Journal: Nature medicine

Article Title: Antigen-independent activation enhances the efficacy of 41BB co-stimulated CD22 CAR T cells

doi: 10.1038/s41591-021-01326-5

Figure Lengend Snippet: a, Pipeline for development and evaluation of new CD22-f2-short CAR. b, Affinity and size of purified CD22-f2-long and short scFvs. c, Expression of CD22 CARs on primary T cells. d, Measurement of secreted IFNγ by CD22-engineered T cells after 24h exposure to CD22+ target cells. e, Progression of Nalm6 disease burden in xenograft mice treated with CD22-f2-short and long T cells (Representative of 4 replicate experiments, n=4–7 mice per condition; see Supplementary Figure 5 for individual animal responses and Supplementary Figure 6 for experimental replicates). f, Survival of Nalm6-bearing xenograft mice after treatment with m971 or CD22-f2 CAR T cells. Data are presented as mean values +/− standard error of the mean (S.E.M.) Statistics reflect differences between CAR22-short and long T cells.

Article Snippet: 40 All animal studies were approved and supervised by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC). scFv design and optimization: To identify novel binders to human CD22 extracellular domain, three rounds of panning were done against recombinant human CD22 (R&D systems, Cat. # 1968-SL-050) using a fully human derived scFv phage library derived internally.

Techniques: Purification, Expressing

Journal: Journal of Translational Medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Observation of CD19-BBζ-CAR expression in relapsed Nalm-6 cells and salvage treatment. A Detection of FMC63 and CD247 transcripts and 4-1BB gene of CAR in CD19 + Nalm-6 (red) and relapsed CD19 − Nalm-6 cells (blue) by qRT-PCR. Data of left bar graph represent the relative quantification using ACTB as the internal reference. Error bars represent s.d. The data are the representative of three independent experiments. B Expression of CD19 and CAR on CD19 + Nalm-6 cells and relapsed CD19 − Nalm-6 cells analyzed by flow cytometry (representative of 3 experiments). Merge Graphs, the blue dots represent CD19 − Nalm-6 cells and the red dots represent Nalm-6 cells. C Confocal imaging of Nalm-6 cells and relapsed CD19 − Nalm-6 cells using Alexa Flour 488-conjugated anti-CD19 antibody (green), Alexa Flour 647-conjugated anti-CAR19 antibody (red), and DAPI (blue). D Lentiviral integration sites of CAR transduced Nalm-6 cells were analyzed by linear-amplification mediated PCR (LAM-PCR) and visualized with Circos plots. The integration sites across the genome and genomic features were shown from outer to inner circle: (1) cytogenetic bands; (2) genes that harbor these integration sites along with a bar chart showing the reads of integration sites; (3) the distribution of integration sites, with colored circles representing different gene functional regions of the host sequence: purple for promoter region, green for intron region, and red for distal intergenic region. E Phenotype changes of Nalm-6 cells transduced with small amount of CD19 CAR lentiviruses detected by flow cytometry over time. Gating was based on the same cells stained with isotype-matched antibody. F Dynamics of CD19 − B phenotype in relapsed cells after co-culture with different ratios (5×, 20×) of Nalm-6 cells. Gating was based on the same cells stained with isotype-matched antibody. G Relapsed CD19 − Nalm-6 cells were tested by qPCR specific for VSV-G sequence. H Comparison of in vitro efficacy of CD19-, CD22-, CD19/CD22- and CD22×CD19- CAR T cells. Cocultures with the relapsed cells were performed at 1:5, 1:1, and 5:1 E: T ratios, and lysis efficacies were detected by the LDH release assay Declarations

Article Snippet: The cells were then washed twice and stained with phycoerythrin (PE) streptavidin (BD bioscience, USA) for 15 min. CART-22 cells and CART-22/19 cells were washed once and incubated with CD22 Fc Alexa Fluor® 647 Protein (R&D Systems, USA) for 15 min. To detect in vitro cytotoxicity of CART-19 cells, transduced and untransduced T cells were co-cultured with Nalm-6 cells (total 1 × 10 6 cells) at E: T ratios (0.2:1, 0.5:1, 1:1, 5:1) for 6, 24 and 72 h. Cells were pipetted to incubate with antibodies for 30 min at room temperature in the dark and washed twice with PBS.

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Flow Cytometry, Imaging, Amplification, Functional Assay, Sequencing, Transduction, Staining, Co-Culture Assay, Comparison, In Vitro, Lysis, Lactate Dehydrogenase Assay

Journal: Frontiers in Pharmacology

Article Title: Deep Mining of Complex Antibody Phage Pools Generated by Cell Panning Enables Discovery of Rare Antibodies Binding New Targets and Epitopes

doi: 10.3389/fphar.2019.00847

Figure Lengend Snippet: Frequency, based on NGS analysis, of discovered clones in the different strategies including the input phage pool. Median frequency is shown with red bars. (A) Antibodies, previously discovered through direct screening with corresponding specificities, CD23 ( n = 107), CD72 ( n = 132), and CD200, CD21, CD32, or HLA-DR ( n = 34). (B) Antibodies, discovered through fishing, binding FCRL5, ROR1, or CD22 ( n = 17). For statistical analysis, Friedman’s test with Dunn’s multiple comparison was done using GraphPad Prism.

Article Snippet: Proteins (CD72 (5405-CD), CD22 (1968-SL), FCRL5 (2078-FC), and CD45 (1430-CD-050) from R&D Systems; CD32b produced in-house; CD200 (10886-H08H), CD21 (10811-H08H), CD23 (10261-H07H), ROR1 (13968-H08H), and LY75 (16490-H08H) from Sino Biological) were coated to ELISA plates overnight.

Techniques: Clone Assay, Binding Assay, Comparison

Journal: Frontiers in Pharmacology

Article Title: Deep Mining of Complex Antibody Phage Pools Generated by Cell Panning Enables Discovery of Rare Antibodies Binding New Targets and Epitopes

doi: 10.3389/fphar.2019.00847

Figure Lengend Snippet: NGS analysis of identified antibodies binding to (A) CD72 ( n = 301) or (B) CD21 ( n = 27) showing the frequency in control (track C) versus antibody blocking (track B1). Yellow-marked clones were analyzed for binding, as scFv displayed on phage, to coated (C) CD72 or (D) CD21 protein in ELISA with or without prior blocking with the pool of IgGs used for blocking in tracks B1 and B2. As control, two additional clones per target, corresponding to sequences showing a decreased frequency in tracks B1 and B2 in the NGS analysis, were included. Binding was detected using an HRP-labeled anti-M13 antibody and a luminescent substrate.

Article Snippet: Proteins (CD72 (5405-CD), CD22 (1968-SL), FCRL5 (2078-FC), and CD45 (1430-CD-050) from R&D Systems; CD32b produced in-house; CD200 (10886-H08H), CD21 (10811-H08H), CD23 (10261-H07H), ROR1 (13968-H08H), and LY75 (16490-H08H) from Sino Biological) were coated to ELISA plates overnight.

Techniques: Binding Assay, Control, Blocking Assay, Clone Assay, Enzyme-linked Immunosorbent Assay, Labeling

Journal: The Journal of Clinical Investigation

Article Title: Perforin-deficient CAR T cells recapitulate late-onset inflammatory toxicities observed in patients

doi: 10.1172/JCI130059

Figure Lengend Snippet: (A) BM samples obtained on day 28 after CAR T cell infusion. H&E-stain shows decreased trilineage hematopoiesis with increased macrophages. CD3 immunohistochemical (IHC) stain highlights extensive T cell infiltration with flow cytometric confirmation of anti-CD22 CAR positivity in 59% of T cells. CD68 IHC stain highlights hemophagocytic macrophages. Giemsa stain of BM aspirate also shows hemophagocytosis. Original magnification, 50× (H&E, CD3, CD68 stains) and 100× (Giemsa stain). (B) Representative chronological changes in serum cytokine levels from patient 52 who had CRS without subsequent HLH. (C) Representative chronological changes in serum cytokine levels from patient 37 who had CRS and subsequent HLH. (D) The percentages of circulating T cells (CD3+) that stained positive for surface CAR expression were assessed by flow cytometry at the indicated time points. (E–G) Peak levels (during the first 28 days) of (E) IFN-γ, (F) IL-1β, and (G) IL-18 in serum/plasma. Data shown in D–G include all patients who were diagnosed with CRS according to previously published criteria (9), and patients who had never been diagnosed with CRS are not included. Data were stratified according to the presence or absence of HLH diagnosis (in addition to CRS) after CAR T cell infusion. Data are reported as the mean ± SD (D–G). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by Mann-Whitney U test (D–G).

Article Snippet: Human anti-CD22 CAR detection was performed using a CD22-Fc chimera protein (R&D Systems) as previously described ( 1 ).

Techniques: Staining, Immunohistochemical staining, Giemsa Stain, Expressing, Flow Cytometry, MANN-WHITNEY