anti murine human psma ab  (Cell Signaling Technology Inc)

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: PSMA antibody, humanized PSMA.CAR10.3, or Cetuximab increases prostate cancer localization of NF-κB p50-deficient immature myeloid cells (p50-IMC) and phagocytosis by their macrophage progeny

doi: 10.1007/s00262-024-03939-4

Figure Lengend Snippet: Development of murine prostate cancer lines expressing hPSMA or hEGFR and their detection by PSMA Ab3.9 or Cetuximab. a Amino acid sequence of wild-type (WT) hPSMA and its NΔ9 variant lacking the MWNLL internalization domain. b Flow cytometry analysis of surface hPSMA on indicated Myc-CaP (MC) lines using commercial hPSMA-APC Ab. c Comparison of surface hPSMA expression in human LNCaP cells and MC/hPSMA(NΔ9) cells. d Detection of surface hPSMA by PSMA Ab3.9, in conjunction with goat anti-murine IgG-PE secondary Ab, on MC/hPSMA(NΔ9) cells. e Detection of surface hEGFR on MC/hEGFR cells and LNCaP cells using hEGFR-Alexa-647 Ab. f Detection of hEGFR on MC/hEGFR cells using Cetuximab, with rat anti-human IgG-PE. These data are each from a single assessment

Article Snippet: Total cellular proteins in Laemmli sample buffer were subjected to Western blotting using anti-murine/human PSMA Ab (#12815, Cell Signaling Technology, Danvers, MA, USA) and β -actin Ab (AC-15, Sigma) as described [ ].

Techniques: Expressing, Sequencing, Variant Assay, Flow Cytometry, Comparison

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: PSMA antibody, humanized PSMA.CAR10.3, or Cetuximab increases prostate cancer localization of NF-κB p50-deficient immature myeloid cells (p50-IMC) and phagocytosis by their macrophage progeny

doi: 10.1007/s00262-024-03939-4

Figure Lengend Snippet: PSMA antibody increases phagocytosis of hPSMA-expressing prostate cancer cells. a Diagram of the Fc domain of an Ab bound to a macrophage via the Fc receptor (FcR). b Lineage-negative (Lin − ) WT or p50 −/− murine bone marrow (mBM) cells were expanded, differentiated to macrophages using M-CSF, M1- or M2-polarized using IFNγ or IL-4, and co-cultured for 3 h with CFSE-labeled MC/PSMA(NΔ9) that had been incubated with PSMA Ab3.9 or isotype IgG control, as diagrammed. c Representative flow cytometry data, previously gating on CD11b + cells. d Results of three experiments (one repetition per experiment) evaluating CFSE + cells as a percentage of CD11b + macrophages. (mean, SD) * p < .05, ** p < .01. e CFSE-labeled macrophages (green) were co-cultured with pHRodo, Red SE-labeled MC/PSMA(NΔ9) cells and PSMA Ab3.9, followed by microscopy (bright field, red, and green channels). Phagocytosed cancer cells are indicated by white arrows

Article Snippet: Total cellular proteins in Laemmli sample buffer were subjected to Western blotting using anti-murine/human PSMA Ab (#12815, Cell Signaling Technology, Danvers, MA, USA) and β -actin Ab (AC-15, Sigma) as described [ ].

Techniques: Expressing, Cell Culture, Labeling, Incubation, Control, Flow Cytometry, Microscopy

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: PSMA antibody, humanized PSMA.CAR10.3, or Cetuximab increases prostate cancer localization of NF-κB p50-deficient immature myeloid cells (p50-IMC) and phagocytosis by their macrophage progeny

doi: 10.1007/s00262-024-03939-4

Figure Lengend Snippet: Development of a fully humanized PSMA.CAR10.3. Diagram of PSMA.CAR10.3, containing a leader sequence from human IgG1, an scFv domain derived from PSMA Ab10.3 by connecting its V H and V L domains with a linker peptide, spacer and trans-membrane (TM) domains from human CD8 (hCD8), and the intracellular (IC) signaling domain from human CD3ζ (hCD3ζ). PSMA Ab10.3 was developed from mice harboring only human immunoglobulin genes. The amino acid sequences of the PSMA.CAR10.3 domains are also shown

Article Snippet: Total cellular proteins in Laemmli sample buffer were subjected to Western blotting using anti-murine/human PSMA Ab (#12815, Cell Signaling Technology, Danvers, MA, USA) and β -actin Ab (AC-15, Sigma) as described [ ].

Techniques: Sequencing, Derivative Assay, Membrane

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: PSMA antibody, humanized PSMA.CAR10.3, or Cetuximab increases prostate cancer localization of NF-κB p50-deficient immature myeloid cells (p50-IMC) and phagocytosis by their macrophage progeny

doi: 10.1007/s00262-024-03939-4

Figure Lengend Snippet: PSMA.CAR10.3 increases phagocytosis of hPSMA-expressing prostate cancer cells. a Diagram of a CAR expressed on a macrophage. b WT or p50 −/− Lin − murine bone marrow cells were expanded and transduced with vector or PSMA.CAR10.3, differentiated to macrophages, M1- or M2-polarized, and co-cultured with CFSE-labeled MC/PSMA(NΔ9) cells, as diagrammed. c Flow cytometry of vector- and PSMA.CAR10.3-transduced IMC, after puromycin-selection, using hPSMA-biotin and SA-APC. d Representative flow cytometry showing phagocytosis by vector versus CAR-expressing macrophages, previously gating on CD11b + cells. e Results of three experiments (one repetition per experiment) evaluating CFSE + cells as a percentage of CD11b + macrophages (mean, SD). * p < .05, ** p < .01, *** p < .001. f CFSE-labeled macrophages expressing PSMA.CAR10.3 (green) were combined with pHRodo, Red SE-labeled MC/PSMA(NΔ9) cells, followed by microscopy (bright field, red, and green channels). Phagocytosed cancer cells are indicated by white arrows

Article Snippet: Total cellular proteins in Laemmli sample buffer were subjected to Western blotting using anti-murine/human PSMA Ab (#12815, Cell Signaling Technology, Danvers, MA, USA) and β -actin Ab (AC-15, Sigma) as described [ ].

Techniques: Expressing, Transduction, Plasmid Preparation, Cell Culture, Labeling, Flow Cytometry, Selection, Microscopy

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: PSMA antibody, humanized PSMA.CAR10.3, or Cetuximab increases prostate cancer localization of NF-κB p50-deficient immature myeloid cells (p50-IMC) and phagocytosis by their macrophage progeny

doi: 10.1007/s00262-024-03939-4

Figure Lengend Snippet: PSMA antibody increases p50-IMC localization to hPSMA-expressing prostate cancer tumors when given after 5-FU. a Lin − p50 −/− murine bone marrow cells were expanded, cultured with M-CSF for one day to obtain p50-IMC, CFSE-labeled, incubated with 100 μg PSMA antibody or isotype IgG control for one hour on ice, and injected into NSG mice bearing subcutaneous tumors derived from MC/CaP-hPSMA(NΔ9) cells in Matrigel, with or without 5-FU (112.5 mg/kg i.p.) given to the mice five days prior to cell injection, followed by tumor flow cytometry at 24 h, as diagrammed. b Representative CFSE/CD11b flow cytometry. c Tumor weight, total tumor CD11b + CSFE + cells, and CD11b + CSFE + cells per mg of tumor in mice that did not receive 5-FU (mean, SE; n = 4 per group, from one experiment). d Tumor weight, total tumor CD11b + CSFE + cells, and CD11b + CSFE + cells per mg of tumor in mice that did receive 5-FU (mean, SD; IgG n = 9 and PSMA Ab n = 10; data are combined from two experiments). e The experiment in d was repeated with removal of excess PSMA Ab or isotype control by centrifugation, supernatant aspiration, and resuspension in HBSS prior to injection ( n = 5 per group, from one experiment). * p < 0.05, ** p < 0.01

Article Snippet: Total cellular proteins in Laemmli sample buffer were subjected to Western blotting using anti-murine/human PSMA Ab (#12815, Cell Signaling Technology, Danvers, MA, USA) and β -actin Ab (AC-15, Sigma) as described [ ].

Techniques: Expressing, Cell Culture, Labeling, Incubation, Control, Injection, Derivative Assay, Flow Cytometry, Centrifugation

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: PSMA antibody, humanized PSMA.CAR10.3, or Cetuximab increases prostate cancer localization of NF-κB p50-deficient immature myeloid cells (p50-IMC) and phagocytosis by their macrophage progeny

doi: 10.1007/s00262-024-03939-4

Figure Lengend Snippet: PSMA.CAR10.3 increases p50-IMC localization to hPSMA-expressing prostate cancer tumors. a Lin − p50 −/− murine bone marrow cells were expanded, transduced with vector or PSMA.CAR10.3, cultured with M-CSF, CFSE-labeled, and injected into NSG mice bearing tumors derived from MC/CaP-hPSMA(NΔ9) cells, and analyzed by flow cytometry 24 h later, as diagrammed. Mice received 5-FU five days prior to p50-IMC injection. b Tumor weight, total tumor CD11b + CFSE + cells, and CD11b + CFSE + cells per mg of tumor (mean, SD; n = 4 for vector and n = 5 for PSMA.CAR10.3, from one experiment)

Article Snippet: Total cellular proteins in Laemmli sample buffer were subjected to Western blotting using anti-murine/human PSMA Ab (#12815, Cell Signaling Technology, Danvers, MA, USA) and β -actin Ab (AC-15, Sigma) as described [ ].

Techniques: Expressing, Transduction, Plasmid Preparation, Cell Culture, Labeling, Injection, Derivative Assay, Flow Cytometry