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

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, Structural models of receptor-binding-deficient PIRYV (ePIRYV RBD ) engineered with K182A and Y352A mutations to eliminate low-density lipoprotein receptor (LDL-R) binding while preserving fusogenic capacity. Receptor-interacting region is highlighted in magenta b, Close up view of the two LDL-R contact residues, glycoprotein (green sticks) form hydrogen bond (dashed lines) with LDL-R (cyan) whereas in the mutant complex, substitutions abolish these bonds. c, Complete structural model of the engineered PIRYV RBD glycoprotein in complex with LDL-R depicting ablation of LDL-R recognition, alongside the four immunogenicity-reducing residues. d , Schematic of lentiviral production and T cell isolation. e, Representative flow cytometry plots of transduction efficiency (%GFP + ) in CD3 + T cells at MOI 2.5 with vector control (VC). ePIRYV wt . ePIRYV RBD or ePIRYV RBD+nbC3/7 . f , Bar graph showing the mean fluorescence intensity (MFI) of GFP + cells. Representative of three independent experiments (n=6 biologically independent samples). g, Humanized scFv design for bispecific BCMA/CD19 CAR construct showing complementarity-determining regions (CDRs) in heavy and light chains. h, Representative contour plots showing comparative CAR expression (PE) in transduced CD4 + (APC, top) and CD8 + (APC, bottom) T cell subsets in activated primary CD3 + T cells from healthy donors (n = 6). Bar graph quantifies the percentage of CAR + cells (using an anti-G4S linker antibody) among CD4 + and CD8 + T cells. i, PBMCs were either left unstimulated (None) or activated with anti-CD3/CD28 (CD3/CD28) Dynabeads before transduction with PIRVYwt or ePIRYRBD +nbC3/7 , followed by analysis of activation. j, T cell activation markers (CD69) measured as MFI following transduction with PIRYV wt or ePIRYV RBD+nbC3/7 at MOI 1 and MOI2.5 after 24 h post-transduction. Each point represents one donor (n = 8). k, l, In vitro cytotoxicity of CAR-T cells generated with PIRY wt or ePIRY RBD+nbC3/7 against BCMA + multiple myeloma targets MM.1S (k) and MM.1R (l), expressed as percentage target-cell survival across the indicated effector-to-target (E:T) ratios after 24 h coculture. Data represents mean ± SEM. ****p < 0.0001. A non-parametric t-test was used for statistical analysis with n = 3 biologically independent samples). See Supplementary Figures 7-11 for extended nanobody characterization and CAR construct optimization .

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: Binding Assay, Preserving, Mutagenesis, Immunopeptidomics, Cell Isolation, Flow Cytometry, Transduction, Plasmid Preparation, Control, Fluorescence, Construct, Expressing, Activation Assay, In Vitro, Generated

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, Experimental timeline of in vivo CAR-T delivery in humanized xenograft model. NSG mice received intravenous engraftment of human hematopoietic stem cells (HSCs) on days −28 with prior busulfan treatment. viroVbot1 (ePIRYV RBD+nbC3/7 encoding bi-CAR transgene) was administered intravenously at doses ranging from 0.25 × 10 6 to 1 × 10 6 TU/mouse on day 0. Blood, was collected at days −4, 7, 14, 21, and 28 post-vector administration and bone marrow (BM), and spleen at the end of the experiment. b , Kinetics of CAR-T cell expansion in peripheral blood. Percentage of BCMA-CAR + CD3 + T cells over time (Day −4 to 28). c , d, CAR-T cell frequency in (c) bone marrow and (d) spleen at Day 28. Each point represents pooled samples from 15 animals (n = 5 from 3 pooled in each group). e, f, CAR-T cell transduction efficiency in peripheral blood cells determined by flow cytometry by collecting the samples at day 7 and day 28. g , Flow cytometry analysis of CD19 + cells among hCD45 + cells showing rapid B cell depletion over time. h, i, Similarly in bone marrow and spleen. j, Representative IHC images of major organs (spleen, liver, lungs, and kidneys) showing CAR expression as indicated by red arrowheads with quantitative analysis (n=6 images); Bar graph shows CAR+ cells per 1×10 3 cells by tissue in visceral tissues. k, Schematic of vector production of the two lentiviral vectors used: (left) ePIRY wt with MHC-I −/− modification and bispecific BCMA/CD19 CAR; (right) viroVbot1 with TcrBM-detargeted envelope (ePIRY RBD+nbC3/7 ), with the same bispecific CAR. Both vectors were used to transduce a panel of 65 distinct human-derived cell lines to assess CAR expression at MOI 2.5. l, CAR expression profile across 65 cell lines (ex vivo transduction assay). Dot plot showing CAR fluorescence (n=3 biologically independent samples). Data represents mean ± SEM. **p < 0.01; *p < 0.05. A non-parametric t-test was used for statistical analysis between groups. Scale bar; d: 200 μm.

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: In Vivo, Plasmid Preparation, Transduction, Flow Cytometry, Expressing, Modification, Derivative Assay, Ex Vivo, Fluorescence

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, Schematic showing receptor-mediated CAR uptake by B cells with wild-type (CD19 wt Raji, BCMA wt multiple myeloma (MM) phenotype and CRISPR-Cas9 knockout (BCMA/CD19 −/− MM, CD19 −/− Raji) phenotype. Cell lines were transduced with viroVbot1 or wild type PIRYV b, Flow cytometry showing ex vivo CAR transduction the wild type and knockout cells (n=6). c, Schematic of receptor competition assay showing that pre-incubation of viroVbot1 with recombinant BCMA (BCMAR) and CD19 (CD19R) extracellular domain proteins before transduction of wild-type Raji and MM.1S cells. d, Transduction efficiency (% CAR + cells) in Raji and MM.1S cells, with viroVbot1 incubated with vehicle (PBS) or viroVbot1 incubated with CD19R or BCMAR (n=8 biologically independent samples). e, Schematic showing CAR glycoprotein displayed on wild-type producer cell line (left) is incorporated onto the lentiviral envelope in contrast to CAR-TRAP producer cell (right) where CD19-ECD-KDEL retains the CAR in ER lumen rather than plasma membrane. f , Representative Immunofluorescence image of HEK293T control cells (left), show robust CD19-CAR (green) throughout the cell surface; CAR-TRAP-expressing cells (right) show minimal surface CD19-CAR fluorescence. Bottom panel is quantification of CD19-CAR intensity density (integrated intensity per cell area) in HEK293T versus CAR-TRAP (n=9 cells). g, Contour plots of CAR (FITC-CD19) surface expression under non-permeabilising conditions with quantitative analysis shown as MFI (n=6), h, Representative Immunofluorescence image showing intracellular ER-localized CAR (anti-CD19-CAR, green) colocalized with calnexin (ER marker, red) in CAR-TRAP cells, along with the lines scans (LS) showing co-localisation of ER signal with CD19 CAR. i, Illustration of viroVbot1 particle produced from wild type producer cell and viroVbot1.1 particle produced from CAR-TRAP cells with bi-CAR as transgene. j, Dot plots showing % BCMA or % CD19 CAR expression in Raji wt or MM.1S wt cells with LVV obtained from HEK293T or CAR-TRAP cells. Quantification bar graphs (right) under same conditions (n= 8). k, BCMA CAR expression in patient multiple myeloma samples (PMM-1 to PMM-12) showing % of BCMA/CAR + cells after transduction with viroVbot1 versus viroVbot1.1. l-m, Similarly, BCMA CAR expression in patient-derived leukemia (PLK-1 to PLK-10) and lymphoma (PLM-1 to PLM-15) samples. Data represents mean ± SEM. ****p < 0.0001. A non-parametric t-test was used for statistical analysis between groups. Scale bar; f: 50 μm, n: 10 μm.

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: CRISPR, Knock-Out, Transduction, Flow Cytometry, Ex Vivo, Competitive Binding Assay, Incubation, Recombinant, Clinical Proteomics, Membrane, Immunofluorescence, Control, Expressing, Fluorescence, Marker, Produced, Derivative Assay

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, Schematic of miR-122-mediated hepatocyte-specific transgene silencing strategy incorporating five tandem miR-122 target sequences (5×miR-122T) in the 3’ untranslated region (UTR) downstream of CD3ζ costimulatory domain of bi-CAR transgene (viroVbot2). b, Flow cytometry analysis of % CAR expression (BCMA CAR-PE) in Huh-7 hepatoma cells transduced with LVV containing miR-122 (viroVbot2) or non-targeting control shRNA (NTC) with bi-CAR as transgene. c, Schematics of CD47 overexpression strategy in CAR-TRAP producer cells for macrophage evasion (viroVbot2.1). d, Flow cytometry analysis of % CAR expression in THP1 cells transduced with either viroVbot2 or viroVbot2.1. e, Schematic flow diagram of PromoterForge pipeline (details of the pipeline are provided in methods) . f, Candidate synthetic promoters fused upstream with GFP reporter cassette in reporter-plasmid format. Each synthetic promoter represents a unique combination of core promoter elements, TFBS motifs and enhancer arrangements. g, Flow cytometric analysis of GFP expression as MFI in primary human T cells transduced with constructs in which GFP is driven by the EF-1α promoter or the indicated synthetic promoters (Syn-T3, Syn-T8, Syn-T15) (n=5 biologically independent samples). h, Schematic of the dual-luciferase reporter assay used to validate promoter activity. i, Relative luciferase activity in T cells transfected with reporter constructs containing EF-1α or the indicated synthetic promoters (Syn-T3, Syn-T8, Syn-T15), normalized to the CMV control (n=6 biologically independent samples). j, Schematic of the viroVbot2.2 construct and LVV design. The transfer vector encodes bi-CAR under the Syn-T8 promoter with mir-122. Producer cells co-express the CAR-TRAP system and CD47, generating viroVbot2.2 particles displaying CD47 on the envelope. k, Flow cytometry analysis of CAR expression on CD3 + T cells in PBMCs from six healthy donors (HD-1 to HD-6) following ex vivo transduction with viroVbot2.1 (top row) and viroVbot2.2 (bottom row). Numbers in gates indicate the percentage of CAR-FITC + cells. The bar graph shows the %CAR + of CD3 + cells per donor. l, Scheme of the humanized mouse model. NCG mice were engrafted with human PBMCs (day −5), inoculated with 1×10 6 MM.1S multiple myeloma cells (day −4), and treated with a single dose of viroVbots (day 0). Peripheral blood was collected and BLI performed on days 7, 14, 21, 28, and 56; all organs were harvested at endpoint (day 56). m Representative BLI images of MM.1S tumor burden in mice treated with PBS control (MM.1S), viroVbot2.1, or viroVbot2.2 at the indicated time points. Red “X” denotes deceased animals. n, Quantification of whole-body BLI radiance (p/sec/cm 2 /sr) over time for each treatment group. Each line represents an individual mouse (n=5 per group). o, Percentage of circulating MM.1S tumor cells per 100 µl of blood over time in mice treated with viroVbot2.1 (orange) or viroVbot2.2 (blue). Data shown as mean ± SEM. p, Percentage of CAR + T cells in peripheral blood over time in the same treatment groups, demonstrating in vivo expansion kinetics and contraction of CAR-T cells. q, Kaplan-Meier survival curves of mice bearing MM.1S tumors and treated with PBS (red), viroVbot2.1 (orange), or viroVbot2.2 (blue); n = mice per group, log-rank test. Data represents mean ± SEM. ****p < 0.0001. A non-parametric t-test was used for statistical analysis between groups.

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: Flow Cytometry, Expressing, Transduction, Control, shRNA, Over Expression, Plasmid Preparation, Construct, Luciferase, Reporter Assay, Activity Assay, Transfection, Ex Vivo, In Vivo

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, Schematic of the screening strategy for selecting a synthetic IL-7R agonist. Three synthetic binding proteins (sBC-P1, sBC-P2, sBC-P3) were paired with full-length IL-7R or minimized IL-7R variants (min1-, min2-, min3-IL7R), expressed in T cells, and assessed for downstream STAT5 phosphorylation using the PathScan Phospho-STAT5 assay. b, Representative flow cytometry histograms of phospho-STAT5 (p-STAT5) in T cells expressing the indicated constructs, compared with isotype and non-transduced (NTP) controls. Anti-p-STAT5 antibody was used followed by Alexafluor 488 and acquired in FITC, channel. c, Quantification of pSTAT5 MFI across conditions, showing comparable STAT5 activation by sBCP3-IL7R and the minimized sBCP3_min3-IL7R receptor relative to NTP-IL7R control (n=8 biologically independent samples). d, Dose-response curves of ligand-induced receptor activation measured by ELISA across a concentration range (mM) for NTP-IL7R, sBCP3-IL7R, and sBCP3_min3-IL7R (n=5 biologically independent samples). e, f Schematic of the viroVbot3 transfer vector and producer cell design. The bi-CAR (BCMA/CD19) cassette is driven by the Syn-Tp promoter, linked via T2A, and detargeted from hepatocytes by 5× miR-122 target sites in the 3′ UTR to co-express the synthetic sBCP3-min3-IL7R (sBCP-mIL7R) cytokine receptor module. g, In vitro cytotoxicity assay showing % MM.1S tumor cell survival at increasing effector-to-target (E:T) ratios following co-culture with untransduced T cells (UT), viroVbot2.2-, or viroVbot3-generated CAR-T cells (n=5 biologically independent samples) after 24 h. h, IL-2 secretion (pg/mL) by CAR-T cells co-cultured with MM.1S target cells at the indicated E:T ratios (n=5 biologically independent samples). i, Similarly, intracellular Granzyme B expression (MFI) in CAR-T cells across the same E:T ratios (n=5). j, Schematic of the serial tumor-rechallenge assay. k, Percentage of Patient MM (PMM) cell survival over time during serial rechallenge in co-cultures with UT, viroVbot2.2, or viroVbot3 CAR-T cells. l, Absolute CAR-T cell counts (5×10 4 ) during serial rechallenge, demonstrating superior expansion and persistence of viroVbot3-generated CAR-T cells. m, Bar graph of frequency of PD1 low (gray) versus PD1 high (orange) populations within CD8 + effector memory (T EM ) cells at days 14, 21, and 28 of co-culture for the three groups. n, Similarly, CD8 + central memory (T cm ) cells at the same time points (n=5 biologically independent samples). o, Memory subset distribution (% of CD8 + T cells); naive/stem-cell memory (T n /T scm ), effector (T eff ), effector memory (T em ), and central memory (T cm ), across UT, viroVbot2.2, and viroVbot3 groups. p, Pie charts showing the relative proportions of T n (gray) and T scm (blue) compartments within CD8 + T cells (n=5 biologically independent samples). Data represents mean ± SEM. ****p < 0.0001. A non-parametric t-test was used for statistical analysis between groups.

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: Binding Assay, Phospho-proteomics, Flow Cytometry, Expressing, Construct, Activation Assay, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Plasmid Preparation, In Vitro, Cytotoxicity Assay, Co-Culture Assay, Generated, Cell Culture

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, NCG mice received MM.1S cells (day −7), human PBMCs (day −5), and viroVbot3 (day 0). On day 45, mice were rechallenged with BCMA negative but expressing GPRC5D (GR) MM.1S (GR + /BCMA − /CD19 − ) and given a second PBMC infusion plus viroVbot-CO or viroVbot-VS on day 47. Blood and BLI were collected at indicated time points; organs were harvested at day 90. b, Representative BLI images of tumor burden over time (days 10-90) in five treatment groups as indicated (n=5 mice in each group). c, Quantification of whole-body BLI radiance (p/sec/cm 2 /sr) over time for each group; each line represents an individual mouse. d, Kaplan-Meier survival curves of the five groups (Group-1 to Group-5) across 12 weeks. Log-rank test (n=10 mice in each group). e, Longitudinal flow cytometric quantification of BCMA + CAR-T cells (% of T cells) in peripheral blood from days 0-90 across Groups 2-5, showing initial expansion and contraction kinetics of the first-line CAR-T population. f, Frequency of GPRC5D + CAR-T cells (% of T cells) in peripheral blood across Groups 2, 4, and 5, demonstrating expansion of the second-line viroVbot-CO/VS-derived CAR-T cells following antigen-loss rechallenge. g, Quantification of BCMA + (left axis) and GPRC5D + (right axis) CAR-T cells in bone marrow (BM) at endpoint (day 90) for Groups 2, 4, and 5. ND, not detected. h, Similarly in spleen. Data represents mean ± SEM. A non-parametric t-test was used for statistical analysis between groups.

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: Expressing, Derivative Assay

Journal: Frontiers in Oncology

Article Title: Clinical implementation of a one-step no-wash flow cytometry method allows for real-time monitoring of patients treated with autologous CAR-T cells

doi: 10.3389/fonc.2026.1774431

Figure Lengend Snippet: Gating strategy for the single-step method. Absolute counting beads were excluded based on scatter and fluorescence properties. Dead cells were excluded using 7-aminoactinomycin D (7-AAD). CD45-positive leukocytes were selected, and lymphocytes were identified according to side scatter (SSC) characteristics. CD3-positive T cells were gated, and CAR-T cells were defined as viable CD45+/CD3+/CAR+ events using directly fluorochrome-conjugated CAR detection reagents (CD19 or BCMA). CD4 and CD8 subpopulations were subsequently identified within the CAR-positive T-cell compartment. Absolute quantification was calculated using TruCount beads according to the manufacturer’s formula.

Article Snippet: New CAR detection reagent (CDR) directly coupled to fluorochrome, either BCMA (BCMA CDR-PE, Miltenyi Biotec 130-133-888) or CD19 (CD19 CDR α-FMC63-PE, Miltenyi Biotec 130-127-342) was added extemporaneously in the analytical tube.

Techniques: Fluorescence, Quantitative Proteomics

Journal: Frontiers in Oncology

Article Title: Clinical implementation of a one-step no-wash flow cytometry method allows for real-time monitoring of patients treated with autologous CAR-T cells

doi: 10.3389/fonc.2026.1774431

Figure Lengend Snippet: Determination of LOD and LLOQ for the single-step method. CAR-T cell absolute counts were measured in 10 negative control samples from patients not treated with CD19 or BCMA CAR-T cells. The blue line represents the mean background signal. LOD was defined as mean + 3 SD and LLOQ (green dashed line) as mean + 10 SD.

Article Snippet: New CAR detection reagent (CDR) directly coupled to fluorochrome, either BCMA (BCMA CDR-PE, Miltenyi Biotec 130-133-888) or CD19 (CD19 CDR α-FMC63-PE, Miltenyi Biotec 130-127-342) was added extemporaneously in the analytical tube.

Techniques: Negative Control

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Timeline of T cell knock-in electroporation workflow, knock-in strategy and designs for BCMA-CAR (1.6 kb integration) across the series of DNA HDR template formats tested. (b) Comparison of BCMA-CAR HDRTs at concentrations 5nM-160nM or 0-100E3 MOI in terms of knock-in efficiency, (c) live cell count per 1e6 edited cells, and (d) knock-in cell count per 1e6 edited cells measured 7 days post electroporation. (e) Knock-in of a logic-gated synNotch circuit (5.6 kb integration) using linear ssDNA + tCTS, circular cssDNA + CTS, and nanoplasmid + CTS templates with corresponding knock-in efficiency, live cell count and knock-in cell count 7 days post electroporation using Cas9 mRNA. Circular cssDNA was produced and provided by Kano Therapeutics. (f) Knock-in strategy and designs for a logic-gated synNotch circuit (5.6 kb integration) at concentrations 5nM-160nM with corresponding knock-in efficiency, live cell count, and knock-in cell count 7 days post electroporation using Cas9 RNP or Cas9 mRNA. Each experiment was performed with T cells from two independent healthy human blood donors represented by individual dots plus mean. CTS, Cas9 target site. RNP, ribonucleoprotein. MOI, multiplicity of infection.

Article Snippet: Knockout and knock-in efficiency were evaluated by staining for the TCR with an anti-TCRα/β antibody (Miltenyi Biotec) and staining for the CAR with recombinant BCMA protein (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems BCA-H82E4_200ug).

Techniques: Knock-In, Electroporation, Comparison, Cell Characterization, Produced, Infection

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Knock-in strategy and designs for BCMA-CAR (1.6 kb integration) at concentrations 5nM-160nM with corresponding (b) knock-in efficiency, (c) live cell count, and (d) knock-in cell count 7 days post electroporation for the following templates respectively: dsDNA, dsDNA + tCTS, ssDNA, ssDNA + tCTS, circular ssDNA + CTS, circular ssDNA + mutated v1 CTS v1, circular ssDNA + mutated v2 CTS. Circular cssDNA was produced and provided by Touchlight. (e) Knock-in strategy and designs for CAR-CARD-11 (3.5 kb integration) using linear ssDNA + tCTS template or a nanoplasmid + CTS template at concentrations 5nM-160nM with corresponding knock-in efficiency, live cell count, and knock-in cell count 7 days post electroporation.

Article Snippet: Knockout and knock-in efficiency were evaluated by staining for the TCR with an anti-TCRα/β antibody (Miltenyi Biotec) and staining for the CAR with recombinant BCMA protein (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems BCA-H82E4_200ug).

Techniques: Knock-In, Cell Characterization, Electroporation, Produced

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Designs for BCMA-CAR (1.6 kb integration) HDRTs variants tested and corresponding template copies per genome measured at 4 hours and 3 days post electroporation (b) and half-life measured by qPCR. (c) Template copies per genome measured over time at 4 hours, 2 days, 3 days, 5 days, 7 days, and 12 days with R2 and half-life values corresponding to each donor. Each experiment was performed with T cells from two independent healthy human blood donors represented by individual dots plus mean. tCTS, truncated Cas9 target site. CTS, Cas9 target site. NP, nanoplasmid. cssDNA, circular cssDNA.

Article Snippet: Knockout and knock-in efficiency were evaluated by staining for the TCR with an anti-TCRα/β antibody (Miltenyi Biotec) and staining for the CAR with recombinant BCMA protein (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems BCA-H82E4_200ug).

Techniques: Electroporation

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Timeline of T cell knock-in electroporation workflow, knock-in strategy and designs for BCMA-CAR (1.6 kb integration) across the series of DNA HDR template formats tested. (b) Comparison of BCMA-CAR HDRTs at concentrations 5nM-160nM or 0-100E3 MOI in terms of knock-in efficiency, (c) live cell count per 1e6 edited cells, and (d) knock-in cell count per 1e6 edited cells measured 7 days post electroporation. (e) Knock-in of a logic-gated synNotch circuit (5.6 kb integration) using linear ssDNA + tCTS, circular cssDNA + CTS, and nanoplasmid + CTS templates with corresponding knock-in efficiency, live cell count and knock-in cell count 7 days post electroporation using Cas9 mRNA. Circular cssDNA was produced and provided by Kano Therapeutics. (f) Knock-in strategy and designs for a logic-gated synNotch circuit (5.6 kb integration) at concentrations 5nM-160nM with corresponding knock-in efficiency, live cell count, and knock-in cell count 7 days post electroporation using Cas9 RNP or Cas9 mRNA. Each experiment was performed with T cells from two independent healthy human blood donors represented by individual dots plus mean. CTS, Cas9 target site. RNP, ribonucleoprotein. MOI, multiplicity of infection.

Article Snippet: To stain for BCMA-CAR, cells were incubated with 0.3 μg BCMA recombinant protein conjugated to biotin (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems, BCA-H82E4_200ug) for 15 minutes at room temperature prior to surface cell staining.

Techniques: Knock-In, Electroporation, Comparison, Cell Characterization, Produced, Infection

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Knock-in strategy and designs for BCMA-CAR (1.6 kb integration) at concentrations 5nM-160nM with corresponding (b) knock-in efficiency, (c) live cell count, and (d) knock-in cell count 7 days post electroporation for the following templates respectively: dsDNA, dsDNA + tCTS, ssDNA, ssDNA + tCTS, circular ssDNA + CTS, circular ssDNA + mutated v1 CTS v1, circular ssDNA + mutated v2 CTS. Circular cssDNA was produced and provided by Touchlight. (e) Knock-in strategy and designs for CAR-CARD-11 (3.5 kb integration) using linear ssDNA + tCTS template or a nanoplasmid + CTS template at concentrations 5nM-160nM with corresponding knock-in efficiency, live cell count, and knock-in cell count 7 days post electroporation.

Article Snippet: To stain for BCMA-CAR, cells were incubated with 0.3 μg BCMA recombinant protein conjugated to biotin (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems, BCA-H82E4_200ug) for 15 minutes at room temperature prior to surface cell staining.

Techniques: Knock-In, Cell Characterization, Electroporation, Produced

Journal: bioRxiv

Article Title: Ultra-large targeted DNA integrations in primary human cells

doi: 10.64898/2026.04.09.717505

Figure Lengend Snippet: (a) Designs for BCMA-CAR (1.6 kb integration) HDRTs variants tested and corresponding template copies per genome measured at 4 hours and 3 days post electroporation (b) and half-life measured by qPCR. (c) Template copies per genome measured over time at 4 hours, 2 days, 3 days, 5 days, 7 days, and 12 days with R2 and half-life values corresponding to each donor. Each experiment was performed with T cells from two independent healthy human blood donors represented by individual dots plus mean. tCTS, truncated Cas9 target site. CTS, Cas9 target site. NP, nanoplasmid. cssDNA, circular cssDNA.

Article Snippet: To stain for BCMA-CAR, cells were incubated with 0.3 μg BCMA recombinant protein conjugated to biotin (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems, BCA-H82E4_200ug) for 15 minutes at room temperature prior to surface cell staining.

Techniques: Electroporation