Journal: Molecular Therapy Oncology

Article Title: TYRP1 directed CAR T cells control tumor progression in preclinical melanoma models

doi: 10.1016/j.omton.2024.200862

Figure Lengend Snippet: Analysis of TYRP1 mRNA expression in tumors (A) TYRP1 RNA-seq expression in 17 TCGA cohorts ( n = 13,325 samples) and Zhang C. et al. data ( n = 57 samples), grouped by cancer type, sorted by median expression. Bolded groups represent melanoma subtypes. The dashed red line depicts the mean expression in the TCGA cohort. Abbreviations are explained in Table S1 . (B) TYRP1 log2 TPM RNA-seq expression in the CCLE, grouped by cell line disease and sorted by median expression. Bolded groups represent melanoma subtypes. Abbreviations are explained in Table S2 .

Article Snippet: Anti-TYRP1 antibody (clone TA99) conjugated to Alexa Fluor 647 (Cat# NBP2-34720AF647) and mouse IgG2a Kappa isotype control conjugated to Alexa Fluor 647 (Cat# NBP1-96981AF647) were purchased from Novus Biologicals (Centennial, CO) and used at a concentration of 5 μL per 10 6 cells.

Techniques: Expressing, RNA Sequencing

Journal: Molecular Therapy Oncology

Article Title: TYRP1 directed CAR T cells control tumor progression in preclinical melanoma models

doi: 10.1016/j.omton.2024.200862

Figure Lengend Snippet: Analysis of TYRP1 mRNA expression in normal tissues and in comparison with melanomas (A) TYRP1 expression in GTEx is shown grouped by detailed tissue type (SMTSD). Expression was measured by RNA-seq and log 2 and TPM normalized. The tissue groups are ordered by median expression. (B) The density of TYRP1 RNA-seq expression in melanomas in the TCGA and Zhang et al. ACMFD tumor tissues (log 2 TPM normalized). The dashed red line depicts the threshold used to separate samples into high and low expressers of TYRP1. (C) TYRP1 RNA-seq expression (log 2 TPM normalized) was compared between melanoma tumor tissues and normal tissues. Three melanoma cohorts from TCGA and Zhang C. et al. were divided into high and low TYRP1 expression using log 2 TPM of 5 (left, in red), and plotted with the 12 top TYRP1-expressing normal tissues grouped by detailed tissue type (SMTSD) from GTEx (right, in blue), sorted by median expression. The dashed red line depicts the mean expression of TYRP1 in all TCGA cohorts.

Article Snippet: Anti-TYRP1 antibody (clone TA99) conjugated to Alexa Fluor 647 (Cat# NBP2-34720AF647) and mouse IgG2a Kappa isotype control conjugated to Alexa Fluor 647 (Cat# NBP1-96981AF647) were purchased from Novus Biologicals (Centennial, CO) and used at a concentration of 5 μL per 10 6 cells.

Techniques: Expressing, Comparison, RNA Sequencing

Journal: Molecular Therapy Oncology

Article Title: TYRP1 directed CAR T cells control tumor progression in preclinical melanoma models

doi: 10.1016/j.omton.2024.200862

Figure Lengend Snippet: IHC for TYRP1 on mouse normal tissues Representative tissues from whole-mouse necropsy were stained for TYRP1. Representative images from positive tissues are shown. Cells staining brown are immunoreactive against the anti-TYRP1 antibody EPR13063. Cells were additionally stained with hematoxylin. Scale bars represent 20 μm.

Article Snippet: Anti-TYRP1 antibody (clone TA99) conjugated to Alexa Fluor 647 (Cat# NBP2-34720AF647) and mouse IgG2a Kappa isotype control conjugated to Alexa Fluor 647 (Cat# NBP1-96981AF647) were purchased from Novus Biologicals (Centennial, CO) and used at a concentration of 5 μL per 10 6 cells.

Techniques: Staining

Journal: Molecular Therapy Oncology

Article Title: TYRP1 directed CAR T cells control tumor progression in preclinical melanoma models

doi: 10.1016/j.omton.2024.200862

Figure Lengend Snippet: Human TA99-CAR T cells have efficacy against human melanomas in vitro (A) Schematic of TA99-CAR harboring a TYRP1 scFv binder based on the murine anti-TYRP1 TA99 antibody, CD28 transmembrane and costimulatory domain, and CD3zeta activation domain in an SFG gamma retroviral vector. (B) Human CAR T cells were co-cultured with TYRP1 + SK-MEL-19 and SK-MEL-188 melanoma cells expressing firefly luciferase at varying effector:T cell ratios and cytotoxicity was assessed using bioluminescence after 24 h. TA99-CAR T cells demonstrated superior antitumor efficacy compared with ovarian-targeted 4H11-CAR and anti-CD19 CAR T cells. (C) Human CAR T cells were co-cultured with CD19 + , TYRP1 − Nalm6 leukemia cells and TYRP1 low SK-MEL-28 human melanoma cells. TA99-CAR T cells showed minimal cytotoxicity compared with the CD1-CAR controls in Nalm6 cells, and no CAR T cells demonstrated cytotoxicity against the SK-MEL-28. For (B) and (C), ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.0005, ∗∗∗∗ p < 0.00005 by unpaired t test adjusted for multiple comparisons. NS = not significant. Means (SDs) are plotted. (D) Cytokine levels of TA99-CAR T cells cultured alone or with various CHO cell lines, demonstrating that CAR T cells only released cytokines in response to CHO cells expressing TYRP1. MT = mock transduced, TYRP1 tr = exogenous expression of truncated (membrane localized) TYRP1. (E) Cytokine levels of various CAR T cells co-cultured with TYRP1 + SK-MEL-19 melanoma cells, demonstrating that TA99-CAR T cells showed increased cytokine release compared with CAR T cells targeted to non-melanoma antigens.

Article Snippet: Anti-TYRP1 antibody (clone TA99) conjugated to Alexa Fluor 647 (Cat# NBP2-34720AF647) and mouse IgG2a Kappa isotype control conjugated to Alexa Fluor 647 (Cat# NBP1-96981AF647) were purchased from Novus Biologicals (Centennial, CO) and used at a concentration of 5 μL per 10 6 cells.

Techniques: In Vitro, Activation Assay, Retroviral, Plasmid Preparation, Cell Culture, Expressing, Luciferase, Membrane

Journal: Molecular Therapy Oncology

Article Title: TYRP1 directed CAR T cells control tumor progression in preclinical melanoma models

doi: 10.1016/j.omton.2024.200862

Figure Lengend Snippet: TA99-CAR T cells have efficacy against human melanomas in vivo (A) Schematic of in vivo experiments. Two million SK-MEL-19 melanoma cells were injected subcutaneously. After 10 days of engraftment, CAR T cells were injected (5 million, with the exception of 0.5 million for one cohort of TA99-CAR T cells). Tumor growth was assessed weekly using bioluminescence and caliper measurements. Image created with the assistance of BioRender.com . (B) Tumor growth of mice treated with various CAR T cells (note that one mouse in the 0.5M TA99 CAR T cell cohort died of unknown causes during week 2 and was excluded from analysis). ∗∗ p = 0.000561 at day 45 by unpaired t test. All mice who received human CAR T cells developed xenogeneic graft vs. host disease (xGvHD); mice were euthanized when clinically indicated per our IACUC protocol and analysis of the cohort was stopped when the first mouse developed xGvHD. (C) Quantitation of the bioluminescence signal from mice in (B). (D) TYRP1 was assessed by IHC in residual tumor tissue from the mice treated in Figures S4 B and S4C. Scale bars represent 50 μm. For (B) and (C), means (SEMs) are plotted.

Article Snippet: Anti-TYRP1 antibody (clone TA99) conjugated to Alexa Fluor 647 (Cat# NBP2-34720AF647) and mouse IgG2a Kappa isotype control conjugated to Alexa Fluor 647 (Cat# NBP1-96981AF647) were purchased from Novus Biologicals (Centennial, CO) and used at a concentration of 5 μL per 10 6 cells.

Techniques: In Vivo, Injection, Quantitation Assay

Journal: Molecular Therapy Oncology

Article Title: TYRP1 directed CAR T cells control tumor progression in preclinical melanoma models

doi: 10.1016/j.omton.2024.200862

Figure Lengend Snippet: Histological analysis of eye tissue from CAR T cell-treated mice (A–D) Hematoxylin-eosin staining of cross sections of eyes from mice treated with either TA99-CAR T cells (A and C) or off-target 4H11-CAR T cells (B and D) (from Figure S4 B) at various magnifications. Note that retinal separation was an artifact of tissue fixation. Scale bars represent 100 μm (A and B) or 20 μm (C and D). (E) IHC for TYRP1 on cross sections from mice treated with TA99-CAR T cells or off-target 4H11-CAR T cells, showing a loss of TYRP1 + cells in the uvea/choroid of mice treated with the TA99 CAR relative to the 4H11 controls. Scale bars represent 500 μm (left), 100 μm (center), and 20 μm (right).

Article Snippet: Anti-TYRP1 antibody (clone TA99) conjugated to Alexa Fluor 647 (Cat# NBP2-34720AF647) and mouse IgG2a Kappa isotype control conjugated to Alexa Fluor 647 (Cat# NBP1-96981AF647) were purchased from Novus Biologicals (Centennial, CO) and used at a concentration of 5 μL per 10 6 cells.

Techniques: Staining

Journal: Molecular Therapy Oncology

Article Title: TYRP1 directed CAR T cells control tumor progression in preclinical melanoma models

doi: 10.1016/j.omton.2024.200862

Figure Lengend Snippet: Loss of TYRP1+ cells in normal tissues of mic treated with TA99 CAR T cells Tissues from mice from cohorts shown in Figure S4 B were collected and stained for TYRP1 using IHC. We noted reduced levels of TYRP1 + cells in thymus, skin, and meninges in mice that received TA99 CAR T cells. Note that the 4H11 cohort are from the same source as images from Figure 3 , shown again for comparison. Scale bars represent 20 μm.

Article Snippet: Anti-TYRP1 antibody (clone TA99) conjugated to Alexa Fluor 647 (Cat# NBP2-34720AF647) and mouse IgG2a Kappa isotype control conjugated to Alexa Fluor 647 (Cat# NBP1-96981AF647) were purchased from Novus Biologicals (Centennial, CO) and used at a concentration of 5 μL per 10 6 cells.

Techniques: Staining, Comparison

Journal: eLife

Article Title: Expression of modified FcγRI enables myeloid cells to elicit robust tumor-specific cytotoxicity

doi: 10.7554/eLife.91999

Figure Lengend Snippet: scFv construct design and preparation process.

Article Snippet: For treatment of established melanoma, C57Bl/6 female mice bearing approximately 20 mm 2 B16F10 tumors were injected s.c. with 2x10 6 BMDC transfected with modified FcγRI and with i.p. injection 250 μg per mouse of TA99 antibody (BioXcell, Lebanon, NH, USA).

Techniques: Construct, Clone Assay, Plasmid Preparation, Ligation, Sequencing, Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing, Control

Journal: eLife

Article Title: Expression of modified FcγRI enables myeloid cells to elicit robust tumor-specific cytotoxicity

doi: 10.7554/eLife.91999

Figure Lengend Snippet: ( A ) Confocal microscopy images of RAW264.7 cells transfected with scFv-based chimeric receptors. ( B–C ) Confocal microscopy images ( B ) and mean expression percentages ( C ) of HEK293FT and DC2.4 cells 24 hr post-transfection with TA99 scFv (n=4). ( D–E ) Confocal microscopy images of HEK293FT and DC 2.4 cells 24 hr post-transfection with αCD19 scFv ( D ) and with CD8-transmembrane portion only ( E ). Results are from one representative experiment out of at least three performed. Statistical significance was calculated using non-parametric t test.

Article Snippet: For treatment of established melanoma, C57Bl/6 female mice bearing approximately 20 mm 2 B16F10 tumors were injected s.c. with 2x10 6 BMDC transfected with modified FcγRI and with i.p. injection 250 μg per mouse of TA99 antibody (BioXcell, Lebanon, NH, USA).

Techniques: Confocal Microscopy, Transfection, Expressing

Journal: eLife

Article Title: Expression of modified FcγRI enables myeloid cells to elicit robust tumor-specific cytotoxicity

doi: 10.7554/eLife.91999

Figure Lengend Snippet: ( A ) Confocal microscopy images of DC 2.4 cells 24 hr post-transfection with αCD19-scFv GFP plasmid. ( B ) Geometric mean of GFP-positive cells 24 hr post-transfection with different αCD19- and TA99- ScFv GFP constructs in DC2.4 (n=3). ( C ) Confocal microscopy images of HEK 293 FT and DC 2.4 cells 24 hr post-transfection with αCD19-variable light chain GFP plasmid. ( D ) Confocal microscopy images of HEK 293 FT and DC 2.4 cells 24 hr post-transfection with αCD19-variable heavy chain GFP plasmid. ( E ) Mean percentages of cells expressing scFv fragments 24 hr post-transfection (n=4). ( F ) Left: Illustration of mutated variable light chain. Right: Confocal microscopy images of HEK 293 FT and DC 2.4 cells 24 hr post-transfection with αCD19- mutated (linear) variable light chain. ( G ) Confocal microscopy images of HEK293FT and DC2.4 cells 24 hr post-transfection with 1/3 fragments of αCD19-variable light chain GFP plasmid. ( H ) Mean percentages of GFP-positive cells 24 hr post-transfection with different fragments of scFv-GFP in DC2.4 and RAW264.7 (n=4). Results are from one representative experiment out of at least three performed. Statistical significance was calculated using non-parametric t test (** denote p<0.01, *** denote p<0.001, **** denote p<0.0001).

Article Snippet: For treatment of established melanoma, C57Bl/6 female mice bearing approximately 20 mm 2 B16F10 tumors were injected s.c. with 2x10 6 BMDC transfected with modified FcγRI and with i.p. injection 250 μg per mouse of TA99 antibody (BioXcell, Lebanon, NH, USA).

Techniques: Confocal Microscopy, Transfection, Plasmid Preparation, Construct, Expressing

Journal: eLife

Article Title: Expression of modified FcγRI enables myeloid cells to elicit robust tumor-specific cytotoxicity

doi: 10.7554/eLife.91999

Figure Lengend Snippet: ( A ) Confocal microscopy imaging of RAW 264.7 24 hr post-transfection with linear mRNA vectors translating to GFP and αCD19-scFv GFP. ( B ) qPCR data showing relative mRNA levels in RAW 264.7 transfected with GFP, Fc receptor-GFP, and αCD19-scFv GFP (n=4). ( C ) Upper: Illustration of plasmid subunits. Lower: Confocal microscopy imaging of HEK293FT and RAW 264.7 24 hr post-transfection with T2A ribosomal skipping plasmid including ScFv. ( D ) Upper: Illustration of plasmid subunits. Lower: Confocal microscopy imaging of HEK293FT and RAW 264.7 24 hr post-transfection with plasmid not containing T2A. ( E ) Confocal microscopy images of RAW264.7 cells at 6 hr and 24 hr post-transfection with αCD19-ScFv GFP plasmid. ( F ) Volcano plot showing differentially expressed proteins αCD19 ScFv GFP and GFP in DC 2.4 cells. ( G ) Confocal microscopy images of DC 2.4 stained with an ER stain, 24 hr post-transfection with GFP, membranous TA99-ScFv GFP. ( H ) Confocal microscopy images of DC 2.4 stained for BiP 24 hr post-transfection. ( I ) Mean levels of phospho-JNK 6 hours following transfection (n=4). ( J ) Percentage of cells expressing GFP or TRP-TCR1 24 hr following transfection (n=3). Results are from one representative experiment out of at least three performed. Statistical significance was calculated using non-parametric t-test (**** denote p<0.0001). Figure 4—source data 1. scFv fragments induce ER stress in myeloid cells. (A) qPCR data showing mRNA levels in RAW 264.7 transfected with GFP, Fc receptor-GFP, and αCD19-ScFv GFP (n=4). (B) LC-MS data results showing protein complexes pulled down using GFP magnetic beads in DC 2.4 6 hr post transfection with αCD19 ScFv GFP and GFP. (C) Flow cytometry data of CFSE dilution in CD8 + and CD4 + T cells following co-culture with ova conjugated scFv.

Article Snippet: For treatment of established melanoma, C57Bl/6 female mice bearing approximately 20 mm 2 B16F10 tumors were injected s.c. with 2x10 6 BMDC transfected with modified FcγRI and with i.p. injection 250 μg per mouse of TA99 antibody (BioXcell, Lebanon, NH, USA).

Techniques: Confocal Microscopy, Imaging, Transfection, Plasmid Preparation, Staining, Expressing, Liquid Chromatography with Mass Spectroscopy, Magnetic Beads, Flow Cytometry, Co-Culture Assay

Journal: eLife

Article Title: Expression of modified FcγRI enables myeloid cells to elicit robust tumor-specific cytotoxicity

doi: 10.7554/eLife.91999

Figure Lengend Snippet:

Article Snippet: For treatment of established melanoma, C57Bl/6 female mice bearing approximately 20 mm 2 B16F10 tumors were injected s.c. with 2x10 6 BMDC transfected with modified FcγRI and with i.p. injection 250 μg per mouse of TA99 antibody (BioXcell, Lebanon, NH, USA).

Techniques: Transfection, Construct, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay