Journal: European Journal of Immunology

Article Title: Engineering Soluble Recombinant BCMA for Ide‐Cel Labeling

doi: 10.1002/eji.70251

Figure Lengend Snippet: Generation of a soluble BCMA‐GFP construct. (A) Scheme of the BCMA‐GFP and BCMAext‐GFP proteins. The plasma membrane of the cell is depicted. (B) Description of the cDNA generated for the production of BCMA‐GFP and BCMAext‐GFP protein. The estimated molecular weight is reported. (C) HEK/293T cells were transfected with pEGFP‐N1 or full‐length BCMA‐GFP, and 24 h after transfection, cells were labelled using a PE‐conjugated anti‐BCMA mAb. The expression level of plasma membrane BCMA was analyzed by flow cytometry. Data are representative of three independently performed experiments. (D)HEK/293T cells were transfected with the indicated constructs, and after 5 days, cells were lysed, and supernatants were harvested. Cell lysates (60 µg) and supernatants (20 µL) were loaded in an SDS‐PAGE, and indicated immunoblots were performed. Data are representative of three independently performed experiments. (E) HEK/293T cells were transfected with the indicated constructs, and 48 h after transfection, confocal microscopy analyses were performed. Golgi was labelled using a mouse IgG1 anti‐GM130 followed by an AlexaFluor 647‐coupled goat anti‐mouse IgG1 antibody. Data are representative of three independently performed experiments. (F) Description of the cDNA generated for the production of IFNα2‐BCMA‐GFP. The estimated molecular weight is reported. (G) HEK/293T cells were transfected with the indicated constructs, and after 5 days, supernatants were harvested. Supernatants (20 µL) were loaded in an SDS‐PAGE, and an anti‐BCMA immunoblot was performed. A long‐time exposure of the immunoblot is depicted to reveal the BCMAext‐GFP construct expression. Data are representative of three independently performed experiments. (H) HEK/293T cells were transfected with the indicated constructs, and after 7 days, supernatants were harvested. Supernatants were subjected to anti‐V5‐mAb immunoprecipitation to purify V5‐tagged BCMA‐GFP and BCMA‐mCherry proteins. O‐glycosylation and N‐glycosylation were analyzed using deglycosylation enzymes following the manufacturer's instructions. N‐glycan removal from IFNα2‐BCMA‐GFP and IFNα2‐BCMA‐mCherry constructs was performed using the PNGase F glycan cleavage kit. O‐glycan removal was performed using O‐glycosidase combined with α‐2‐3,6,8,9 neuraminidase A. O‐ and N‐glycans were removed simultaneously using protein deglycosylation Mix II. IFNα2‐BCMA‐GFP and IFNα2‐BCMA‐mCherry constructs treated or untreated with glycohydrolases were subjected to western blot analysis. The green arrow indicates the untreated IFNα2‐BCMA‐GFP construct, while the red arrow shows the untreated IFNα2‐BCMA‐mCherry reagent. The white arrows depict degraded IFNα2‐BCMA‐mCherry products, and these constructs are labeled in the anti‐BCMA immunoblot. Upper panel: deglycosylation was analyzed by immunoblot using an anti‐V5 antibody. Lower panel: deglycosylation was analyzed by immunoblot using an anti‐human BCMA antibody. (I) HEK/293T cells were transfected with IFNα2‐BCMA‐GFP‐encoding vector, and 48 h after transfection, confocal microscopy analyses were performed. Golgi was labelled using a mouse IgG1 anti‐GM130 followed by an AlexaFluor 647‐coupled goat anti‐mouse IgG1 antibody. Data are representative of three independently performed experiments.

Article Snippet: Biotinylated BCMA CAR Detection Reagent (BCMA‐Fc‐biotin) (#130‐126‐090), APC‐conjugated anti‐biotin (#130‐111‐069), and Whitlow/218 Linker‐PE (#130‐137‐251) were from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Construct, Clinical Proteomics, Membrane, Generated, Molecular Weight, Transfection, Expressing, Flow Cytometry, SDS Page, Western Blot, Confocal Microscopy, Immunoprecipitation, Glycoproteomics, Labeling, Plasmid Preparation

Journal: European Journal of Immunology

Article Title: Engineering Soluble Recombinant BCMA for Ide‐Cel Labeling

doi: 10.1002/eji.70251

Figure Lengend Snippet: A new soluble BCMA to stain ide‐cel CAR. (A)Scheme of the ide‐cel CAR. The different domains of the light and heavy variable chain (VL and VH) are represented with the intracellular domains of 4‐1BB and CD3z proteins. (B) HEK/293T cells were transduced with the indicated lentiviruses, and 48 h after transduction, cells were labelled with BCMA‐biotin and revealed with an Alexa488‐conjugated streptavidin or with IFNα2‐BCMA‐GFP. The expression level of plasma membrane CAR ide‐cel was analyzed by flow cytometry. Data are representative of three independently performed experiments. (C) Jurkat cells were transduced with the indicated lentivirus and cloned by limiting dilutions. Clones were amplified and screened for ide‐cel CAR expression. The selected clones were labeled with BCMA‐Fc‐biotin/Alexa488‐conjugated streptavidin or with IFNα2‐BCMA‐GFP. Data are representative of three independently performed experiments. (D) Description of the cDNA generated for the production of IFNα2‐BCMA‐Cherry. The estimated molecular weight is reported. (E) HEK/293T cells were transfected with pEGFP‐N1, IFNα2‐BCMA‐GFP, or IFNα2‐BCMA‐Cherry, and after 48 h, cells were stained with the Golgi marker, GM130, using a secondary antibody conjugated with either an AlexaFluor 647‐coupled goat anti‐mouse IgG1 antibody or an AlexaFluor 488‐coupled goat anti‐mouse IgG1 antibody. Data are representative of three independently performed experiments. (F) HEK/293T cells were transfected with the indicated constructs, and after 7 days, supernatants were harvested. The raw, ultracentrifuged, and concentrated supernatants (20 µL) were loaded in an SDS‐PAGE, and anti‐BCMA and anti‐V5 immunoblots were performed. Data are representative of three independently performed experiments. (G) Left panel: ultracentrifuged and concentrated supernatants (20 µL) in F were loaded in a BN‐PAGE, and an anti‐V5 immunoblot was performed. Data are representative of three independently performed experiments. Right panel: for each soluble BCMA construct, a densitometric analysis was performed on the two bands observed in BN‐PAGE. Data are representative of three independently performed experiments. Data represent mean ± SD of three independent experiments. (H) The three ide‐cel‐expressing Jurkat clones shown in C were labelled with indicated concentrations of IFNα2‐BCMA‐Cherry, IFNα2‐BCMA‐GFP, or BCMA‐Fc‐Biotin (Miltenyi), and the mean of fluorescence (MFI) was assessed using flow cytometry. Data represent mean ± SD of three independently performed experiments. (I) CAR‐T cells were monitored in the blood of patients (between n = 3 and 7) using BCMA‐Fc‐Biotin or a whitlow antibody‐PE and compared with BCMA‐Fc‐GFP. The chart represents the percentage of positive CAR‐T cells in n = 3–7 patients with MM treated with ide‐cel CAR‐T cells. Data represent mean ± SD; * p < 0.05 and ** p < 0.01, using two‐tailed Mann–Whitney test. (J) Correlations between the percentage of CAR‐T cells detected in the blood of patients with MM using IFNα2‐BCMA‐GFP (1 µg/mL), BCMA‐Fc‐Biotin, and an anti‐whitlow antibody‐PE. Correlation between each labeling was analyzed using a nonparametric Spearman correlation assay (* p < 0.05 and ** p < 0.01). (K) Dot plots are representative of the IFNα2‐BCMA‐GFP staining with or without AF488‐conjugated anti‐GFP mAb. Fluorescence minus one (FMO) control is the sample that contains all the fluorophores in the multicolor panel except IFNα2‐BCMA‐GFP.

Article Snippet: Biotinylated BCMA CAR Detection Reagent (BCMA‐Fc‐biotin) (#130‐126‐090), APC‐conjugated anti‐biotin (#130‐111‐069), and Whitlow/218 Linker‐PE (#130‐137‐251) were from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Staining, Transduction, Expressing, Clinical Proteomics, Membrane, Flow Cytometry, Clone Assay, Amplification, Labeling, Generated, Molecular Weight, Transfection, Marker, Construct, SDS Page, Western Blot, Fluorescence, Two Tailed Test, MANN-WHITNEY, Two-Photon Excitation Fluorescence Cross-Correlation Assay, Control