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Fetal bovine serum (FBS) or human serum is widely used in the production of chimeric antigen receptor (CAR) T‑cells. In order to overcome a lot‑to‑lot inconsistency, the use of chemically defined medium that is free of animal-components would be highly desirable. The present study compared three serum‑free media [Prime‑XV™ T Cell CDM, Fujifilm™ (FF), LymphoONE™ T‑Cell Expansion Xeno‑Free Medium, Takara Bio™ (TB) and TCM GMP‑Prototype, CellGenix™ (CG)] to the standard CAR T‑cell medium containing FBS (RCF). After 12 days of CD19.CAR T‑cell culture, the expansion, viability, transduction efficiency and phenotype were assessed using flow cytometry. The functionality of CAR T‑cells was evaluated using intracellular staining, a chromium release assay and a long‑term co‑culture assay. Expansion and viability did not differ between the CAR T‑cells generated in serum‑free media compared to the standard FBS‑containing medium. The CG CAR T‑cells had a statistically significant higher frequency of IFNγ + and IFNγ + TNF‑α + CAR T‑cells than the CAR T‑cells cultured with FBS (22.5 vs. 7.6%, P=0.0194; 15.3 vs. 6.2%, P=0.0399, respectively) as detected by intracellular cytokine staining. The CAR T‑cells generated with serum‑free media exhibited a higher cytotoxicity than the CAR T‑cells cultured with FBS in the evaluation by chromium release assay [CG vs. RCF (P=0.0182), FF vs. RCF (P=0.0482) and TB vs. RCF (P=0.0482)]. Phenotyping on day 12 of CAR T‑cell production did not reveal a significant difference in the expression of the exhaustion markers, programmed cell death protein 1, lymphocyte‑activation gene 3 and T‑cell immunoglobulin and mucin‑domain containing‑3. The CAR T‑cells cultured in FF had a higher percentage of central memory CAR T‑cells (40.0 vs. 14.3%, P=0.0470) than the CAR T‑cells cultured with FBS, whereas the CAR T‑cells in FF (6.2 vs. 24.2%, P=0.0029) and CG (11.0% vs. 24.2%, P=0.0468) had a lower frequency of naïve CAR T‑cells. On the whole, the present study demonstrates that in general, the functionality and expansion of CAR T cells are maintained in serum‑free media. Given the advantages of freedom from bovine material and consistent quality, serum‑free media hold promise for the future development of the field of GMP manufacturing of CAR T‑cells.

Chimeric antigen receptor (CAR) T cell therapy with axicabtagene ciloleucel, tisagenlecleucel and brexucabtagen ciloleucel has been adopted as the standard of care for patients with refractory and/or relapsed CD19‑positive lymphoid malignancies. Monitoring of kinetics of CAR T cells after administration is crucial for patient follow‑up and important to guide clinical decisions for patients subjected to CAR T cell therapy. Information of transgene copies within a CAR T cell product prior to administration, i.e. vector copy numbers, is of high importance to warrant patient safety. However, experimental assays for quantitative CAR T cell monitoring in the open domain are currently lacking. Several institutions have established in‑house assays to monitor CAR T cell frequencies. In the present study, the quantitative (q)PCR assay established at the Heidelberg University Hospital (Heidelberg, Germany), i.e. single copy gene‑based duplex qPCR, was compared with the digital droplet PCR assay established at the University Medical Center Hamburg‑Eppendorf (Hamburg, Germany). Both methods that were independently developed enable accurate and comparable CAR T cell frequency assessment and are useful in the clinical setting.

Chimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell transplantation. Multiple myeloma still constitutes an incurable disease. CAR-T-cell therapy that targets BCMA (B-cell maturation antigen) is currently revolutionizing the treatment of those patients. To monitor and improve treatment outcomes, methods to detect CAR-T cells in human peripheral blood are highly desirable. In this study, three different detection reagents for staining BCMA-CAR-T cells by flow cytometry were compared. Moreover, a quantitative polymerase chain reaction (qPCR) to detect BCMA-CAR-T cells was established. By applying a cell-titration experiment of BCMA-CAR-T cells, both methods were compared head-to-head. In flow-cytometric analysis, the detection reagents used in this study could all detect BCMA-CAR-T cells at a similar level. The results of false-positive background staining differed as follows (standard deviation): the BCMA-detection reagent used on the control revealed a background staining of 0.04% (±0.02%), for the PE-labeled human BCMA peptide it was 0.25% (±0.06%) and for the polyclonal anti-human IgG antibody it was 7.2% (±9.2%). The ability to detect BCMA-CAR-T cells down to a concentration of 0.4% was similar for qPCR and flow cytometry. The qPCR could detect even lower concentrations (0.02-0.01%). In summary, BCMA-CAR-T-cell monitoring can be reliably performed by both flow cytometry and qPCR. In flow cytometry, reagents with low background staining should be preferred.

Chimeric antigen receptor T (CAR-T) cells targeting CD19 came into clinical practice for the treatment of B cell lymphoma in 2018. However, patients being treated for B cell lymphoma often suffer from comorbidities such as chronic pain, cardiovascular diseases and arthritis. Thus, these patients frequently receive concomitant medications that include nonsteroidal anti-inflammatory drugs (NSAIDs) like cyclooxygenase (COX) inhibitors. Celecoxib, a selective COX-2 inhibitor, and aspirin, a non-selective COX-1 and COX-2 inhibitor, are being used as anti-inflammatory, analgesic and anti-pyretic drugs. In addition, several studies have also focused on the anti-neoplastic properties of COX-inhibitors. As the influence of COX-inhibitors on CD19.CAR-T cells is still unknown, we investigated the effect of celecoxib and aspirin on the quantity and quality of CD19.CAR-T cells at different concentrations with special regard to cytotoxicity, activation, cytokine release, proliferation and exhaustion. A significant effect on CAR-T cells could be observed for 0.1 mmol/L of celecoxib and for 4 mmol/L of aspirin. At these concentrations, we found that both COX-inhibitors could induce intrinsic apoptosis of CD19.CAR-T cells showing a significant reduction in the ratio of JC-10 red to JC-10 green CAR-T cells from 6.46 ± 7.03 (mean ± SD) to 1.76 ± 0.67 by celecoxib and to 4.41 ± 0.32 by aspirin, respectively. Additionally, the ratios of JC-10 red to JC-10 green Daudi cells were also decreased from 3.41 ± 0.30 to 0.77 ± 0.06 by celecoxib and to 1.26 ± 0.04 by aspirin, respectively. Although the cytokine release by CD19.CAR-T cells upon activation was not hampered by both COX-inhibitors, activation and proliferation of CAR-T cells were significantly inhibited via diminishing the NF-ĸB signaling pathway by a significant down-regulation of expression of CD27 on CD4 + and CD8 + CAR-T cells, followed by a clear decrease of phosphorylated NF-ĸB p65 in both CD4 + and CD8 + CAR-T cells by a factor of 1.8. Of note, COX-inhibitors hampered expansion and induced exhaustion of CAR-T cells in an antigen stress assay. Collectively, our findings indicate that the use of COX-inhibitors is a double-edged sword that not only induces apoptosis in tumor cells but also impairs the quantity and quality of CAR-T cells. Therefore, COX-inhibitors should be used with caution in patients with B cell lymphoma under CAR-T cell therapy., Competing Interests: MS received funding for collaborative research from Apogenix, Hexal and Novartis, travel grants from Hexal and Kite, he received financial support for educational activities and conferences from bluebird bio, Kite and Novartis, he is a board member for MSD and (co-)PI of clinical trials of MSD, GSK, Kite and BMS, as well as co-founder and shareholder of TolerogenixX Ltd. AS received travel grants from Hexal and Jazz Pharmaceuticals, research grant from Therakos/Mallinckrodt and is co-founder of TolerogenixX Ltd. AS and LW are part- or full-time employers of TolerogenixX Ltd. LS was employed by Takeda Pharma Vertrieb GmbH & Co. KG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yang, Wang, Ni, Neuber, Wang, Gong, Sauer, Schubert, Hückelhoven-Krauss, Xia, Ge, Kleist, Eckstein, Sellner, Müller-Tidow, Dreger, Schmitt and Schmitt.)

Chimeric antigen receptor (CAR) T cell therapy has shown promising responses in patients with refractory or relapsed aggressive B-cell malignancies that are resistant to conventional chemotherapy or stem cell transplantation. A potentially combinatorial therapeutic strategy may be the inhibition of anti-apoptotic Bcl-2 family proteins, overexpressed in most cancer cells. In this study we investigated the combination of 3rd-generation CD19.CAR-T cells and the BH3 mimetics venetoclax, a Bcl-2 inhibitor, or S63845, a Mcl-1 inhibitor, under three different treatment conditions: pre-sensitization of cancer cells with BH3 mimetics followed by CAR-T cell treatment, simultaneous combination therapy, and the administration of BH3 mimetics after CAR-T cell treatment. Our results showed that administration of CAR-T cells and BH3 mimetics had a significant effect on the quantity and quality of CD19.CAR-T cells. The administration of BH3 mimetics prior to CAR-T cell therapy exerted an enhanced cytotoxic efficacy by upregulating the CD19 expression and pro-apoptotic proteins in highly sensitive tumor cells, and thereby improving both CD19.CAR-T cell cytotoxicity and persistence. In simultaneous and post-treatment approaches, however, the quantity of CAR-T cells was adversely affected. Our findings indicate pre-sensitization of highly sensitive tumor cells with BH3 mimetics could enhance the cytotoxic efficacy of CAR-T cell treatment., Competing Interests: MS received funding for collaborative research from Apogenix, Hexal and Novartis, travel grants from Hexal and Kite, received financial support for educational activities and conferences from bluebird bio, Kite and Novartis, is a board member for MSD and (co-)PI of clinical trials of MSD, GSK, Kite, and BMS, as well as a co-founder and shareholder of TolerogenixX Ltd. AS received travel grants from Hexal and Jazz Pharmaceuticals, research grant from Therakos/Mallinckrodt, and is a co-founder of TolerogenixX LtD. AS and LW are part- or full-time employers of TolerogenixX Ltd. LS was employed by Takeda Pharma Vertrieb GmbH & Co. KG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past collaboration with one of the authors TS., (Copyright © 2020 Yang, Wang, Ni, Neuber, Wang, Gong, Sauer, Sellner, Schubert, Hückelhoven-Krauss, Hong, Zhu, Kleist, Eckstein, Müller-Tidow, Dreger, Schmitt and Schmitt.)

Despite encouraging results with chimeric antigen receptor T (CART) cells, outcome can still be improved by optimization of the CART cell generation process. The proportion of less-differentiated T cells within the transfused product is linked to enhanced in vivo CART cell expansion and long-term persistence. The clinically approved PI3Kδ inhibitor idelalisib is well established in the treatment of B cell malignancies. Besides B cell receptor pathway inhibition, idelalisib can modulate T cell differentiation and function. Here, detailed longitudinal analysis of idelalisib-induced effects on T cell phenotype and function was performed during CART cell production. A third generation CD19.CAR.CD28.CD137zeta CAR vector system was used. CART cells were generated from peripheral blood mononuclear cells of healthy donors (HDs) and chronic lymphocytic leukemia (CLL) patients. Idelalisib-based CART cell generation resulted in an enrichment of less-differentiated naïve-like T cells (CD45RA+CCR7+), decreased expression of the exhaustion markers PD-1 and Tim-3, as well as upregulation of the lymph node homing marker CD62L. Idelalisib increased transduction efficiency, but did not impair viability and cell expansion. Strikingly, CD4:CD8 ratios that were altered in CART cells from CLL patients were approximated to ratios in HDs by idelalisib. Furthermore, in vivo efficacy of idelalisib-treated CART cells was validated in a xenograft mouse model. Intracellular TNF-α and IFN-γ production decreased in presence of idelalisib. This effect was reversible after resting CART cells without idelalisib. In summary, PI3Kδ inhibition with idelalisib can improve CART cell products, particularly when derived from CLL patients. Further studies with idelalisib-based CART cell generation protocols are warranted., (© 2019 UICC.)

Introduction: Chimeric antigen receptor (CAR) T cells spark hope for patients with CD19+ B cell neoplasia, including relapsed or refractory (r/r) acute lymphoblastic leukaemia (ALL) or r/r non-Hodgkin's lymphoma (NHL). Published studies have mostly used second-generation CARs with 4-1BB or CD28 as costimulatory domains. Preclinical results of third-generation CARs incorporating both elements have shown superiority concerning longevity and proliferation. The University Hospital of Heidelberg is the first institution to run an investigator-initiated trial (IIT) CAR T cell trial (Heidelberg Chimeric Antigen Receptor T cell Trial number 1 [HD-CAR-1]) in Germany with third-generation CD19-directed CAR T cells., Methods and Analysis: Adult patients with r/r ALL (stratum I), r/r NHL including chronic lymphocytic leukaemia, diffuse large B-cell lymphoma, follicular lymphoma or mantle cell lymphoma (stratum II) as well as paediatric patients with r/r ALL (stratum III) will be treated with autologous T-lymphocytes transduced by third-generation RV-SFG.CD19.CD28.4-1BB zeta retroviral vector (CD19.CAR T cells). The main purpose of this study is to evaluate safety and feasibility of escalating CD19.CAR T cell doses (1-20×10 6 transduced cells/m 2 ) after lymphodepletion with fludarabine (flu) and cyclophosphamide (cyc). Patients will be monitored for cytokine release syndrome (CRS), neurotoxicity, i.e. CAR-T-cell-related encephalopathy syndrome (CRES) and/or other toxicities (primary objectives). Secondary objectives include evaluation of in vivo function and survival of CD19.CAR T cells and assessment of CD19.CAR T cell antitumour efficacy.HD-CAR-1 as a prospective, monocentric trial aims to make CAR T cell therapy accessible to patients in Europe. Currently, HD-CAR-1 is the first and only CAR T cell IIT in Germany. A third-generation Good Manufacturing Practice (GMP) grade retroviral vector, a broad spectrum of NHL, treatment of paediatric and adult ALL patients and inclusion of patients even after allogeneic stem cell transplantation (alloSCT) make this trial unique., Ethics and Dissemination: Ethical approval and approvals from the local and federal competent authorities were granted. Trial results will be reported via peer-reviewed journals and presented at conferences and scientific meetings., Trial Registration Number: Eudra CT 2016-004808-60; NCT03676504; Pre-results., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Chimeric antigen receptor T cell (CART) therapy is currently one of the most promising treatment approaches in cancer immunotherapy. However, the immunosuppressive nature of the tumor microenvironment, in particular increased reactive oxygen species (ROS) levels, provides considerable limitations. In this study, we aimed to exploit increased ROS levels in the tumor microenvironment with prodrugs of ROS accelerators, which are specifically activated in cancer cells. Upon activation, ROS accelerators induce further generation of ROS. This leads to an accumulation of ROS in tumor cells. We hypothesized that the latter cells will be more susceptible to CARTs. CD19-specific CARTs were generated with a CD19.CAR.CD28.CD137zeta third-generation retroviral vector. Cytotoxicity was determined by chromium-51 release assay. Influence of the ROS accelerators on viability and phenotype of CARTs was determined by flow cytometry. The combination of CARTs with the ROS accelerator PipFcB significantly increased their cytotoxicity in the Burkitt lymphoma cell lines Raji and Daudi, as well as primary chronic lymphocytic leukemia cells. Exposure of CARTs to PipFcB for 48 h did not influence T cell exhaustion, viability, or T cell subpopulations. In summary, the combination of CARTs with ROS accelerators may improve adoptive immunotherapy and help to overcome tumor microenvironment-mediated treatment resistance.

Background: Chimeric antigen receptor engineered T (CAR-T) cell therapy is a promising approach currently revolutionizing the field of cancer immunotherapy. However, data concerning clinical-grade CAR-T cell stability and functionality after months of cryopreservation have not been released by companies so far. To investigate the effect of cryopreservation on CAR-T cells and to further optimize the potency assays, we performed this study., Methods: A third generation of CD19 CAR-T cells was manufactured according to Good Manufacturing Practice (GMP) requirements, which is applied to patients in an ongoing clinical phase 1 study. Quality control tests for sterility, endotoxin and mycoplasma were performed for each batch. Stability in terms of viability, recovery, transduction efficiency and functional capacity was determined using microscopy, multiparametric flow cytometry as well as chromium-51 release tests., Results: Up to 90days of cryopreservation had no influence on viability, recovery and transduction efficiency of CAR-T cells. However, higher cell concentration for cryopreservation could alter the cell viability and recovery but not the transduction efficiency. Moreover, directly after thawing, both the quantity and quality of the functionality of CAR-T cells were transiently hampered by the negative effects of cryopreservation. Notably, the impaired functionality could be fully restored and even strengthened after an overnight resting process., Discussion: Cryopreservation is a challenge for the functional activity of CAR-T cells. However, CAR-T cells regain their potency by overnight incubation at 37°C, which mimics the clinical application setting. Therefore, an overnight resting step should be included in in vitro potency assays., (Copyright © 2019 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)