Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Automated generation of gene-edited CAR T cells at clinical scale

doi: 10.1016/j.omtm.2020.12.008

Figure Lengend Snippet: Automated generation of TCRα/β-free CAR T cells (A) Schematic overview of the automated process on the CliniMACS Prodigy platform. Day 0, T cell selection and activation. Day 1, T cell transduction with CD19-CAR encoding lentiviral particles. Day 3, electroporation of transduced T cells with TRAC -targeting TALEN. Days 9–13, expansion of engineered T cells followed by TCRα/β depletion, harvesting, and cryopreservation. (B and C) Cell expansion and cell viability. Cell numbers (B) and cell viability (C) were determined using an automated cell counter (NucleoCounter). (D and E) Cellular composition. Representative samples were taken from the culture at indicated cell processing steps, and cellular composition was determined by flow cytometry (see ). Shown are the single data points and the averages of three runs. T, T cells; B, B cells; M, monocytes; G, granulocytes; NK, natural killer cells; NKT, natural killer T cells; Tn/Tscm, T cell naive or T stem cell memory; Tcm, T cell central memory; Tem, T cell effector memory; Teff, T cell effector; UT, untreated T cells. ∗, the depletion process started with 50% of T cells from the pre-depletion step.

Article Snippet: During the TCRα/β depletion process, the cells were labeled and magnetically selected with CliniMACS TCRα/β-biotin and CliniMACS anti-biotin reagents (Miltenyi Biotec) and finally formulated in CliniMACS PBS/EDTA buffer (Miltenyi Biotec).

Techniques: Selection, Activation Assay, Transduction, Electroporation, Flow Cytometry

Journal: Nutrients

Article Title: Dietary Fatty Acids in Postprandial Triglyceride-Rich Lipoproteins Modulate Human Monocyte-Derived Dendritic Cell Maturation and Activation

doi: 10.3390/nu12103139

Figure Lengend Snippet: In vitro expression of dendritic cell (DC) gene markers in monocyte-derived DCs (moDCs) after stimulation with TRL-SFAs, TRL-MUFAs and TRL-PUFAs (TRL, triglyceride-rich lipoprotein) at 100 µg of TGs/mL for 6 days and in presence of GM-CSF and interleukin-4 (IL-4). DC maturation markers: ( a ) CD123 and ( b ) CCR7. DC pro-inflammatory activation markers: ( c ) CD80 and ( d ) CD86. DC tolerogenic activation markers: ( e ) PD-L1 and ( f ) PD-L2. Control means non-treated cells in the presence of GM-CSF and IL-4. Values are presented as means ± SD ( n = 6) and those marked with different letters are significantly different ( p < 0.05).

Article Snippet: Degree of differentiation of the resulting population was determined for CD123 antigen using anti-human CD123 monoclonal antibody (Miltenyi Biotec) by flow cytometry analysis (more than 95% of cells were positive for CD123) [ , , ].

Techniques: In Vitro, Expressing, Derivative Assay, Activation Assay, Control

Journal: Cancers

Article Title: Preclinical Evaluation of Novel Folate Receptor 1-Directed CAR T Cells for Ovarian Cancer

doi: 10.3390/cancers16020333

Figure Lengend Snippet: Ultrahigh-content imaging enables the identification of FOLR1 as potential target for high-grade serous epithelial OvCa. ( a ) Co-expression of EPCAM (cyan) and FOLR1 (magenta) in HGSOC samples (each number indicates an individual patient sample). Individual stainings are shown in . Scale bar represents 100 µm. ( b ) Co-expression of EPCAM (cyan) and FOLR1 (magenta) in a selection of healthy tissues. Individual stainings and additional healthy samples are presented in . Scale bar represents 100 µm. ( c ) Quantification of FOLR1 expression on a single-cell level on epithelial cells from primary OvCa tissue. ( d ) Quantification of FOLR1 expression on a single-cell level on epithelial cells from healthy tissue.

Article Snippet: On day 12 after transduction, CAR T cells were used in in vitro assays, and the number of viable CAR T cells was determined by staining T cells with 7-AAD (Miltenyi Biotec) and biotinylated human FOLR1 protein (Miltenyi Biotec).

Techniques: Imaging, Expressing, Selection

Journal: Cancers

Article Title: Preclinical Evaluation of Novel Folate Receptor 1-Directed CAR T Cells for Ovarian Cancer

doi: 10.3390/cancers16020333

Figure Lengend Snippet: In vitro evaluation of novel CAR T cell candidates. ( a ) Schematic diagram of the anti-FOLR1 CAR library designed with varying hinges (IgG4 or CD8α), scFv sequences, and scFv orientations. FOLR1 binding scFvs are derived from either MORAb-003 (anti-FOLR1 CAR candidates A–D) or M9346A4 (anti-FOLR1 CAR candidates E–H). ( b ) Anti-FOLR1 CAR expression was detected via anti-LNGFR staining and flow cytometry. Each bar represents the mean ± SEM of three donors ( n = 3). ( c ) Representative killing kinetics of the different anti-FOLR1 CAR T cell candidates with OV-90 wt or FOLR1 KO cells in co-culture at an effector-to-target ratio of 2:1 (additional T cell donors are shown in ). Each data point represents mean ± SEM ( n = 3). ( d ) Activation markers CD25, CD69, and 4-1BB expressed by CAR T cells after 48 h of co-culture assay. ( e ) Secreted cytokines GM-CSF, IFN-γ, IL-2, and TNF-α after 24 h of co-culture with OV-90 wt or FOLR1 KO cells. ( f ) Expressions of Granzyme B by anti-FOLR1 CAR T cells were analyzed after 24 h of co-culture with OV-90 wt or FOLR1 KO cells. Intracellular Granzyme B was detected via flow cytometry. Each bar represents the mean value ± SEM of three replicates from three donors ( n = 9).

Article Snippet: On day 12 after transduction, CAR T cells were used in in vitro assays, and the number of viable CAR T cells was determined by staining T cells with 7-AAD (Miltenyi Biotec) and biotinylated human FOLR1 protein (Miltenyi Biotec).

Techniques: In Vitro, Binding Assay, Derivative Assay, Expressing, Staining, Flow Cytometry, Co-Culture Assay, Activation Assay, Co-culture Assay

Journal: Cancers

Article Title: Preclinical Evaluation of Novel Folate Receptor 1-Directed CAR T Cells for Ovarian Cancer

doi: 10.3390/cancers16020333

Figure Lengend Snippet: Anti-FOLR1 CAR T candidates efficiently eradicate OV-90 wt tumors in vivo. ( a ) Scheme of this study evaluating intravenously injected CAR T cell efficacy against subcutaneous OV-90 wt xenograft tumors. ( b ) Representative bioluminescence images of individual OV-90 xenograft tumor-bearing NSG mice post CAR T cell injection. ( c ) Quantification of bioluminescence as total flux in photons per second (p/s) of the different experimental groups after CAR T cell injection over 21 d. Data points represent mean ± SEM. Human leucocyte expansion in peripheral blood of injected mice was analyzed by flow cytometry over time. ( d ) Human CD45 expression and ( e ) CAR-positive cells relative to mouse CD45 cells in blood samples collected at day 7, 14, and 21 post T cell injection were measured. Data are shown as mean ± SEM. On day 21 post T cell injection, mice were sacrificed, and the respective organs were collected and dissociated for ex vivo analysis by flow cytometry. ( f ) Human CD4 and CD8 frequencies as well as ( g ) CAR expression (detected as LNGFR positive) among human CD45 in mouse bone marrow, spleen, lung, and tumor were analyzed, respectively. Each data point represents an individual mouse and horizontal lines represent the mean of the respective group. ( h ) Secretion of human cytokines was analyzed in peripheral blood samples over time. Plasma was isolated from blood samples collected on days 7, 14, and 21 post T cell injection, and cytokine levels were subsequently determined with the human MACSplex Cytokine 12 Kit. Data are shown as mean ± SEM.

Article Snippet: On day 12 after transduction, CAR T cells were used in in vitro assays, and the number of viable CAR T cells was determined by staining T cells with 7-AAD (Miltenyi Biotec) and biotinylated human FOLR1 protein (Miltenyi Biotec).

Techniques: In Vivo, Injection, Flow Cytometry, Expressing, Ex Vivo, Clinical Proteomics, Isolation

Journal: Cancers

Article Title: Preclinical Evaluation of Novel Folate Receptor 1-Directed CAR T Cells for Ovarian Cancer

doi: 10.3390/cancers16020333

Figure Lengend Snippet: In vitro evaluation of optimized anti-FOLR1 CAR T cells. ( a ) Schematic diagram of the second generation anti-FOLR1 CAR T cell designed with CD8α hinge and CD8α transmembrane domain and scFv in Vh-Vl orientation without truncated LNGFR reporter gene. Anti-FOLR1 CAR candidate A or untransduced T cells (Mock) produced with the CliniMACS Prodigy ® TCT process were co-cultured for 48 h with OvCa cell lines at an effector cell-to-target cell ratio of 2:1. ( b ) Co-culture experiments of GFP-expressing target cell lines OV-90 FOLR1 KO, OV-90 wt, Caov-3, SKOV-3, and OVCAR-3 with anti-FOLR1 CAR T cell candidate A. Data points of each group are normalized to baseline (defined as 100% confluency at the first timepoint (T = 0 h)), and each data point represents mean ± SEM ( n = 3). Anti-FOLR1 CAR T cells express various levels of activation markers. ( c ) After 48 h of co-culture with the different target cells, CAR T cells were assessed by flow cytometry for expressions of activation markers CD25, CD69, and CD137. Each bar represents the mean ± SEM ( n = 3). ( d ) Anti-FOLR1 CAR T cells antigen-dependently secrete cytokines in co-culture with OV-90 wt, Caov-3, SKOV-3, and OVCAR-3 cells, whereas OV-90 FOLR1 KO cells did not induce cytokine secretion by CAR T cells. Supernatants were collected after 24 h of co-culture, and cytokine concentration was subsequently determined with the human MACSplex Cytokine 12 Kit. Each bar represents the mean ± SEM ( n = 3). ( e ) Anti-FOLR1 CAR T cells express Granzyme B antigen dependently in co-culture with OV-90 wt, Caov-3, SKOV-3, and OVCAR-3 cells, whereas OV-90 FOLR1 KO cells did not induce Granzyme B expression in CAR T cells. T cells were collected after 24 h of co-culture and overnight block of protein secretion by incubation with Brefeldin A (1 μg/mL). Cells were subsequently fixed, stained for CD8 surface marker, and permeabilized, and finally, intracellular Granzyme B was detected via flow cytometry. Each bar represents the mean ± SEM ( n = 3).

Article Snippet: On day 12 after transduction, CAR T cells were used in in vitro assays, and the number of viable CAR T cells was determined by staining T cells with 7-AAD (Miltenyi Biotec) and biotinylated human FOLR1 protein (Miltenyi Biotec).

Techniques: In Vitro, Produced, Cell Culture, Co-Culture Assay, Expressing, Activation Assay, Flow Cytometry, Concentration Assay, Blocking Assay, Incubation, Staining, Marker

Journal: Cancers

Article Title: Preclinical Evaluation of Novel Folate Receptor 1-Directed CAR T Cells for Ovarian Cancer

doi: 10.3390/cancers16020333

Figure Lengend Snippet: Evaluation of CAR T cells in advanced in vitro assays. ( a ) FOLR1-directed CAR and untransduced T cells were co-cultured with GFP-expressing OV-90 spheroids for 16 h. Spheroids were analyzed for GFP expression and CD3 cell presence. The scale bar represents 100 µm for each condition. ( b ) FOLR1-directed CAR and untransduced T cells were co-cultured with GFP-expressing OV-90 spheroids for 4 h. Spheroids were analyzed by light sheet microscopy for CAR T cell infiltration. Light sheet microscopy of OV-90 (left graph) shows FOLR1 expression in OV-90 spheroid. Middle and right graph color coding for anti-CD3 stains indicates distance of CD3 cells to surface. Number of infiltrated CAR T cells and penetration depth were quantified. Fold change is based on N/µm 3 measurements. Patient-derived OvCa samples were dissociated, and FOLR1 expression was analyzed by flow cytometry. ( c ) FOLR1-directed CAR T cells were co-cultured with dissociated primary OvCa tumor for 16 h. Thereafter, the frequency of FOLR1-expressing tumor cells was analyzed by flow cytometry. ( d ) After co-culture with OvCa tumor cells, FOLR1 CAR T cells were analyzed for expression of the indicated surface markers by flow cytometry. ( e ) Concentration of secreted cytokines was determined in the co-culture supernatant.

Article Snippet: On day 12 after transduction, CAR T cells were used in in vitro assays, and the number of viable CAR T cells was determined by staining T cells with 7-AAD (Miltenyi Biotec) and biotinylated human FOLR1 protein (Miltenyi Biotec).

Techniques: In Vitro, Cell Culture, Expressing, Microscopy, Derivative Assay, Flow Cytometry, Co-Culture Assay, Concentration Assay

Journal: Cancers

Article Title: Preclinical Evaluation of Novel Folate Receptor 1-Directed CAR T Cells for Ovarian Cancer

doi: 10.3390/cancers16020333

Figure Lengend Snippet: Optimized anti-FOLR1 CAR T cells efficiently and specifically eradicate tumors in vivo. ( a ) Representative bioluminescence images of individual OV-90 xenograft tumor-bearing NSG mice post CAR T cell injection. ( b ) Quantification of bioluminescence as total flux in photons per second (p/s) of the different experimental groups after CAR T cell injection over 21 d. Data points represent mean ± SEM. Human leucocyte expansion in peripheral blood of injected mice was analyzed by flow cytometry over time. ( c ) Quantification of tumor size of the different experimental groups before and after CAR T cell injection. Data points represent mean ± SEM. ( d ) Human CD45 expressions in blood samples collected at days 7, 14, and 21 post T cell injection were measured. Data are shown as mean ± SEM. ( e ) CAR T cell count among murine CD45 in mouse blood, bone marrow, spleen, and tumor was analyzed, respectively. On day 21 post T cell injection, mice were sacrificed, and the respective organs were collected and dissociated for ex vivo analysis by flow cytometry. Each data point represents an individual mouse, and horizontal lines represent the mean of the respective group.

Article Snippet: On day 12 after transduction, CAR T cells were used in in vitro assays, and the number of viable CAR T cells was determined by staining T cells with 7-AAD (Miltenyi Biotec) and biotinylated human FOLR1 protein (Miltenyi Biotec).

Techniques: In Vivo, Injection, Flow Cytometry, Cell Counting, Ex Vivo

Journal: Biomedicines

Article Title: Evaluation of Anti-CAR Linker mAbs for CAR T Monitoring after BiTEs/bsAbs and CAR T-Cell Pretreatment

doi: 10.3390/biomedicines12081641

Figure Lengend Snippet: Schematical representation of CAR T-cell detection with the classical ligand-based assay and anti-CAR linker mAb. ( A ) A schematical representation of CD19 and BCMA tumor antigens and their targeting CARs are shown. The recombinantly produced and biotinylated ligands and APC-conjugated anti-biotin Abs are used to visualize CAR expression on the surface of T-cells. ( B ) The general structure of a chimeric antigen receptor (CAR) is depicted as an illustration. The single-chain variable fragment (scFv) of CARs contains either a Whitlow linker (GSTSGSGKPGSGEGSTKG) or a (G 4 S) 3 linker (GGGGSGGGGSGGGGS), targeted by anti-Whitlow mAb or by anti-G 4 S monoclonal antibody (mAb), respectively. Biotinylated CAR-linker mABs and APC-conjugated anti-biotin Abs are used for the universal visualization of CAR expression on the surface of T-cells.

Article Snippet: Biotin-labeled BCMA CAR detection reagent, biotin-labeled CD19 CAR detection reagent, and anti-biotin APC were obtained from Miltenyi Biotec (Bergisch-Gladbach, Germany).

Techniques: Produced, Expressing

Journal: Biomedicines

Article Title: Evaluation of Anti-CAR Linker mAbs for CAR T Monitoring after BiTEs/bsAbs and CAR T-Cell Pretreatment

doi: 10.3390/biomedicines12081641

Figure Lengend Snippet: Anti-CAR linker mAbs allows for specific and sensitive CAR T monitoring in patients treated with any clinically approved CAR T therapies except Cilta-cel. ( A ) The structures of BCMA- and CD19-specific CARs are depicted in the illustration. CD19 antigen-specific CARs: Brexu-cel, Liso-cel, Axi-cel, and Tisa-cel; BCMA-specific CARs: Ide-cel and Cilta-cel. ( B ) Flow cytometry-based CAR detection analyses are shown, performed with biotinylated anti-CAR linker mAbs and APC-conjugated anti-biotin Abs to all approved BCMA and CD19 CAR T-cell products using isolated lymphocyte specimens from patients’ blood (n = 3). ( C ) Flow cytometry-based CAR detection analyses are shown, performed with Alexa 647-conjugated anti-CAR linker mAbs to all approved BCMA and CD19 CAR T-cell products using isolated lymphocyte specimens from patients’ blood (n = 3). The CAR-positive cells were determined from the CD3-positive cells and significances calculated based on BCMA- and CD19-antigen-based CAR detection reagent (Miltenyi = 100%). Statistical analysis was performed using Student’s t -test. * p ≤ 0.05%; ** p ≤ 0.01%; *** p ≤ 0.001%.

Article Snippet: Biotin-labeled BCMA CAR detection reagent, biotin-labeled CD19 CAR detection reagent, and anti-biotin APC were obtained from Miltenyi Biotec (Bergisch-Gladbach, Germany).

Techniques: Flow Cytometry, Isolation

Journal: Biomedicines

Article Title: Evaluation of Anti-CAR Linker mAbs for CAR T Monitoring after BiTEs/bsAbs and CAR T-Cell Pretreatment

doi: 10.3390/biomedicines12081641

Figure Lengend Snippet: Anti-CAR linker mAbs allows for specific CAR T monitoring in patients pretreated with BiTE/bsAb, targeting the same antigen as the CAR. ( A ) The structure of the blinatumomab (CD19×CD3 BiTE) and teclistamab (BCMA×CD3 bsAb) are illustrated. ( B ) Shown is a schematic overview of CAR T-cell detection strategies based on the CD19 or BCMA CAR detection reagent (Miltenyi) that leads to a false positive staining in patients pretreated with BiTE/bsAb targeting the same antigen as the CAR. The structure of CD19- or BCMA-specific CAR and CD3 on the cell surface of a T-cell is depicted as an illustration. ( C ) Shown is a schematic overview of CAR T-cell detection strategies based on anti-CAR linker mAbs. The specificity of the CAR detection is not affected by BiTE/bsAb targeting the same antigen as the CAR. ( D ) Detection of CAR-positive cells in CD3-positive blood T-lymphocytes from patients, treated either with Brexu-cel, Liso-cel, and Axi-cel (anti-CD19 CAR T-cells) simultaneously in the presence or absence of blinatumomab BiTE (CD19×CD3) or treated either with Ide-cel (anti-BCMA CAR T-cells) simultaneously in the presence or absence of teclistamab bsAb (BCMA×CD3). Representative clinical blood specimens of a Lymphoma or MM patient are shown as dot plots. CAR-negative and CAR-positive cells are visualized using blue and green dots, respectively. Green and red checkmarks denote for specific or artificial CAR T-cell detection, respectively. Shown are the CD3-positive cells. Note that CD19- and BCMA-based CAR detection reagents (Miltenyi) interfere with blinatumomab and teclistamab, respectively, and bind to CD3 on the surface of all T-cells, irrespective of whether they carry a CAR or not. This leads to false positive results (denoted by dots shown in orange or red). The problem is solved using anti-CAR linker mAbs targeting the artificial linker sequence between the variable heavy- and light-chain domains of the scFv.

Article Snippet: Biotin-labeled BCMA CAR detection reagent, biotin-labeled CD19 CAR detection reagent, and anti-biotin APC were obtained from Miltenyi Biotec (Bergisch-Gladbach, Germany).

Techniques: Staining, Sequencing