Production of anti-cd19 car t cells to support multicenter trials evaluating Kte- c19 in b cell malignancies

The treatment of cancer patients with autologous T cells expressing a chimeric antigen receptor (CAR) is one of the most promising adoptive cellular therapy approaches. Reproducible manufacturing of high-quality, clinical-grade CAR-T cell products is required for the wide utilization of such technology. The manufacturing process established at Kite Pharma to support phase I/II clinical trials utilizing KTE-C19 in hematological malignancies aimed to improve productivity, maximize manufacturing success rate and minimize timing between leukapheresis collection and KTE-C19 administration at bedside. The manufacturing process initiates with the enrolled subjects undergoing to leukapehersis procedure to target collection of mononuclear cells (MNCs). After collection, leukapehersis material is shipped to the central manufacturing site and processed. T cells are activated, transduced with a gamma retroviral vector that encodes the CAR gene and further expanded for 3 to 5 days to achieve the target dose of 2 x 106 CAR positive T cells/kg body weight (minimum of 1 x 106). Final KTE-C19 product is washed, formulated, cryopreserved and tested for identity, potency, and safety. After acceptance criteria were met, the KTE-C19 was shipped to the clinical site using a validated cryoshipper. The manufacturing process has been able to successfully generate KTE-C19 products for patients enrolled in Kite sponsored trials allowing a rapid turnaround from patient leukapheresis collection to product administration

Development Of A High-Dose Engineered Tcr T Cell Manufacturing Process Using Automated Semi-Continuous Perfusion Bioreactors

Clinical activity with engineered TCR (eTCR) T cell products directed against solid tumor indications may require doses up to two orders of magnitude greater than those investigated during CAR-T studies in hematological malignancies.1 Novel methods for optimizing productivity and production times are required to progress the industrial feasibility of high-dose cell therapies. We developed an automated semi-continuous perfusion culture method suitable for rapidly generating high T cell densities with the aid of risk-based process models. The result is a robust manufacturing process capable of generating a target dose with high certainty, and minimal operational complexity and variability. Please click Additional Files below to see the full abstract

Removal Of Myeloid Cells From Autologous Leukocytes Used For Chimeric Antigen Receptor (Car) T Cell Manufacturing Improves Final Product Consistency And Yields

Early phase clinic trails of T cells genetically engineered to express Chimeric Antigen Receptors (CAR) have been promising. CD19-CAR T cells have been used successfully in a number of clinical trials to treat non-Hodgkin’s lymphoma and acute lymphocytic leukemia (ALL) and clinical trials of GD2-CAR T cells for the treatment of osteosarcoma and neuroblastoma are underway. Most CAR T cell manufacturing protocols make use of autologous peripheral blood mononuclear cell (PBMC) concentrates collected by apheresis, however, the lymphocyte-rich PBMC concentrates are also enriched for monocytes and contain small but variable quantities of red blood cells, platelets and neutrophils and prior to beginning CAR T cell manufacturing the PBMC concentrates are generally enriched for lymphocytes or CD3+ cells. We initially manufactured CD19- and GD2-CAR T cells using autologous PBMC concentrates enriched for T cells by selection with the anti-CD3/CD28 beads. These same anti-CD3/CD28 beads were used to stimulated T cell expansion. While the method was, in general, effective, we found that the quantities of GD2-CAR T cells produced were less than the quantities of CD19 CAR T cells produced. In addition, T cells from some patients failed to expand at all. Further investigation found that the presence of large quantities of monocytes or granulocytes in some PBMC concentrates which was associated with poor in vitro CAR T cell expansion. Myeloid derived suppressor cells (MDSCs) that inhibit T cell proliferation are present in sarcoma and ALL patients. These MDSCs may have a monocyte or neutrophil phenotype. We hypothesized that MDSCs in the PBSC concentrates bound non-specifically to the anti-CD3/CD28 beads and more rigorous enrichment of the starting material for lymphocytes would improve CAR T cell yields and reduce the incidence of manufacturing failures. We modified the T cell enrichment method to include a monocyte-depleting plastic adherence step. This change improved T cell expansion, but it was not completely effective at removing contaminating monocytes and granulocytes and did not completely eliminate manufacturing failures. To provide better depletion of monocytes and granulocytes we subjected PBMC concentrates to counter flow elutriation instrument. We manufactured 8 CD19- and 5 GD2-CAR T cell products from elutriated lymphocytes. All 13 CAR T cell manufacturing procedures yielded sufficient quantities of T cells to meet the dose criteria. The 13 CAR T cell products contained 2,166±1,113 x106 CD3+ cells and 1,064±877 x106 transduced CD3+ T cells. The CAR T cell products manufactured from elutriated lymphocytes yielded significantly more CD3+ cells and transduced CD3+ cells than that of those manufactured from anti-CD3/CD28 bead selected and plastic adherence selected cells. These results show CAR T cell manufacturing yields are greater and more consistent when manufacturing is initiated with lymphocytes that have few contaminating myeloid cells

"Sequencing-grade" screening for BRCA1 variants by oligo-arrays

The need for fast, efficient, and less costly means to screen genetic variants associated with disease predisposition led us to develop an oligo-nucleotide array-based process for gene-specific single nucleotide polymorphism (SNP) genotyping. This cost-effective, high-throughput strategy has high sensitivity and the same degree of accuracy as direct sequencing, the current gold standard for genetic screening. We used the BRCA1 breast and ovarian cancer predisposing gene model for the validation of the accuracy and efficiency of our strategy. This process could detect point mutations, insertions or deletions of any length, of known and unknown variants even in heterozygous conditions without affecting sensitivity and specificity. The system could be applied to other disorders and can also be custom-designed to include a number of genes related to specific clinical conditions. This system is particularly useful for the screening of long genomic regions with relatively infrequent but clinically relevant variants, while drastically cutting time and costs in comparison to high-throughput sequencing

15 kDa Granulysin versus GM-CSF for monocytes differentiation: analogies and differences at the transcriptome level

<p>Abstract</p> <p>Background</p> <p>Granulysin is an antimicrobial and proinflammatory protein with several isoforms. While the 9 kDa isoform is a well described cytolytic molecule with pro-inflammatory activity, the functions of the 15 kDa isoform is less well understood. Recently it was shown that 15 kDa Granulysin can act as an alarmin that is able to activate monocytes and immature dendritic cells. Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) is a growth factor widely used in immunotherapy both for <it>in vivo </it>and <it>ex vivo </it>applications, especially for its proliferative effects.</p> <p>Methods</p> <p>We analyzed gene expression profiles of monocytes cultured with 15 kDa Granulysin or GM-CSF for 4, 12, 24 and 48 hours to unravel both similarities and differences between the effects of these stimulators.</p> <p>Results</p> <p>The analysis revealed a common signature induced by both factors at each time point, but over time, a more specific signature for each factor became evident. At all time points, 15 kDa Granulysin induced immune response, chemotaxis and cell adhesion genes. In addition, only 15 kDa Granulsyin induced the activation of pathways related to fundamental dendritic cell functions, such as co-stimulation of T-cell activation and Th1 development. GM-CSF specifically down-regulated genes related to cell cycle arrest and the immune response. More specifically, cytokine production, lymphocyte mediated immunity and humoral immune response were down-regulated at late time points.</p> <p>Conclusion</p> <p>This study provides important insights on the effects of a novel agent, 15 kDa granulysin, that holds promise for therapeutic applications aimed at the activation of the immune response.</p

Intra-subject variability in human bone marrow stromal cell (BMSC) replicative senescence: Molecular changes associated with BMSC senescence

AbstractThe outcomes of clinical trials using bone marrow stromal cell (BMSC) are variable; the degree of the expansion of BMSCs during clinical manufacturing may contribute to this variability since cell expansion is limited by senescence. Human BMSCs from aspirates of healthy subjects were subcultured serially until cell growth stopped. Phenotype and functional measurements of BMSCs from two subjects including senescence-associated beta-galactosidase staining and colony formation efficiency changed from an early to a senescence pattern at passage 6 or 7. Transcriptome analysis of 10 early and 15 late passage BMSC samples from 5 subjects revealed 2122 differentially expressed genes, which were associated with immune response, development, and cell proliferation pathways. Analysis of 57 serial BMSC samples from 7 donors revealed that the change from an early to senescent profile was variable among subjects and occurred prior to changes in phenotypes. BMSC age expressed as a percentage of maximum population doublings (PDs) was a good indicator for an early or senescence transcription signature but this measure of BMSC life span can only be calculated after expanding BMSCs to senescence. In order to find a more useful surrogate measure of BMSC age, we used a computational biology approach to identify a set of genes whose expression at each passage would predict elapsed age of BMSCs. A total of 155 genes were highly correlated with BMSC age. A least angle regression algorithm identified a set of 24 BMSC age-predictive genes. In conclusion, the onset of senescence-associated molecular changes was variable and preceded changes in other indicators of BMSC quality and senescence. The 24 BMSC age predictive genes will be useful in assessing the quality of clinical BMSC products

Efficient clinical-grade γ-retroviral vector purification by high-speed centrifugation for CAR T cell manufacturing

γ-Retroviral vectors (γ-RV) are powerful tools for gene therapy applications. Current clinical vectors are produced from stable producer cell lines which require minimal further downstream processing, while purification schemes for γ-RV produced by transient transfection have not been thoroughly investigated. We aimed to develop a method to purify transiently produced γ-RV for early clinical studies. Here, we report a simple one-step purification method by high-speed centrifugation for γ-RV produced by transient transfection for clinical application. High-speed centrifugation enabled the concentration of viral titers in the range of 107-108 TU/mL with >80% overall recovery. Analysis of research-grade concentrated vector revealed sufficient reduction in product- and process-related impurities. Furthermore, product characterization of clinical-grade γ-RV by BioReliance demonstrated two-logs lower impurities per transducing unit compared with regulatory authority-approved stable producer cell line vector for clinical application. In terms of CAR T cell manufacturing, clinical-grade γ-RV produced by transient transfection and purified by high-speed centrifugation was similar to γ-RV produced from a clinical-grade stable producer cell line. This method will be of value for studies using γ-RV to bridge vector supply between early- and late-stage clinical trials

OR1.3 Reportistica dei survey effettuati nel Mar Tirreno sud-orientale e Canale di Sicilia: dati disponibili per OR1

In questo report vengono presentati i dati disponibili per la realizzazione delle attività di OR1 del progetto Marine Hazard (geofisica, campionature di sedimento e dati chimico-fisici della colonna d’acqua). I datasets presentati derivano da campagne oceanografiche eseguite dal CNR nel mar Tirreno SE (Semounts Marsili e Palinuro, alti relativi del fondo mare dell’Arco Eoliano) con focus sulla catena vulcanica del Palinuro (Seamounts Palinuro, Glabro, Enotrio, Ovidio). Vengono introdotte, quindi, le rappresentazioni cartografiche dei datasets batimetrici, side scan sonar, campionature del fondo, dati magnetici e dati sismici a riflessione già disponibile e in fase di elaborazione