102 The deep phenotype characterization of ‘Off-the-Shelf’ CD19-chimeric antigen receptor (CAR) T cells allows to identify their subset complexity and to optimize their manufacturing

BackgroundUmbilical cord blood (UCB) represents a promising source of T cells for the generation of ‘off-the-shelf’ T cells engineered to express a chimeric antigen receptor (CAR). This study is aimed at understanding the composition of T cell subsets within UCB-CAR-T cells.MethodsT cells, either from UCB or peripheral mononuclear cells (PBMCs) of healthy donors, were activated in vitro with CD3/CD28 mAbs either conjugated to magnetic beads (Dynabeads) or to a colloidal polymeric nanomatrix (TransAct; Miltenyi Biotec). T cells were then transduced with lentiviral vectors encoding for CD19-CD28z or CD19-4-1BBz CARs. The deep phenotype analyses of the CD19-CAR-T cells (N=32) was performed through a multidimensional flow cytometry to assess the expression/co-expression of T cell-associated markers (N=29). The NGFR was utilized as probe for the expression of CD19-CAR. To select the pertinent markers characterising the different groups, we applied a machine learning technique called L0-regularized logistic regression,1 2 and implemented in the R packageL0Learn. 5-fold cross-validation (CV) was used to select the optimal values of the tuning parameters. CD19-CAR-T cells have been also characterized for the transcriptomic profile by parallel quantitative PCR using the high throughput BioMark HD platform and for cytokines, perforin and granzyme B release upon the co-culture with CD19 expressing or not target cells.ResultsT lymphocytes UCB showed efficient expression of the CARs (40–70% of positive cells). Different T cell subsets could discriminate the composition of T cells activated with either Beads or TranAct. CD4+NGFR+CD45RA+ or CD8+NGFR+CD45RA+ T cells associated with different combinations of CCR7, CD62L, LAG3, CD57, CD56 could discriminate between cells activated with Beads vs. TranAct (figures 2–3). CD8+NGFR+CD45RO+CD279−CD152+ T cells were also differentially expressed in TranAct vs. Beads. The PCA analyses also highlighted differences in terms of CD19-CAR-T cell subsets (such as CD8+NGFR+CD45RO+CD62L+, CD8+NGFR+CD45RO+CCR7+, CD8+NGFR+CD45RO+CD272+TIM−3+, CD8+NGFR+CD45RO+CD272+TIM−3+, CD8+NGFR+CD45RA+CD272+TIM−3− and CD4+NGFR+CD45RA+CD272−TIM−3+) in PBMCs vs. UCBs (figure 1). In addition, bystander T cells with different phenotype not expressing the CARs were also detected within the populations of T cells with different origins. Similarly, different T subsets were found in relationship with the sources of T cells. These CD19-CAR-T cells were also characterized for the anti-tumor activity and transcriptomic profiling.Abstract 102 Figure 1PCA of CAR-T cells from UCB vs. PBMCsAbstract 102 Figure 2PCA of CAR-T cells from UCB to compare TransAct vs. beadsAbstract 102 Figure 3PCA of CD19-CAR-T cells to compare TransAct vs. Beads irrespective of the source of the T cellsConclusionsThe combination of deep phenotype characterization with novel statistical tools allowed to identify the complexity of subsets in the engineered T cells in relationship with the starting material and the methods for the activation of the lymphocytes. These findings have important implications for the optimization of the manufacturing of CD19-CAR-T cells.ReferencesAntoine Dedieu, Hussein Hazimeh, and Rahul Mazumder. Learningsparse classifiers: Continuous and mixed integer optimization perspectives. Journal of Machine Learning Research 2021.Hussein Hazimeh and Rahul Mazumder. Fast best subset selection: Coordinatedescent and local combinatorial optimization algorithms. Operations Research 2020;68(5):1517–1537.Ethics ApprovalSidra Medicine’s Ethics Board approval, #1812044429

Multiple Congenital Ocular Anomalies in a silver coat Missouri Fox Trotter stallion

This is the first description of Multiple Congenital Ocular Anomalies (MCOA) in a silver coat Missouri Fox Trotter determined to be heterozygous for the Silver PMEL17 missense mutation associated with MCOA and a silver coat in other breeds. The stallion was treated for meningoencephalitis and bilateral uveitis of unknown origin. A complete ophthalmic examination and ocular ultrasonography were performed. As an incidental finding, the patient exhibited bilateral cystic lesions restricted to the temporal anterior uvea consistent with the Cyst phenotype and was genotyped heterozygous for the Silver mutation. Additionally, 4 other non-silver colored Missouri Fox Trotters were genotyped homozygous for the wild-type allele. Screening for PMEL17 mutation in Missouri Fox Trotters accompanied by ophthalmic phenotype characterization is recommended to determine the allelic frequency and facilitate informed breeding decisions since the silver coat color is particularly popular.

Electrophysiological Phenotype Characterization of Human iPSC-Derived Neuronal Cell Lines by Means of High-Density Microelectrode Arrays

Recent advances in the field of cellular reprogramming have opened a route to study the fundamental mechanisms underlying common neurological disorders. High-density microelectrode-arrays (HD-MEAs) provide unprecedented means to study neuronal physiology at different scales, ranging from network through single-neuron to subcellular features. In this work, we used HD-MEAs in vitro to characterize and compare human induced-pluripotent-stem-cell (iPSC)-derived dopaminergic and motor neurons, including isogenic neuronal lines modeling Parkinson’s disease and amyotrophic lateral sclerosis. We established reproducible electrophysiological network, single-cell and subcellular metrics, which were used for phenotype characterization and drug testing. Metrics such as burst shapes and axonal velocity enabled the distinction of healthy and diseased neurons. The HD-MEA metrics could also be used to detect the effects of dosing the drug retigabine to human motor neurons. Finally, we showed that the ability to detect drug effects and the observed culture-to-culture variability critically depend on the number of available recording electrodes.