who received 2 cycles of treatment and 85% and 82%, respectively, for those who received 4 cycles.
No cases of CRS, neurotoxicity, or GVHD were reported in the study; even infusion-related reactions were considered infrequent. Moderate neutropenia and thrombocytopenia were seen with the lymphodepleting chemotherapy.
The safety didnt come as a surprise; we expected that. What really came as a surprise was the high level of activity in the heavily pretreated patients with refractory tumors we treated, said Nieto, a professor in the Department of Stem Cell Transplantation at The University of Texas MD Anderson Cancer Center in Houston during an interview with Targeted Therapies in Oncology. He added that 6 of 7 patients who had a response subsequently consolidated with a stem cell transplant remained in CR at more than 18 months, making it an effective bridging therapy.
In September 2023, AFM13 in combination with allogeneic NK cells (AB-101) received an FDA fast track designation for the treatment of patients with relapsed/refractory Hodgkin lymphoma.12 Going forward, AFM13 is being explored in combination with AB-101 in patients with relapsed/refractory Hodgkin lymphoma and CD30-positive peripheral T-cell lymphoma in a phase 2 trial (LuminICE-203; NCT05883449). The combination is expected to augment the innate immunity of AFM13 alone to boost the antitumor cytotoxicity in patients with CD30-positive tumors.12,13 Nieto also explained that the AFM13 and NK-cell therapy model can be extrapolated to treat other malignancies by choosing an alternate bispecific antibody for the tumor type.
An ongoing phase 1 trial (NCT05182073) of FT576, a multiplex-engineered, BCMA-targeted CAR NK-cell therapy, in patients with relapsed/refractory multiple myeloma has shown early promise and safety for the iPSC-derived agent as a monotherapy and in combination with daratumumab (Darzalex).14 No cases of CRS, ICANS, or GVHD were reported in the trial at any dose level with or without added daratumumab. Additionally, no dose-limiting toxicities or serious treatment-related AEs were observed.
Among 9 patients treated as of the interim report, responses were seen in 33% of patients and stable disease in 55%. One patient treated with FT576 monotherapy, who had received 5 prior lines of therapy and was triple refractory, achieved a very good partial response.
As of now, efficacy with NK-cell therapies is considered similar to that of CAR T-cell therapies, although the studies of these agents is in the early stages. However, current constructs of first-generation CAR NK-cell therapies have limited long-term efficacy due to shorter in vivo persistence and cell exhaustion. To potentially improve the efficacy beyond that of autologous approaches and even to generate efficacy in solid tumors, newer approaches are being considered, including engaging different targets, transducing T-cell receptor (TCR)expressing NK cells, multiplexed engineering, and combination regimens.2,15
The potential is huge [for] what we can achieve with these cells. With our increasing understanding of NK biology, of access to big data, and also the engineering tools that we have available to usenot just CAR transduction, but for instance with TCR into NK cells, with CRISPR [clustered regularly interspaced short palindromic repeats] gene editing of your NK cells to make them more resistant to the impact of the tumor microenvironment, and combination with various drugsI think the field could see major advances in a relatively short period of time, Rezvani said.
Targeting CD70 is showing promise as it is expressed in primary AML samples. Investigators created a number of second-generation CAR constructs to determine the most optimal one and found that anti- CD70 CAR NK cells with IL-15 allowed for the most superior antitumor activity of the various constructs in aggressive CD70-positive AML models.16 The construct is now being used in an ongoing phase 1/2 basket trial in patients with CD70-expressing hematologic malignancies (NCT05092451) and being explored across 3 dose levels.2
To date, NK-cell therapies have not seen the same success in solid tumors as in hematologic malignancies. This is believed to be because of the immunosuppressive tumor microenvironment that hampers NK cell activation and function. Advances in understanding these barriers and developing strategies to overcome them are critical for enhancing the therapeutic potential of NK-cell therapies in solid tumors.1,2,15
TROP2 is a target of interest with NK cells to treat patients with solid tumors as it is overexpressed in many epithelial cancers but not in healthy tissues.2 The FDA has approved investigational new drug applications for the study of CAR TROP2/IL-15 NK cells delivered intravenously to patients with advanced solid tumors (NCT06066424) and delivered intraperitoneally to patients with ovarian cancer and pancreatic cancer (NCT05922930).
Genetically engineered New York esophageal squamous cell carcinoma 1 (NY-ESO-1)targeted, TCR/IL-15expressing cord bloodderived NK cells are being investigated in a phase 1/1b trial of patients with advanced synovial sarcoma and myxoid/round cell liposarcoma (NCT06083883) as well as in a phase 1 trial for patients with NY-ESO-1positive relapsed/refractory multiple myeloma or plasma cell leukemia (NCT06066359). NY-ESO-1 is considered highly immunogenic and is expressed in many cancer cells but not in healthy tissue, making it an attractive target.
The phase 1b ADVENT-AML trial is exploring the use of allogeneic NK cells in combination with azacitidine and venetoclax (Venclexta) in patients with newly diagnosed AML (NCT05834244). The synergy of the regimen allows for upregulation of silenced NKG2D ligands, priming of leukemia cells, and reduction of disease burden.17
Multiplexed CRISPR gene-edited therapies are being created to design the safest and most effective products for patients. By employing CRISPR/Cas9 technology, multiple genes within NK cells can be simultaneously edited to enhance their persistence, cytotoxicity, and ability to navigate immunosuppressive barriers.2,15
A phase 1 trial is exploring the treatment of patients with recurrent glioblastoma with multiplex CRISPR gene-edited NK cells with deleted TGFBR2 and NR3C1 (NCT04991870). With new, innovative approaches and clinical trials quickly emerging, the field of NK-cell therapies is surely one to watch.
Excerpt from:
Emerging Frontiers in Immunotherapy: The Promise of NK-Cell Therapies - Targeted Oncology
AUSTIN, Texas, March 25, 2024 (GLOBE NEWSWIRE) -- Cassava Sciences, Inc. (Nasdaq: SAVA), a biotechnology company focused on Alzheimer’s disease, today announced the completion of another interim safety review of simufilam in on-going Phase 3 clinical trials in patients with Alzheimer’s disease. A routine, scheduled meeting of a Data and Safety Monitoring Board (DSMB) recommended that both of Cassava Sciences’ on-going Phase 3 studies continue as planned, without modification.
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Cassava Sciences Announces Completion of an Interim Safety Review of Oral Simufilam On-going Phase 3 Trials
THE WOODLANDS, Texas, March 25, 2024 (GLOBE NEWSWIRE) -- Lexicon Pharmaceuticals, Inc. (Nasdaq: LXRX) today announced that four data presentations related to sotagliflozin, an inhibitor of two sodium glucose transport proteins (SGLT2 and SGLT1), will be delivered during the American College of Cardiology 73rd Annual Scientific Session & Expo being held April 6 - 8, 2024 in Atlanta, Georgia, including results from a post-hoc evaluation of the efficacy of sotagliflozin in reducing stroke events in patients with type 2 diabetes, chronic kidney disease (CKD), and high cardiovascular (CV) risk in the SCORED Phase 3 clinical trial.
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Clinical Data on the Impact of Sotagliflozin on Stroke and Heart Attack Risk Among Four Lexicon-Sponsored Presentations at the American College of...