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The search for target antigens for CAR-T cell therapy against multiple myeloma defined the B-cell maturation antigen (BCMA) as an interesting candidate. Several studies with BCMA-directed CAR-T cell therapy showed promising results. Second-generation point-of-care BCMA.CAR-T cells were manufactured to be of a GMP (good manufacturing practice) standard using the CliniMACS Prodigy® device. Cytokine release in BCMA.CAR-T cells after stimulation with BCMA positive versus negative myeloma cell lines, U266/HL60, was assessed via intracellular staining and flow cytometry. The short-term cytotoxic potency of CAR-T cells was evaluated by chromium-51 release, while the long-term potency used co-culture (3 days/round) at effector/target cell ratios of 1:1 and 1:4. To evaluate the activation and exhaustion of CAR-T cells, exhaustion markers were assessed via flow cytometry. Stability was tested through a comparison of these evaluations at different timepoints: d0 as well as d + 14, d + 90 and d + 365 of cryopreservation. As results, (1) Killing efficiency of U266 cells correlated with the dose of CAR-T cells in a classical 4 h chromium-release assay. There was no significant difference after cryopreservation on different timepoints. (2) In terms of endurance of BCMA.CAR-T cell function, BCMA.CAR-T cells kept their ability to kill all tumor cells over six rounds of co-culture. (3) BCMA.CAR-T cells released high amounts of cytokines upon stimulation with tumor cells. There was no significant difference in cytokine release after cryopreservation. According to the results, BCMA.CAR-T cells manufactured under GMP conditions exerted robust and specific killing of target tumor cells with a high release of cytokines. Even after 1 year of cryopreservation, cytotoxic functions were maintained at the same level. This gives clinicians sufficient time to adjust the timepoint of BCMA.CAR-T cell application to the patient's course of the underlying disease. [ABSTRACT FROM AUTHOR]

Background: Third-generation chimeric antigen receptor (CAR)-engineered T cells (CARTs) might improve clinical outcome of patients with B cell malignancies. This is the first report on a third-generation CART dose-escalating, phase-1/2 investigator-initiated trial treating adult patients with refractory and/or relapsed (r/r) acute lymphoblastic leukemia (ALL). Methods: Thirteen patients were treated with escalating doses of CD19-directed CARTs between 1 × 106 and 50 × 106 CARTs/m2. Leukapheresis, manufacturing and administration of CARTs were performed in-house. Results: For all patients, CART manufacturing was feasible. None of the patients developed any grade of Immune effector cell-associated neurotoxicity syndrome (ICANS) or a higher-grade (≥ grade III) catokine release syndrome (CRS). CART expansion and long-term CART persistence were evident in the peripheral blood (PB) of evaluable patients. At end of study on day 90 after CARTs, ten patients were evaluable for response: Eight patients (80%) achieved a complete remission (CR), including five patients (50%) with minimal residual disease (MRD)-negative CR. Response and outcome were associated with the administered CART dose. At 1-year follow-up, median overall survival was not reached and progression-free survival (PFS) was 38%. Median PFS was reached on day 120. Lack of CD39-expression on memory-like T cells was more frequent in CART products of responders when compared to CART products of non-responders. After CART administration, higher CD8 + and γδ-T cell frequencies, a physiological pattern of immune cells and lower monocyte counts in the PB were associated with response. Conclusion: In conclusion, third-generation CARTs were associated with promising clinical efficacy and remarkably low procedure-specific toxicity, thereby opening new therapeutic perspectives for patients with r/r ALL. Trial registration This trial was registered at www.clinicaltrials.gov as NCT03676504. [ABSTRACT FROM AUTHOR]

The transcription factor SOX11 is a tumor-associated antigen with low expression in normal cells, but overexpression in glioblastoma (GBM). So far, conventional surgery, chemotherapy, and radiotherapy have not substantially improved the dismal prognosis of relapsed/refractory GBM patients. Immunotherapy is considered a promising strategy against GBM, but there is a fervent need for better immunotargets in GBM. To this end, we performed an in silico prediction study on SOX11, which primarily yielded ten promising HLA-A*0201-restricted peptides derived from SOX11. We defined a novel peptide FMACSPVAL, which had the highest score according to in silico prediction (6.02 nM by NetMHC-4.0) and showed an exquisite binding affinity to the HLA-A*0201 molecule in the peptide-binding assays. In the IFN-γ ELISPOT assays, FMACSPVAL demonstrated a high efficiency for generating SOX11-specific CD8+ T cells. Nine out of thirty-two healthy donors showed a positive response to SOX11, as assessed by the ELISPOT assays. Therefore, this novel antigen peptide epitope seems to be promising as a target for T cell-based immunotherapy in GBM. The adoptive transfer of in vitro elicited SOX11-specific CD8+ T cells constitutes a potential approach for the treatment of GBM patients. [ABSTRACT FROM AUTHOR]

• ECP was able to increase Tfh cells to ameliorate GvHD. • Upregulation of Th22 cells was observed in aGvHD patients with CR. • Tim-3 expression was downregulated on effector T cells by ECP. • ECP shows immunomodulatory effects in GvHD setting. Extracorporeal photopheresis (ECP), a personalized cellular immunotherapy, constitutes a promising treatment for steroid-refractory/-resistant graft-versus-host disease (SR-GvHD), with encouraging clinical response rates. To further investigate its mechanism of action, ECP's effects on T helper (Th) cells as well as on expression of immune checkpoint (PD-1 and Tim-3) and apoptotic (Fas receptor [FasR]) molecules were investigated in 27 patients with SR-GvHD. Our data show that GvHD patients had significantly higher levels of Th2, Th17, Th22 and granulocyte-macrophage colony-stimulating factor (GM-CSF)-positive Th (ThG) cells and clearly lower levels of T follicular helper (Tfh) cells, including Th1- and Th2-like cells, compared with healthy donors. ECP therapy for GvHD was effective through the modulation of different Th subsets: increases of Th22 (1.52-fold) and Tfh cells (1.48-fold) in acute GvHD (aGvHD) and increases of Th2-like Tfh cells (1.74-fold) in chronic GvHD (cGvHD) patients were associated with clinical response. Expression of FasR was further upregulated in CD4+CD8+ T cells. Additionally, Tim-3–expressing effector T cells associated with the severity of GvHD were reduced. Taken together, these data show that ECP therapy exerts immunomodulatory effects by promoting a balanced immune reconstitution and inducing immune tolerance. Therefore it represents an attractive option for the treatment of GvHD. [ABSTRACT FROM AUTHOR]

BACKGROUNDPreclinical experiments have shown that donor blood cells, modified in vitro by an alkylating agent (modified immune cells [MICs]), induced long-term specific immunosuppression against the allogeneic donor.METHODSIn this phase I trial, patients received either 1.5 × 106 MICs per kg BW on day -2 (n = 3, group A), or 1.5 × 108 MICs per kg BW on day -2 (n = 3, group B) or day -7 (n = 4, group C) before living donor kidney transplantation in addition to post-transplantation immunosuppression. The primary outcome measure was the frequency of adverse events (AEs) until day 30 (study phase) with follow-up out to day 360.RESULTSMIC infusions were extremely well tolerated. During the study phase, 10 treated patients experienced a total of 69 AEs that were unlikely to be related or not related to MIC infusion. No donor-specific human leukocyte antigen Abs or rejection episodes were noted, even though the patients received up to 1.3 × 1010 donor mononuclear cells before transplantation. Group C patients with low immunosuppression during follow-up showed no in vitro reactivity against stimulatory donor blood cells on day 360, whereas reactivity against third-party cells was still preserved. Frequencies of CD19+CD24hiCD38hi transitional B lymphocytes (Bregs) increased from a median of 6% before MIC infusion to 20% on day 180, which was 19- and 68-fold higher, respectively, than in 2 independent cohorts of transplanted controls. The majority of Bregs produced the immunosuppressive cytokine IL-10. MIC-treated patients showed the Immune Tolerance Network operational tolerance signature.CONCLUSIONMIC administration was safe and could be a future tool for the targeted induction of tolerogenic Bregs.TRIAL REGISTRATIONEudraCT number: 2014-002086-30; ClinicalTrials.gov identifier: NCT02560220.FUNDINGFederal Ministry for Economic Affairs and Technology, Berlin, Germany, and TolerogenixX GmbH, Heidelberg, Germany. [ABSTRACT FROM AUTHOR]

The anti-tumor efficacy of TCR-engineered T cells in vivo depends largely on less-differentiated subsets such as T cells with naïve-like T cell (TN) phenotypes with greater expansion and long-term persistence. To increase these subsets, we compared the generation of New York esophageal squamous cell carcinoma-1 (NY-ESO-1)-specific T cells under supplementation with either IL-2 or IL-7/IL-15. PBMCs were transduced with MS3II-NY-ESO-1-siTCR retroviral vector. T cell generation was adapted from a CD19-specific CART cell production protocol. Comparable results in viability, expansion and transduction efficiency of T cells under stimulation with either IL-2 or IL-7/IL-15 were observed. IL-7/IL-15 led to an increase of CD4+ T cells and a decrease of CD8+ T cells, enriched the amount of TN among CD4+ T cells but not among CD8+ T cells. In a 51Cr release assay, similar specific lysis of NY-ESO-1-positive SW982 sarcoma cells was achieved. However, intracellular cytokine staining revealed a significantly increased production of IFN-γ and TNF-α in T cells generated by IL-2 stimulation. To validate these unexpected findings, NY-ESO-1-specific T cell production was evaluated in another protocol originally established for TCR-engineered T cells. IL-7/IL-15 increased the proportion of TN. However, the absolute number of TN did not increase due to a significantly slower expansion of T cells with IL-7/IL-15. In conclusion, IL-7/IL-15 does not seem to be superior to IL-2 for the generation of NY-ESO-1-specific T cells. This is in sharp contrast to the observations in CD19-specific CART cells. Changes of cytokine cocktails should be carefully evaluated for individual vector systems. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

After solid-organ transplantation, reactivation of the cytomegalovirus (CMV) is often observed in seronegative patients and associated with a high risk of disease and mortality. CMV-specific T cells can prevent CMV reactivation. In a phase 1 trial, CMV-seronegative patients with end-stage renal disease listed for kidney transplantation were subjected to CMV phosphoprotein 65 (CMVpp65) peptide vaccination and further investigated for T-cell responses. To this end, CMV-specific CD8+ T cells were characterized by bulk T-cell-receptor (TCR) repertoire sequencing and combined single-cell RNA and TCR sequencing. In patients mounting an immune response to the vaccine, a common SYE(N)E TCR motif known to bind CMVpp65 was detected. CMV-peptide-vaccination-responder patients had TCR features distinct from those of non-responders. In a non-responder patient, a monoclonal inflammatory T-cell response was detected upon CMV reactivation. The identification of vaccine-induced CMV-reactive TCRs motifs might facilitate the development of cellular therapies for patients wait-listed for kidney transplantation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Chimeric-antigen-receptor-T (CAR-T) cells are currently revolutionizing the field of cancer immunotherapy. Therefore, there is an urgent need for CAR-T cell monitoring by clinicians to assess cell expansion and persistence in patients. CAR-T cell manufacturers and researchers need to evaluate transduction efficiency and vector copy number for quality control. Here, CAR expression was analyzed in peripheral blood samples from patients and healthy donors by flow cytometry with four commercially available detection reagents and on the gene level by quantitative polymerase chain reaction (qPCR). Flow cytometric analysis of CAR expression showed higher mean CAR expression values for CD19 CAR detection reagent and the F(ab')2 antibody than Protein L and CD19 Protein. In addition, the CD19 CAR detection reagent showed a significantly lower median background staining of 0.02% (range 0.007–0.06%) when compared to the F(ab')2 antibody, CD19 protein and Protein L with 0.80% (range 0.47–1.58%), 0.65% (range 0.25–1.35%) and 0.73% (range 0.44–1.23%). Furthermore, flow cytometry-based CAR-T cell frequencies by CD19 CAR detection reagent showed a good correlation with qPCR results. In conclusion, quality control of CAR-T cell products can be performed by FACS and qPCR. For the monitoring of CAR-T cell frequencies by FACS in patients, CAR detection reagents with a low background staining are preferable. [ABSTRACT FROM AUTHOR]

NY-ESO-1-specific T cells have shown promising activity in the treatment of soft tissue sarcoma (STS). However, standardized protocols for their generation are limited. Particularly, cost-effectiveness considerations of cell production protocols are of importance for conducting clinical studies. In this study, two different NY-ESO-1-specific T cell production protocols were compared. Major differences between protocols 1 and 2 include culture medium, interleukin-2 and retronectin concentrations, T cell activation strategy, and the transduction process. NY-ESO-1-specific T cells generated according to the two protocols were investigated for differences in cell viability, transduction efficiency, T cell expansion, immunophenotype as well as functionality. NY-ESO-1-specific T cells showed similar viability and transduction efficiency between both protocols. Protocol 1 generated higher absolute numbers of NY-ESO-1-specific T cells. However, there was no difference in absolute numbers of NY-ESO-1-specific T cell subsets with less-differentiated phenotypes accounting for efficient in vivo expansion and engraftment. Furthermore, cells generated according to protocol 1 displayed higher capacity of TNF-α generation, but lower cytotoxic capacities. Overall, both protocols provided functional NY-ESO-1-specific T cells. However, compared to protocol 1, protocol 2 is advantageous in terms of cost-effectiveness. Cell production protocols should be designed diligently to achieve a cost-effective cellular product for further clinical evaluation. [ABSTRACT FROM AUTHOR]

Simple Summary: To monitor patients after CAR T cell treatment, measuring frequencies of chimeric antigen receptor (CAR) T cells is crucial. However, experimental assays to quantify CAR T cells are lacking. Here, we describe a quantitative single copy gene-based PCR approach to measure frequencies of CAR T cells based on the FMC63 single chain variable fragment (scFv) including commercially available CAR T cell products. Besides enabling to monitor development of CAR T cells after treatment and guide further therapeutic decisions, this quantification assay proved highly useful for diagnosis of CAR T cell associated neurotoxic side effects. Overall, this quantification approach contributes significantly to the better monitoring and safety of treatment of patients with CAR T cells. Chimeric antigen receptor (CAR) T cell (CART) therapy has been established as a treatment option for patients with CD19-positive lymphoid malignancies in both the refractory and the relapsed setting. Displaying significant responses in clinical trials, two second-generation CART products directed against CD19, axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel), have been approved and integrated into the clinical routine. However, experimental assay for quantitative monitoring of both of these CART products in treated patients in the open domain are lacking. To address this issue, we established and validated a quantitative single copy gene (SCG)-based duplex (DP)-PCR assay (SCG-DP-PCR) to quantify CARTs based on the FMC63 single chain variable fragment (scFv), i.e., axi-cel and tisa-cel. This quantitative PCR (qPCR) approach operates without standard curves or calibrator samples, offers a tool to assess cellular kinetics of FMC63 CARTs and allows direct comparison of CART-copies in axi-cel versus tisa-cel patient samples. For treating physicians, SCG-DP-PCR is an important tool to monitor CARTs and guide clinical decisions regarding CART effects in respective patients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]