Supplementary Appendix. All-trans retinoic acid works synergistically with the γ- secretase inhibitor crenigacestat to augment BCMA on multiple myeloma and the efficacy of BCMA-CAR T cells

Supplement Figure 1: ATRA treatment does not affect the viability of myeloma cell lines. MM.1S, OPM-2 and NCI-H929 cells were treated with ATRA for up to 72 hours. Cell viability was measured by flow cytometry and 7AAD staining (n=6). Bar diagrams show mean values +SD.Supplement Figure 2: ATRA plus crenigacestat treatment enhance BCMA expression on myeloma cell lines. Bar diagram shows BCMA expression on OPM-2 cells (n=3) after treatment with 100 nM ATRA and/or 10 nM GSI crenigacestat for 72 hours. Bar diagram shows mean values +SD. P-values between indicated groups were calculated using unpaired t-test. *p<0.05, **p<0.01.Supplement Figure 3: ATRA treatment leads to increased BCMA transcripts in OPM-2 myeloma cells. BCMA RNA levels in OPM-2 were analyzed by quantitative reverse transcription PCR (qRT-PCR) assay after incubation with increasing doses of ATRA for 48 hours (n=3). Bar diagram shows mean values +SD. P-values between indicated groups were calculated using unpaired t-test. *p<0.05.Supplement Figure 4: ATRA treatment leads to enhanced BCMA expression on primary myeloma cells. Representative flow cytometric analysis of BCMA expression on primary myeloma cells that had been cultured in the absence or presence of ATRA at different concentrations for 72 hours. 7-AAD was used to exclude dead cells from analysis.Supplement Figure 5: ATRA treatment does not impair viability of primary myeloma cells. Viability of primary myeloma cells with or without 72 hours of ATRA treatment was analyzed by flow cytometry and 7-AAD staining (n=5 biological replicates). Bar diagram shows mean values +SD.Supplement Figure 6: sBCMA does not impair BCMA CAR T cell functionality. CD8+ BCMA-CAR T-cells were co-cultured with MM.1S target cells in absence or presence of 150 ng/ml of soluble BCMA. After 4 hours, cytotoxicity was evaluated by bioluminescence- based assay. Diagram shows mean values +/-SD.Supplement Figure 7: ATRA treatment does not increase shedding of sBCMA. sBCMA concentration in the supernatant of OPM-2 and NCI-H929 after incubation with increasing doses of ATRA was analyzed by ELISA. Cell lines were cultured at 1x106/well (n=3 technical replicates). Bar diagrams show mean values +SD, P-values between indicated groups were calculated using 2way ANOVA. n.s. = not significant, *p<0.05, **p<0.01.Supplement Figure 8: BCMA-CAR T-cells confer enhanced cytotoxicity against ATRA plus crenigacestat-treated OPM-2 cells in vitro. OPM-2 cells were incubated with 100 nM ATRA and/or 10 nM GSI for 72 hours or were left untreated. Cytolytic activity of CD8+ BCMA- CAR T-cells was determined in a bioluminescence-based assay after 4h of co-incubation with target cells. Assay was performed in triplicate wells with 5,000 target cells per well. Data are presented as mean values +SD (n=4 biological replicates). P-values between indicated groups were calculated using unpaired t-test. n.s. = not significant, *p<0.05.Supplement Figure 9: Patient-derived BCMA-CAR T-cells confer enhanced cytotoxicity against ATRA-treated MM.1S cells. MM.1S cells were incubated with 50 nM ATRA for 72 hours or were left untreated. Cytolytic activity of MM patient-derived CD8+ BCMA-CAR T-cells was determined in a bioluminescence-based assay after 4h of co-incubation with target cells. Data are presented as mean values +SD of triplicate wells. P-values between indicated groups were calculated using unpaired t-test. *p<0.05, **p<0.01.Peer reviewe

Antibody‐Based CAR T Cells Produced by Lentiviral Transduction

One promising approach to treat hematologic malignancies is the usage of patient‐derived CAR T cells. There are continuous efforts to improve the function of these cells, to optimize their receptor, and to use them for the treatment of additional types of cancer and especially solid tumors. In this protocol, an easy and reliable approach for CAR T cell generation is described. T cells are first isolated from peripheral blood (here: leukoreduction system chambers) and afterwards activated for one day with anti‐CD3/CD28 Dynabeads. The gene transfer is performed by lentiviral transduction and gene transfer rate can be verified by flowcytometric analysis. Six days after transduction, the stimulatory Dynabeads are removed. T cells are cultured in interleukin‐2 conditioned medium for several days for expansion. There is an option to expand CAR T cells further by co‐incubation with irradiated, antigen‐expressing feeder cell lines. The CAR T cells are ready to use after 10 (without feeder cell expansion) to 24 days (with feeder cell expansion)

A Clinical Case of COVID-19-Associated Pulmonary Aspergillosis (CAPA), Illustrating the Challenges in Diagnosis (Despite Overwhelming Mycological Evidence)

The COVID-19 pandemic has resulted in large numbers of patients requiring critical care management. With the established association between severe respiratory virus infection and invasive pulmonary aspergillosis (7.6% for COVID-19-associated pulmonary aspergillosis (CAPA)), the pandemic places a significant number of patients at potential risk from secondary invasive fungal disease. We described a case of CAPA with substantial supporting mycological evidence, highlighting the need to employ strategic diagnostic algorithms and weighted definitions to improve the accuracy in diagnosing CAPA

A clinical case of COVID-19-associated pulmonary aspergillosis (CAPA), illustrating the challenges in diagnosis (despite overwhelming mycological evidence)

The COVID-19 pandemic has resulted in large numbers of patients requiring critical care management. With the established association between severe respiratory virus infection and invasive pulmonary aspergillosis (7.6% for COVID-19-associated pulmonary aspergillosis (CAPA)), the pandemic places a significant number of patients at potential risk from secondary invasive fungal disease. We described a case of CAPA with substantial supporting mycological evidence, highlighting the need to employ strategic diagnostic algorithms and weighted definitions to improve the accuracy in diagnosing CAPA

CARAMBA: a first-in-human clinical trial with SLAMF7 CAR-T cells prepared by virus-free Sleeping Beauty gene transfer to treat multiple myeloma

Clinical development of chimeric antigen receptor (CAR)-T-cell therapy has been enabled by advances in synthetic biology, genetic engineering, clinical-grade manufacturing, and complex logistics to distribute the drug product to treatment sites. A key ambition of the CARAMBA project is to provide clinical proof-of-concept for virus-free CAR gene transfer using advanced Sleeping Beauty (SB) transposon technology. SB transposition in CAR-T engineering is attractive due to the high rate of stable CAR gene transfer enabled by optimized hyperactive SB100X transposase and transposon combinations, encoded by mRNA and minicircle DNA, respectively, as preferred vector embodiments. This approach bears the potential to facilitate and expedite vector procurement, CAR-T manufacturing and distribution, and the promise to provide a safe, effective, and economically sustainable treatment. As an exemplary and novel target for SB-based CAR-T cells, the CARAMBA consortium has selected the SLAMF7 antigen in multiple myeloma. SLAMF7 CAR-T cells confer potent and consistent anti-myeloma activity in preclinical assays in vitro and in vivo. The CARAMBA clinical trial (Phase-I/IIA; EudraCT: 2019-001264-30) investigates the feasibility, safety, and anti-myeloma efficacy of autologous SLAMF7 CAR-T cells. CARAMBA is the first clinical trial with virus-free CAR-T cells in Europe, and the first clinical trial that uses advanced SB technology worldwide

Electroporated Antigen-Encoding mRNA Is Not a Danger Signal to Human Mature Monocyte-Derived Dendritic Cells

For therapeutic cancer vaccination, the adoptive transfer of mRNA-electroporated dendritic cells (DCs) is frequently performed, usually with monocyte-derived, cytokine-matured DCs (moDCs). However, DCs are rich in danger-sensing receptors which could recognize the exogenously delivered mRNA and induce DC activation, hence influencing the DCs’ immunogenicity. Therefore, we examined whether electroporation of mRNA with a proper cap and a poly-A tail of at least 64 adenosines had any influence on cocktail-matured moDCs. We used 16 different RNAs, encoding tumor antigens (MelanA, NRAS, BRAF, GNAQ, GNA11, and WT1), and variants thereof. None of those RNAs induced changes in the expression of CD25, CD40, CD83, CD86, and CD70 or the secretion of the cytokines IL-8, IL-6, and TNFα of more than 1.5-fold compared to the control condition, while an mRNA encoding an NF-κB-activation protein as positive control induced massive secretion of the cytokines. To determine whether mRNA electroporation had any effect on the whole transcriptome of the DCs, we performed microarray analyses of DCs of 6 different donors. None of 60,000 probes was significantly different between mock-electroporated DCs and MelanA-transfected DCs. Hence, we conclude that no transcriptional programs were induced within cocktail-matured DCs by electroporation of single tumor-antigen-encoding mRNAs

Impaired FADD/BID signaling mediates cross-resistance to immunotherapy in Multiple Myeloma

Abstract The treatment landscape in multiple myeloma (MM) is shifting from genotoxic drugs to immunotherapies. Monoclonal antibodies, immunoconjugates, T-cell engaging antibodies and CART cells have been incorporated into routine treatment algorithms, resulting in improved response rates. Nevertheless, patients continue to relapse and the underlying mechanisms of resistance remain poorly understood. While Impaired death receptor signaling has been reported to mediate resistance to CART in acute lymphoblastic leukemia, this mechanism yet remains to be elucidated in context of novel immunotherapies for MM. Here, we describe impaired death receptor signaling as a novel mechanism of resistance to T-cell mediated immunotherapies in MM. This resistance seems exclusive to novel immunotherapies while sensitivity to conventional anti-tumor therapies being preserved in vitro. As a proof of concept, we present a confirmatory clinical case indicating that the FADD/BID axis is required for meaningful responses to novel immunotherapies thus we report impaired death receptor signaling as a novel resistance mechanism to T-cell mediated immunotherapy in MM