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Background: Chimeric antigen receptor T (CAR-T) cells have significantly advanced the treatment of cancers such as leukemia and lymphoma. Traditionally, T cells are collected from patients through leukapheresis, an expensive and potentially invasive process that requires specialized equipment and trained personnel. Although whole blood collections are much more technically straightforward, whole blood starting material has not been widely utilized for clinical CAR-T cell manufacturing, in part due to lack of manufacturing processes designed for use in a good manufacturing practice (GMP) environment. Collecting cellular starting material from whole blood without leukapheresis could reduce manufacturing complexity and cost, thereby improving accessibility to CAR-T cell therapy., Methods: Whole blood samples were collected from eight healthy donors and one pediatric B-cell acute lymphoblastic leukemia (B-ALL) patient. These samples were processed using the Sepax C-Pro (Cytiva) instrument to isolate mononuclear cells (MNCs) via density gradient separation. CAR-T cells were then manufactured from the isolated MNCs using a GMP-compliant 7-day protocol, whereby T cells were activated with anti-CD3 and IL-2, transduced with GMP lentiviral vector encoding a CD19/CD22 bispecific CAR, and expanded in gas permeable cell culture bags. The resulting CAR-T cells were then evaluated for their phenotypic and functional properties using flow cytometry, cytokine release and cytotoxicity assays., Results: From an average 77.7 mL of whole blood from healthy donors (range = 29-96 mL), we isolated an average of 42.2 × 10 6 CD3⁺ T cells (range 7.3-63.0) postprocessing. CAR-T cell cultures were initiated from thaw using 1-10 × 10 6 starting CD3 + T cells, yielding a median T cell number of 105 × 10 6 cells on day 7 (range 61-188 × 10 6 ). We observed 66 ± 11% mean transduction efficiency and produced a mean of 77.4 × 10 6 transduced CAR-T cells (range 30.8-143.5 × 10 6 ). Similar results were obtained when using a blood sample (28mL) obtained from a patient with relapsed B-ALL who had received recent chemotherapy., Conclusions: Therapeutically relevant doses of CD19/CD22 CAR-T cells can be successfully manufactured from whole blood. On average, 80 mL of whole blood yields enough CAR-T cells to create a single dose for a pediatric patient (50 kg) at a dosage of 1 × 10 6 CAR-T cells/kg. For larger patients, scaling up is straightforward by collecting a larger blood volume. This method also demonstrates a cost-effective approach to T cell activation and expansion which, alongside a more straightforward collection of whole blood, makes it more widely accessible especially for middle- and low-income countries. By reducing costs and labor, this strategy has the potential to significantly expand global access to CAR-T cell therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Published by Elsevier Inc.)

With investigators looking to expand engineered T cell therapies such as CAR-T to new tumor targets and patient populations, a variety of cell manufacturing platforms have been developed to scale manufacturing capacity using closed and/or automated systems. Such platforms are particularly useful for solid tumor targets, which typically require higher CAR-T cell doses. Although T cell phenotype and function are key attributes that often correlate with therapeutic efficacy, how manufacturing platforms influence the final CAR-T cell product is currently unknown. We compared 4 commonly used T cell manufacturing platforms (CliniMACS Prodigy, Xuri W25 rocking platform, G-Rex gas-permeable bioreactor, static bag culture) using identical media, stimulation, culture length, and donor starting material. Selected CD4 + CD8 + cells were transduced with lentiviral vector incorporating a CAR targeting FGFR4, a promising target for pediatric sarcoma. We observed significant differences in overall expansion over the 14-day culture; bag cultures had the highest capacity for expansion while the Prodigy had the lowest (481-fold versus 84-fold, respectively). Strikingly, we also observed considerable differences in the phenotype of the final product, with the Prodigy significantly enriched for CCR7 + CD45RA + naïve/stem central memory (T n/scm )-like cells at 46% compared to bag and G-Rex with 16% and 13%, respectively. Gene expression analysis also showed that Prodigy CAR-Ts are more naïve, less cytotoxic and less exhausted than bag, G-Rex, and Xuri CAR-Ts, and pointed to differences in cell metabolism that were confirmed via metabolic assays. We hypothesized that dissolved oxygen level, which decreased substantially during the final 3 days of the Prodigy culture, may contribute to the observed differences in T cell phenotype. By culturing bag and G-Rex cultures in 1% O 2 from day 5 onward, we could generate >60% T n/scm -like cells, with longer time in hypoxia correlating with a higher percentage of T n/scm -like cells. Intriguingly, our results suggest that oxygenation is responsible, at least in part, for observed differences in T cell phenotype among bioreactors and suggest hypoxic culture as a potential strategy prevent T cell differentiation during expansion. Ultimately, our study demonstrates that selection of bioreactor system may have profound effects not only on the capacity for expansion, but also on the differentiation state of the resulting CAR-T cells., Competing Interests: Declaration of Competing Interest The authors declare no competing interests. The views expressed by the authors do not necessarily represent the views of the NIH, Department of Health and Human Services, or the US Federal Government., (Published by Elsevier Inc.)

Chimeric antigen receptor (CAR) T-cell toxicities resembling hemophagocytic lymphohistiocytosis (HLH) occur in a subset of patients with cytokine release syndrome (CRS). As a variant of conventional CRS, a comprehensive characterization of CAR T-cell-associated HLH (carHLH) and investigations into associated risk factors are lacking. In the context of 59 patients infused with CD22 CAR T cells where a substantial proportion developed carHLH, we comprehensively describe the manifestations and timing of carHLH as a CRS variant and explore factors associated with this clinical profile. Among 52 subjects with CRS, 21 (40.4%) developed carHLH. Clinical features of carHLH included hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, coagulopathy, hepatic transaminitis, hyperbilirubinemia, severe neutropenia, elevated lactate dehydrogenase, and occasionally hemophagocytosis. Development of carHLH was associated with preinfusion natural killer(NK) cell lymphopenia and higher bone marrow T-cell:NK cell ratio, which was further amplified with CAR T-cell expansion. Following CRS, more robust CAR T-cell and CD8 T-cell expansion in concert with pronounced NK cell lymphopenia amplified preinfusion differences in those with carHLH without evidence for defects in NK cell mediated cytotoxicity. CarHLH was further characterized by persistent elevation of HLH-associated inflammatory cytokines, which contrasted with declining levels in those without carHLH. In the setting of CAR T-cell mediated expansion, clinical manifestations and immunophenotypic profiling in those with carHLH overlap with features of secondary HLH, prompting consideration of an alternative framework for identification and management of this toxicity profile to optimize outcomes following CAR T-cell infusion., (© 2021 by The American Society of Hematology.)

While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19 - disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials ( NCT02588456 and NCT02650414 ) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.

Purpose: Patients with B-cell acute lymphoblastic leukemia who experience relapse after or are resistant to CD19-targeted immunotherapies have limited treatment options. Targeting CD22, an alternative B-cell antigen, represents an alternate strategy. We report outcomes on the largest patient cohort treated with CD22 chimeric antigen receptor (CAR) T cells., Patients and Methods: We conducted a single-center, phase I, 3 + 3 dose-escalation trial with a large expansion cohort that tested CD22-targeted CAR T cells for children and young adults with relapsed/refractory CD22 + malignancies. Primary objectives were to assess the safety, toxicity, and feasibility. Secondary objectives included efficacy, CD22 CAR T-cell persistence, and cytokine profiling., Results: Fifty-eight participants were infused; 51 (87.9%) after prior CD19-targeted therapy. Cytokine release syndrome occurred in 50 participants (86.2%) and was grade 1-2 in 45 (90%). Symptoms of neurotoxicity were minimal and transient. Hemophagocytic lymphohistiocytosis-like manifestations were seen in 19/58 (32.8%) of subjects, prompting utilization of anakinra. CD4/CD8 T-cell selection of the apheresis product improved CAR T-cell manufacturing feasibility as well as heightened inflammatory toxicities, leading to dose de-escalation. The complete remission rate was 70%. The median overall survival was 13.4 months (95% CI, 7.7 to 20.3 months). Among those who achieved a complete response, the median relapse-free survival was 6.0 months (95% CI, 4.1 to 6.5 months). Thirteen participants proceeded to stem-cell transplantation., Conclusion: In the largest experience of CD22 CAR T-cells to our knowledge, we provide novel information on the impact of manufacturing changes on clinical outcomes and report on unique CD22 CAR T-cell toxicities and toxicity mitigation strategies. The remission induction rate supports further development of CD22 CAR T cells as a therapeutic option in patients resistant to CD19-targeted immunotherapy.

Background: Most chimeric antigen receptor T (CAR-T) cells and other adoptive T-cell therapies (ACTs) are currently manufactured by ex vivo expansion of patient lymphocytes in culture media supplemented with human plasma from group AB donors. As lymphocytes do not express A or B antigens, the isoagglutinins of non-AB plasmas are unlikely to cause deleterious effects on lymphocytes in culture., Study Design and Methods: Seeding cultures with peripheral blood mononuclear cell (PBMNC) concentrates from group A 1 donors and using a CAR-T culture protocol, parallel cultures were performed, each with unique donor plasmas as media supplements (including group O plasmas with high-titer anti-A and group AB plasmas as control). An additional variable, a 3% group A 1 red blood cell (RBC) spike, was added to simulate a RBC-contaminated PBMNC collection. Cultures were monitored by cell count, viability, flow cytometric phenotype, gene expression analysis, and supernatant chemokine analysis., Results: There was no difference in lymphocyte expansion or phenotype when cultured with AB plasma or O plasma with high-titer anti-A. Compared to controls, the presence of contaminating RBCs in lymphocyte culture led to poor lymphocyte expansion and a less desirable phenotype-irrespective of the isoagglutinin titer of the plasma supplement used., Conclusions: This study suggests that ABO incompatible plasma may be used as a media supplement when culturing cell types that do not express ABO antigens-such as lymphocytes for CAR-T or other ACT. The presence of contaminating RBCs in culture was disadvantageous independent of isoagglutinin titer., (Published 2020. This article is a U.S. Government work and is in the public domain in the USA.)

As clinical applications for chimeric antigen receptor T cell (CART) therapy extend beyond early phase trials, commercial manufacture incorporating cryopreservation steps becomes a logistical necessity. The effect of cryopreservation on CART characteristics is unclear. We retrospectively evaluated the effect of cryopreservation on product release criteria and in vivo characteristics in 158 autologous CART products from 6 single-center clinical trials. Further, from 3 healthy donor manufacturing runs, we prospectively identified differentially expressed cell surface markers and gene signatures among fresh versus cryopreserved CARTs. Within 2 days of culture initiation, cell viability of the starting fraction (peripheral blood mononuclear cells [PBMNCs]) decreased significantly in the cryo-thawed arm compared to the fresh arm. Despite this, PBMNC cryopreservation did not affect final CART fold expansion, transduction efficiency, CD3%, or CD4:CD8 ratios. In vivo CART persistence and clinical responses did not differ among fresh and cryopreserved final products. In healthy donors, compared to fresh CARTs, early apoptotic cell-surface markers were significantly elevated in cryo-thawed CARTs. Cryo-thawed CARTs also demonstrated significantly elevated expression of mitochondrial dysfunction, apoptosis signaling, and cell cycle damage pathways. Cryopreservation during CART manufacture is a viable strategy, based on standard product release parameters. The clinical impact of cryopreservation-related subtle micro-cellular damage needs further study., (Published by Elsevier Inc.)

Genetically engineered T cells expressing CD19-specific chimeric antigen receptors (CAR) have shown impressive activity against B-cell malignancies, and preliminary results suggest that T cells expressing a first-generation disialoganglioside (GD2)-specific CAR can also provide clinical benefit in patients with neuroblastoma. We sought to assess the potential of GD2-CAR therapies to treat pediatric sarcomas. We observed that 18 of 18 (100%) of osteosarcomas, 2 of 15 (13%) of rhabdomyosarcomas, and 7 of 35 (20%) of Ewing sarcomas expressed GD2. T cells engineered to express a third-generation GD2-CAR incorporating the 14g2a-scFv with the CD28, OX40, and CD3ζ signaling domains (14g2a.CD28.OX40.ζ) mediated efficient and comparable lysis of both GD2 + sarcoma and neuroblastoma cell lines in vitro However, in xenograft models, GD2-CAR T cells had no antitumor effect against GD2 + sarcoma, despite effectively controlling GD2 + neuroblastoma. We observed that pediatric sarcoma xenografts, but not neuroblastoma xenografts, induced large populations of monocytic and granulocytic murine myeloid-derived suppressor cells (MDSC) that inhibited human CAR T-cell responses in vitro Treatment of sarcoma-bearing mice with all-trans retinoic acid (ATRA) largely eradicated monocytic MDSCs and diminished the suppressive capacity of granulocytic MDSCs. Combined therapy using GD2-CAR T cells plus ATRA significantly improved antitumor efficacy against sarcoma xenografts. We conclude that retinoids provide a clinically accessible class of agents capable of diminishing the suppressive effects of MDSCs, and that co-administration of retinoids may enhance the efficacy of CAR therapies targeting solid tumors. Cancer Immunol Res; 4(10); 869-80. ©2016 AACR., Competing Interests: The authors report no conflict of interest exists., (©2016 American Association for Cancer Research.)