The mesenchymal stromal cell (MSC) field continues to rapidly progress with a number of clinical trials initiated and completed, with some reported successes in multiple clinical indications, and a ...growing number of companies established. The field, nevertheless, faces several challenges. Persistent issues include the definition of a MSC and comparability between MSC preparations. This is because of inherent cell heterogeneity, the absence of markers that are unique to MSCs, and the difficulty in precisely defining them by developmental origin. Differences in the properties of MSCs also depend on the site of tissue harvest, phenotypic and genotypic characteristics of the donor and the isolation, and storage and expansion methods used. These differences may be sufficient to ensure that attributes of the final MSC product could differ in potentially significant ways. Since there are currently no gold standards, we propose using a reference material to establish methods of comparability among MSC preparations. We suggest four possible "ruler scenarios" and a method for global distribution. We further suggest that critical to establishing a reference material is the need to define protocols for comparing cells. The main purpose of this article is to solicit input in establishing a consensus-based comparison. A comparative approach will be critical to all stages of translation to better clarify mechanisms of MSC actions, define an optimal cell manufacturing process, ensure best practice clinical investigations, extend the use of an MSC product for new indications, protect an MSC product from imitators, and develop uniform reimbursement policies. Importantly, a reference material may enable a consensus on a practical definition of MSCs.
Abstract
Background
Cytokine release syndrome (CRS) is a strong immune system response that can occur as a result of the reaction of a cellular immunotherapy with malignant cells. While the frequency ...and management of CRS in CAR T-cell therapy has been well documented, there is emerging interest in pre-emptive treatment to reduce CRS severity and improve overall outcomes. Accordingly, identification of genomic determinants that contribute to cytokine release may lead to the development of targeted therapies to prevent or abrogate the severity of CRS.
Methods
Forty three clinical CD22 CAR T-cell products were collected for RNA extraction. 100 ng of mRNA was used for Nanostring assay analysis which is based on the nCounter platform. Several public datasets were used for validation purposes.
Results
We found the expression of the PFKFB4 gene and glycolytic pathway activity were upregulated in CD22 CAR T-cells given to patients who developed CRS compared to those who did not experience CRS. Moreover, these results were further validated in cohorts with COVID-19, influenza infections and autoimmune diseases, and in tumor tissues. The findings were similar, except that glycolytic pathway activity was not increased in patients with influenza infections and systemic lupus erythematosus (SLE).
Conclusion
Our data strongly suggests that PFKFB4 acts as a driving factor in mediating cytokine release in vivo by regulating glycolytic activity. Our results suggest that it would beneficial to develop drugs targeting PFKFB4 and the glycolytic pathway for the treatment of CRS.
We translated two cancer vaccine strategies from mice into human clinical trials. (1) In preclinical studies on TARP, an antigen expressed in most prostate cancers, we mapped epitopes presented by ...HLA-A*0201, modified them to increase affinity and immunogenicity in HLA transgenic mice, and induced human T cells that killed human cancer cells (“epitope enhancement”). In a clinical trial, HLA-A2
+
prostate cancer patients with PSA biochemical recurrence (Stage D0) were vaccinated with two peptides either in Montanide-ISA51 or on autologous dendritic cells (DCs). In stage D0, the Prostate-Specific Antigen (PSA) slope is prognostic of time to radiographic evidence of metastases and death. With no difference between arms, 74% of combined subjects had a decreased PSA slope at 1 year compared to their own baseline slopes (
p
= 0.0004). For patients vaccinated with DCs, response inversely correlated with a tolerogenic DC signature. A randomized placebo-controlled phase II trial is underway. (2) HER2 is a driver surface oncogene product expressed in multiple tumors. We made an adenoviral vector vaccine expressing the extracellular and transmembrane domains of HER2 and cured mice with large established HER2
+
tumors, dependent on antibodies to HER2, not T cells. The mechanism differed from that of trastuzumab. We tested a human version in advanced metastatic cancer patients naïve to HER2-directed therapies. At the second and third dose levels, 45% of evaluable patients showed clinical benefit. Circulating tumor cells also declined in some vaccinated patients. Thus, cancer vaccines developed in mice were successfully translated to humans with promising early results.
PURPOSE OF REVIEWSeveral advances have been made in characterization of the molecule that carries human neutrophil antigen (HNA)-2a, NB1 glycoprotein, the gene that encodes NB1 glycoprotein, CD177, ...and the role of antibodies to HNA-2a in transfusion reactions.
RECENT FINDINGSNB1 glycoprotein binds to the endothelial cell adhesion molecule, platelet endothelial cell adhesion molecule-1 (PECAM-1), and participates in neutrophil transmigration. The overexpression of neutrophil CD177 mRNA has become a useful, but nonspecific biomarker of myeloproliferative diseases, especially polycythemia vera. CD177 mRNA overexpression is also a biomarker of a subset of patients with essential thrombocythemia who are at increased risk of thromboembolic complications. In patients with myeloproliferative disorders CD177 mRNA overexpression is secondary to a gain-of-function mutation in JAK2, JAK2 V617F. NB1 glycoprotein is co-localized on neutrophil plasma membranes with proteinase 3 and a complex of NB1 glycoprotein and proteinase 3 may initiate the activation of neutrophils by antineutrophil cytoplasmic antibodies in patients with Wegenerʼs granulomatosis. The inadvertent transfusion of antibodies to HNA-2a with blood components frequently causes pulmonary transfusion reactions.
SUMMARYThe expression of CD177 is an important biomarker of myeloproliferative diseases, NB1 glycoprotein is a ligand for PECAM-1 and it may have a role in Wegenerʼs granulomatosis, and antibodies to HNA-2a frequently cause pulmonary transfusion reactions.
Bone marrow stromal cells (BMSCs) have classically been cultured in media supplemented with fetal bovine serum (FBS). As an alternative to FBS, pooled solvent detergent apheresis platelets, HPGF-C18, ...was evaluated for BMSC culture.
A comparison of passage 2 BMSC growth revealed that 10% HPGF-C18 produced similar cell numbers as 20% FBS. Marrow aspirates from 5 healthy subjects were cultured for 4 passages in 10% HPGF-C18 or 20% FBS and were analyzed for proliferation, colony formation efficiency (CFE), surface marker expression, suppression of mixed lymphocyte reactions (MLRs), global gene and microRNA expression analysis. BMSC supernatant cytokine and growth factor concentrations were also compared.
Primary cultures of marrow aspirates in 10% HPGF-C18 and 20% FBS yielded similar numbers and CFE. After 4 passages, 10% HPGF-C18 and 20% FBS yielded similar numbers of BMSCs, surface marker expression patterns and immunosuppression effects. Gene and microRNA expression analysis revealed that BMSCs cultured under the two conditions had distinct expression profiles. Gene Set Enrichment Analysis (GSEA) revealed HPGF-C18-cultured BMSCs were enriched in metabolic processing and biosynthetic pathways, cell proliferation and cell cycle pathways, and immune response pathways. FBS-cultured BMSCs were enriched in MAPK signaling, TGF-beta signaling, cell adhesion and extracellular matrix pathways. Differently expressed microRNAs were related to the osteogenesis of BMSCs. The supernatant of HPGF-C18 BMSCs had higher levels of PEDF and TGFB1 and lower levels of IL6, VEGF, SDF1 and PLGF.
Traditional measures, expansion, surface marker expression and inhibition of MLRs suggest that BMSC cultured in HPGF-C18 and FBS were similar, but analysis at the molecular level revealed many differences. BMSCs cultured in HPGF-C18 should be assessed in specific functional assays that reflect application-specific potency before substituting FBS with HPGF-C18.
Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are manufactured using many different methods, but little is known about the spectrum of ...manufacturing methods used and their effects on BMSC characteristics and function. Seven centers using, and one developing, Good Manufacturing Practices (GMP) processes were surveyed as to their production methods. Among the seven centers, all used marrow aspirates as the starting material, but no two centers used the same manufacturing methods. Two to four BMSC lots from each center were compared using global gene expression. Among the twenty-four BMSC lots from the eight centers intra-center transcriptome variability was low and similar among centers. Principal component analysis and unsupervised hierarchical clustering analysis separated all the lots from five centers into five distinct clusters. BMSCs from six of the eight centers were tested for their ability to form bone and support hematopoiesis by in vivo transplantation (defining features of BMSCs). Those from all six centers tested formed bone, but the quantity formed was highly variable and BMSCs from only three centers supported hematopoiesis. These results show that differences in manufacturing resulted in variable BMSC characteristics including their ability to form bone and support hematopoiesis.
Bone marrow stromal or stem cells (BMSCs) remain a promising potential therapy for ischemic cardiomyopathy. The primary objective of this study was to evaluate the safety and feasibility of direct ...intramyocardial injection of autologous BMSCs in patients undergoing transmyocardial revascularization (TMR) or coronary artery bypass graft surgery (CABG).
A phase I trial was conducted on adult patients who had ischemic heart disease with depressed left ventricular ejection fraction and who were scheduled to undergo TMR or CABG. Autologous BMSCs were expanded for 3 weeks before the scheduled surgery. After completion of surgical revascularization, BMSCs were directly injected into ischemic myocardium. Safety and feasibility of therapy were assessed. Cardiac functional status and changes in quality of life were evaluated at 1 year.
A total of 14 patients underwent simultaneous BMSC and surgical revascularization therapy (TMR+BMSCs = 10; CABG+BMSCs = 4). BMSCs were successfully expanded, and no significant complications occurred as a result of the procedure. Regional contractility in the cell-treated areas demonstrated improvement at 12 months compared with baseline (TMR+BMSCs Δ strain: −4.6% ± 2.1%; P = .02; CABG+MSCs Δ strain: −4.2% ± 6.0%; P = .30). Quality of life was enhanced, with substantial reduction in angina scores at 1 year after treatment (TMR+BMSCs: 1.3 ± 1.2; CABG+MSCs: 1.0 ± 1.4).
In this phase I trial, direct intramyocardial injection of autologous BMSCs in conjunction with TMR or CABG was technically feasible and could be performed safely. Preliminary results demonstrate improved cardiac function and quality of life in patients at 1 year after treatment.
BACKGROUND
When manufacturing chimeric antigen receptor (CAR) T cells using anti‐CD3/anti‐CD28 beads, ex vivo T‐cell expansion is dependent on the composition of leukocytes used in the manufacturing ...process. We investigated the effects of leukocyte composition on CAR T‐cell expansion and characteristics using an alternative manufacturing method.
METHODS
Anti–B‐cell maturation antigen and CD19‐CAR T cells were manufactured using autologous peripheral blood mononuclear cell (PBMNC) concentrates. The PBMNCs were enriched for lymphocytes using density gradient separation, which were used for CAR T‐cell culture initiation. T‐cell expansion was stimulated with soluble anti‐CD3 and interleukin‐2.
RESULTS
Fifty‐one CAR T‐cell products were evaluated; 28 anti–B‐cell maturation antigen (BCMA) CAR T cells produced for 24 patients and 27 CD19 CAR T cells produced for 24 patients. CAR T‐cell expansion was reduced when greater quantities of monocytes were present in the post–density gradient separation PBMNCs. In addition, the ratio of CD4 to CD8 cells in the CAR T‐cell products after 7 days of culture was dependent on the quantity of monocytes, RBCs, and neutrophils in the post–density gradient separation PBMNCs. Greater quantities of monocytes and RBCs were associated with a greater proportion of CD4+ cells and greater quantities of neutrophils were associated with a greater proportion of CD8+ cells.
CONCLUSIONS
The composition of leukocytes used to manufacture CAR T cells can affect cell expansion and the composition of CAR T‐cell products. More uniform or complete lymphocyte enrichment of PBMNCs improves the consistency of final CAR T‐cell products.
BACKGROUND
Efforts are underway to eliminate fetal bovine serum from mammalian cell cultures for clinical use. An emerging, viable replacement option for fetal bovine serum is human platelet lysate ...(PL) as either a plasma‐based or serum‐based product.
STUDY DESIGN AND METHODS
Nine industrial‐scale, serum‐based PL manufacturing runs (i.e., lots) were performed, consisting of an average ± standard deviation volume of 24.6 ± 2.2 liters of pooled, platelet‐rich plasma units that were obtained from apheresis donors. Manufactured lots were compared by evaluating various biochemical and functional test results. Comprehensive cytokine profiles of PL lots and product stability tests were performed. Global gene expression profiles of mesenchymal stromal cells (MSCs) cultured with plasma‐based or serum‐based PL were compared to MSCs cultured with fetal bovine serum.
RESULTS
Electrolyte and protein levels were relatively consistent among all serum‐based PL lots, with only slight variations in glucose and calcium levels. All nine lots were as good as or better than fetal bovine serum in expanding MSCs. Serum‐based PL stored at −80°C remained stable over 2 years. Quantitative cytokine arrays showed similarities as well as dissimilarities in the proteins present in serum‐based PL. Greater differences in MSC gene expression profiles were attributable to the starting cell source rather than with the use of either PL or fetal bovine serum as a culture supplement.
CONCLUSION
Using a large‐scale, standardized method, lot‐to‐lot variations were noted for industrial‐scale preparations of serum‐based PL products. However, all lots performed as well as or better than fetal bovine serum in supporting MSC growth. Together, these data indicate that off‐the‐shelf PL is a feasible substitute for fetal bovine serum in MSC cultures.
Advances in T-cell Immunotherapies Stroncek, David F; Reddy, Opal; Highfill, Steven ...
Hematology/oncology clinics of North America,
10/2019, Letnik:
33, Številka:
5
Journal Article
Recenzirano
Cell therapies have become an important part of clinical hematology and oncology. Cell therapy laboratories were first established in academic health centers to process ABO-incompatible marrow ...grafts. These laboratories now produce a wide variety of cell and gene therapies. Some of the most widely used and clinically important cell therapies are T-cell immunotherapies. These therapies include donor lymphocyte infusions, tumor-infiltrating lymphocytes, T-cell receptor-engineered T cells, chimeric antigen receptor T cells, and virus-specific T cells. The clinical application and methods used to manufacture these adoptive cell therapies are reviewed.
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