Journal: Cell Reports Medicine

Article Title: Chimeric antigen receptor-induced antigen loss protects CD5.CART cells from fratricide without compromising on-target cytotoxicity

doi: 10.1016/j.xcrm.2024.101628

Figure Lengend Snippet: T cells resistant to CD5.CART cell elimination are enriched for CD8 + effector T cells (A) Percentage of residual targets following coculture of CD5.CART cells with resting T cells, CCRF-CEM, or Jurkat tumor cells for 48 h at an E:T ratio of 1:3. Resting T cells were CD3 + MACS isolated from fresh peripheral blood mononuclear cells. p values calculated using one-way ANOVA with Dunnett’s correction. n = 3 donors. Data are represented as mean ± SD. (B) Freshly isolated T cells were cocultured for 36 h with autologous CD19.CART cells or CD5.CART cells. Residual target cells were sort-purified and subjected to CITE-Seq. ssRNA-seq clustering by Seurat is shown. (C) Jaccard Similarity Index of Seurat UMAP of autologous T cells cocultured with either CD19.CART cells (left) or CD5.CART cells (center). Table denoting enriched and depleted clusters (right). (D) CD4, CD8, CD45RA, and CD62L surface expression of T cells (CITE-Seq) cultured with either CD19.CART cells or CD5.CART cells overlaid on Seurat UMAP. Gradient denotes log UMI counts of surface transcripts from CITE-seq. (E) CD5 protein surface expression (orange, top) and CD5 transcript levels (green, bottom) in residual T cells after coculture with either CD19.CART cells or CD5.CART cells overlaid on Seurat UMAP. Gradient denotes log UMI counts of surface and gene transcripts from CITE-Seq and RNA-seq, respectively. (F) Heatmap denoting fold change of genes in enriched clusters 3, 4, and 5, or depleted cluster 0 and 2 vs. all other cells; clustering by Seurat. (G) Differential abundance neighborhood groups. Color denotes significantly upregulated/downregulated genes with fold change differences above in CD5 CART treatment (red) or below compared to CD19 CART treatment (blue) with half log threshold using Milo. (H) Differentially expressed genes between CD19.CART treatment (left) and CD5.CART treatment (right) using Milo. Genes upregulated under CD5.CART treatment on the right.

Article Snippet: Mouse anti-human CD3 FiTC (clone SK7) , BD Biosciences , Cat#349201; RRID: AB_400405.

Techniques: Isolation, Purification, Expressing, Cell Culture, RNA Sequencing Assay

Journal: Cell Reports Medicine

Article Title: Chimeric antigen receptor-induced antigen loss protects CD5.CART cells from fratricide without compromising on-target cytotoxicity

doi: 10.1016/j.xcrm.2024.101628

Figure Lengend Snippet: Long-term resistance of normal T cells to CD5.CART cells is mediated by the loss of CD5 gene expression (A) Gating strategy (top). CD5 expression of T cells in patient #5 treated with CD5.CART cells that produced an abbreviated expansion (bottom). (B) CD3 + T cells in peripheral blood of patients with T cell lymphoma (TCL) or T cell acute lymphoblastic leukemia (T-ALL) treated with CD5.CART cells (NCT03081910). (C) CD5 expression of T cells in patient #7 (top), #8 (middle), and #9 (bottom) with durable CD5.CART cell persistence and achieved complete response. (D) CD5 mRNA expression of healthy T cells isolated from a lymph node biopsy at week 5 in patient #7 (left) and week 4 in patient #8 (right) normalized to respective donors’ T cells pre-lymphodepletion. (E) T cell phenotype of healthy T cells based on CCR7 and CD45RA expression of patient #7, #8, and #9. Naive (CCR7 + /CD45RA + ), central memory (CCR7 + /CD45RA − ), effector memory (CCR7 − /CD45RA − ), TEMRA (CCR7 − /CD45RA + ), and CD4/CD8 T cell distributions in healthy circulating T cells in patient #7, #8, and #9.

Article Snippet: Mouse anti-human CD3 FiTC (clone SK7) , BD Biosciences , Cat#349201; RRID: AB_400405.

Techniques: Expressing, Produced, Isolation

Journal: Cell Reports Medicine

Article Title: Chimeric antigen receptor-induced antigen loss protects CD5.CART cells from fratricide without compromising on-target cytotoxicity

doi: 10.1016/j.xcrm.2024.101628

Figure Lengend Snippet:

Article Snippet: Mouse anti-human CD3 FiTC (clone SK7) , BD Biosciences , Cat#349201; RRID: AB_400405.

Techniques: Enzyme-linked Immunosorbent Assay, Antibody Labeling, Western Blot, Sequencing, CRISPR, Software

Journal: Oncology Letters

Article Title: Cetuximab intensifies the ADCC activity of adoptive NK cells in a nude mouse colorectal cancer xenograft model

doi: 10.3892/ol.2016.4835

Figure Lengend Snippet: The ratio of CD3 − CD56 + natural killer cells after 14 days of culture. CD, cluster of differenatiation; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Article Snippet: The antibodies used in the present study were mouse anti-human CD3-fluorescein isothiocyanate (FITC; monoclonal; 1:100; cat. no. 55539; BD Biosciences, Franklin Lakes, NJ, USA), mouse anti-human CD56-phycoerythrin (PE) (monoclonal; 1:100; cat. no. 555516; BD Biosciences); human immunoglobulin G (hIgG; polyclonal; 1:200; cat. no. bs-0297P; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China), rabbit anti-human EGFR (polyclonal; 1:200; cat. no. Sc-03AC; Santa Cruz Biotechnology, Inc.) rabbit anti-human Ki-67 (monoclonal; 1:200; cat. no ZA-0502; OriGene Technologies, Inc., Beijing, China).

Techniques:

Journal: British Journal of Cancer

Article Title: Identification of a high-risk immunogenic prostate cancer patient subset as candidates for T-cell engager immunotherapy and the introduction of a novel albumin-fused anti-CD3 × anti-PSMA bispecific design

doi: 10.1038/s41416-022-01994-1

Figure Lengend Snippet: a – c Schematic representation of the AlproTox panel containing an anti-PSMA human domain antibody (Green) linked to an anti-CD3 scFv (Blue) and HSA (Red) with amino acid point mutations (yellow dots) providing different FcRn affinity (NB: K500A, H510Q, HB: E505Q, T527M, K573P). d – f Representative AFM images of particle height and histograms showing distribution and average height of AlproTox WT, NB and HB, respectively. Scale bar is 200 nm and intensity scale −6–6 nm. g – j Sensorgrams showing binding of serial dilutions of protein to immobilised human FcRn at pH 5.5. Red curves display fit. g Binding curves of albumin WT. h Binding curves of AlproTox WT. i Binding curves of albumin HB. j Binding curves of AlproTox HB. k rHSA detection in the supernatant following a cellular recycling assay. l AlproTox detection in the supernatant following a cellular recycling assay. Statistical analysis was made in Graphpad prism v.8 using ANOVA one-way with Tukey Post hoc correction. Mean ± SEM shown, N = 3 independent experiments.

Article Snippet: LNCaP, C4-2, Du-145, PC3 or Jurkat cells were cultivated, and 1 × 10 5 cells were seeded into a U-bottom Nunclon TM plate and incubated on ice with dilution series of ice-cold assay buffer (PBS + 1% FBS) supplemented with either AlproTox, mouse anti-human CD3 FITC-conjugated antibody (Immunotools, Friesoythe, Germany #21850033), or PE-conjugated mouse monoclonal anti-PSMA antibody (Abcam, #ab77228).

Techniques: Binding Assay

Journal: British Journal of Cancer

Article Title: Identification of a high-risk immunogenic prostate cancer patient subset as candidates for T-cell engager immunotherapy and the introduction of a novel albumin-fused anti-CD3 × anti-PSMA bispecific design

doi: 10.1038/s41416-022-01994-1

Figure Lengend Snippet: Measured binding affinities of rHSA and AlproTox variants to hFcRn (K D , K on and K off ) at pH 5.5.

Article Snippet: LNCaP, C4-2, Du-145, PC3 or Jurkat cells were cultivated, and 1 × 10 5 cells were seeded into a U-bottom Nunclon TM plate and incubated on ice with dilution series of ice-cold assay buffer (PBS + 1% FBS) supplemented with either AlproTox, mouse anti-human CD3 FITC-conjugated antibody (Immunotools, Friesoythe, Germany #21850033), or PE-conjugated mouse monoclonal anti-PSMA antibody (Abcam, #ab77228).

Techniques: Binding Assay

Journal: British Journal of Cancer

Article Title: Identification of a high-risk immunogenic prostate cancer patient subset as candidates for T-cell engager immunotherapy and the introduction of a novel albumin-fused anti-CD3 × anti-PSMA bispecific design

doi: 10.1038/s41416-022-01994-1

Figure Lengend Snippet: It shows the panel of AlproTox WT, HB and NB fusions antigen-specific binding at 1 µg/mL in PSMA-positive (LNCaP, C4-2) and CD3-positive cells (Jurkat) detected by an anti-human serum antibody ( b – d ). No shifts in fluorescence were detected in non-expressing CD3 or PSMA (PC3, Du-145) cells. Secondary antibody without primary antibody was used to determine background intensity. PSMA and CD3 expression was verified ( a ) with an anti-PSMA PE-conjugated antibody (ab77228) and an anti-CD3 FITC-conjugated antibody (21850033), respectively.

Article Snippet: LNCaP, C4-2, Du-145, PC3 or Jurkat cells were cultivated, and 1 × 10 5 cells were seeded into a U-bottom Nunclon TM plate and incubated on ice with dilution series of ice-cold assay buffer (PBS + 1% FBS) supplemented with either AlproTox, mouse anti-human CD3 FITC-conjugated antibody (Immunotools, Friesoythe, Germany #21850033), or PE-conjugated mouse monoclonal anti-PSMA antibody (Abcam, #ab77228).

Techniques: Binding Assay, Fluorescence, Expressing

Journal: British Journal of Cancer

Article Title: Identification of a high-risk immunogenic prostate cancer patient subset as candidates for T-cell engager immunotherapy and the introduction of a novel albumin-fused anti-CD3 × anti-PSMA bispecific design

doi: 10.1038/s41416-022-01994-1

Figure Lengend Snippet: An increasing dose of tested AlproTox fusion was co-incubated with Jurkat cells and PC cell lines (PSMA-positive closed symbols, PSMA negative open symbols) followed by flow cytometry analysis quantifying the expression of CD69 ( a – c ) Cell killing was quantified using an LDH-endpoint assay determining the percentage of lysed PC cell lines when co-incubated with increasing levels of AlproTox and isolated PBMCs ( d – f ). Statistical analysis was made in Graphpad prism v.8 with Mean ± SEM shown, N = 3 independent experiments, (LNCaP N = 2 independent experiments).

Article Snippet: LNCaP, C4-2, Du-145, PC3 or Jurkat cells were cultivated, and 1 × 10 5 cells were seeded into a U-bottom Nunclon TM plate and incubated on ice with dilution series of ice-cold assay buffer (PBS + 1% FBS) supplemented with either AlproTox, mouse anti-human CD3 FITC-conjugated antibody (Immunotools, Friesoythe, Germany #21850033), or PE-conjugated mouse monoclonal anti-PSMA antibody (Abcam, #ab77228).

Techniques: Incubation, Flow Cytometry, Expressing, End Point Assay, Isolation

Journal: Nature Communications

Article Title: Tumor response and endogenous immune reactivity after administration of HER2 CAR T cells in a child with metastatic rhabdomyosarcoma

doi: 10.1038/s41467-020-17175-8

Figure Lengend Snippet: a Histological examination of the bone marrow 4 weeks after the salvage chemotherapy (ARST0921) and prior to initiating CAR T-cell infusions showing hypocellularity and presence of rhabdomyosarcoma (RMS) cells on hematoxylin and eosin (H&E) staining and immunoreactivity to desmin and myogenin, b complete disease response (CR1), evidenced by recovery of trilineage hematopoiesis and absence of immunoreactivity to desmin and myogenin after three HER2 CAR T-cell infusions. Panels ( a , b ) show representative microscopic images; scale bar 100 µm. c Representative image from positron emission tomography–computed tomography (PET-CT) with no evidence of FDG-avid disease in bone marrow or other sites 6 weeks after the third HER2 CAR T-cell infusion. d Detection of HER2 CAR-expressing T cells in the peripheral blood 7 days after the second infusion using flow cytometry. HER2 CAR was specifically recognized using HER2.Fc chimeric protein followed by a goat anti-human Fc conjugated with PE as a secondary antibody. SSC side scatter. e The proportion of CD3+ HER2 CAR-expressing T cells on day +7 after each infusion during the induction period. f Histograms showing the PD-1 and LAG3 surface expression in CAR-positive CD8+ (in blue) in comparison to CAR-negative CD8+ T cells (in black) at peak expansion (day +7) after each infusion during induction, and g the corresponding median fluorescence intensity (MFI) of PD-1 and LAG3 surface expression in CAR-positive and CAR-negative CD8+ T cells.

Article Snippet: The following monoclonal antibodies were used: mouse anti-human CD3 FITC (stock solution, 5 μL/100 μL test at 1:20 dilution, clone: SK7, Cat# 349201, Lot# 7221481, Becton Dickinson Biosciences, San Jose, CA), mouse anti-human CD8 Pacific Blue (stock solution, 5 μL/100 μL test at 1:20 dilution, clone: B9.11, Cat# A82791, Lot# 38, Beckman Coulter, Indianapolis, IN), mouse anti-human CD279 (PD-1) APC (stock solution, 5 μL/100 μL test at 1:20 dilution, clone: MIH4, Cat# 558694, Lot# 8012764, Becton Dickinson Biosciences, San Jose, CA), mouse anti-human CD223 (LAG3) FITC (stock solution, 5 μL/100 μL test at 1:20 dilution, clone: 3DS223H, Cat# 11-2239-42, Lot# 4337363, Thermo Fisher Scientific, Waltham, MA).

Techniques: Staining, Positron Emission Tomography, Computed Tomography, Positron Emission Tomography-Computed Tomography, Expressing, Flow Cytometry, Comparison, Fluorescence