Journal: Cell Reports Medicine

Article Title: Preclinical development of a chimeric antigen receptor T cell therapy targeting FGFR4 in rhabdomyosarcoma

doi: 10.1016/j.xcrm.2023.101212

Figure Lengend Snippet: FGFR4 is highly expressed in RMS and other cancers, with low expression in healthy tissue (A) High expression of FGFR4 mRNA is found in both FP-RMS and FN-RMS tumors and cell lines compared with other pediatric cancers and healthy tissues. Expression levels measured as FPKM (fragments per kilobase of transcript per million mapped reads) for FGFR4 are summarized in violin plots with medians and quartiles. ASPS, alveolar soft part sarcoma; CCSK, clear cell sarcoma of the kidney; DSRCT, desmoplastic small round cell tumor; EWS, Ewing sarcoma; HBL, hepatoblastoma; ML, melanoma; NB, neuroblastoma; OS, osteosarcoma; SS, synovial sarcoma; UDS, undifferentiated sarcoma; WT, Wilms tumor; YST, yolk sac tumor). (B) FGFR4 mRNA expression in TCGA data shows highest expression in liver hepatocellular carcinoma (LIHC), cholangiocarcinoma (CHOL), and individual tumors of other types. Abbreviations are as per TCGA ( https://gdc.cancer.gov/resources-tcga-users/tcga-code-tables/tcga-study-abbreviations ). (C) Representative images of immunohistochemistry (IHC) for FGFR4 show high expression in RMS, with minimum or no expression in healthy organs. H score displayed in bottom right corner, and scale bars, 200 μm. (D) Summary of membrane-staining H score of FGFR4 IHC of RMS and healthy tissues. Values represent mean ± SEM (error bars). (E) Representative flow cytometry plots show differential levels of FGFR4 expression on FP-RMS or FN-RMS cell lines. Mean fluorescence intensity (MFI) of FGFR4 on indicated RMS cells is listed in the right table, stained with phycoerythrin (PE)-conjugated anti-human FGFR4 antibody or mouse IgG1 isotype control.

Article Snippet: Recombinant mouse FGFR4 , Sino Biological , 50194-M08H.

Techniques: Expressing, Wilms Tumor Assay, Immunohistochemistry, Membrane, Staining, Flow Cytometry, Fluorescence

Journal: Cell Reports Medicine

Article Title: Preclinical development of a chimeric antigen receptor T cell therapy targeting FGFR4 in rhabdomyosarcoma

doi: 10.1016/j.xcrm.2023.101212

Figure Lengend Snippet: PAX3-FOXO1 establishes a super-enhancer at the FGFR4 locus, and RMSs are dependent on FGFR4 for survival (A) PAX3-FOXO1 (top) and H3K27ac (bottom) ChIP-seq at the FGFR4 locus in FP-RMS cell lines and tumors (orange), FN-RMS cell lines and tumors (blue), and human skeletal muscle cell lines and tissues (gold). (B) Top: FGFR4 expression is induced in fibroblasts after introduction of PAX3-FOXO1. Bottom: ChIP-seq demonstrates that PAX3-FOXO1 protein opens chromatin and establishes a super-enhancer at the FGFR4 locus. Open chromatin was assayed by DNase hypersensitivity; binding of PAX3-FOXO1 and BRD4 as well as chromatin H3K27ac status were assayed by ChIP-seq in human fibroblasts with or without PAX3-FOXO1. (C) Average dependency score of RMS for FGFR4 was found to be the lowest among all human cancers, suggesting the highest dependency of RMS on this receptor for survival.

Article Snippet: Recombinant mouse FGFR4 , Sino Biological , 50194-M08H.

Techniques: ChIP-sequencing, Expressing, Binding Assay

Journal: Cell Reports Medicine

Article Title: Preclinical development of a chimeric antigen receptor T cell therapy targeting FGFR4 in rhabdomyosarcoma

doi: 10.1016/j.xcrm.2023.101212

Figure Lengend Snippet: Development and characterization of a specific FGFR4 binder from the monoclonal murine antibody 3A11 (A) Structure of the mouse anti-FGFR4 mouse monoclonal antibody (mAb) 3A11. (B) Representative flow cytometry plots show the specificity of 3A11 mAb by staining with FGFR4 + cell lines, FGFR4 KO RH30, or FGFR4 − cell 7250. Mouse IgG (msIgG) is used as isotype control for this mouse antibody 3A11. (C) Structure of anti-FGFR4 antibody 3A11 in scFvFc format fused to the human IgG1 Fc region. (D) Representative flow cytometry plots show 3A11 scFvFc chimeric antibody specifically binds to FGFR4 + cell lines RH30, RH4, and RMS559 but not to the RH30 FGFR4-KO, the RH4 FGFR4-KO, or fibroblast 7250 lines. Human IgG (huIgG) as an isotype control for 3A11 scFvFc. MFI fold change shown as orange font calculated by formula [ M F I ( s c F v F c ) − M F I ( h u I g G i s o t y p e ) ] / M F I ( h u I g G i s o t y p e ) . (E) Binding avidity of FGFR4 scFvFc, using 2-to-1 binding model and global fitting analysis, demonstrates the dissociation constant (K D ) of 3A11 scFvFc against FGFR4 ECD is 4.17 nM. (F) ELISA shows 3A11 scFvFc only recognizes human FGFR4 but not human FGFR1–3 or mouse FGFR4. (G) Flow cytometry using 3A11 scFvFc shows FGFR4 is expressed in several RMS cell lines at various levels with higher expression in FP-RMS compared with in FN-RMS. MFI of 3A11 scFvFc or isotype control huIgG staining on above cells is shown in the table on the right.

Article Snippet: Recombinant mouse FGFR4 , Sino Biological , 50194-M08H.

Techniques: Flow Cytometry, Staining, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cell Reports Medicine

Article Title: Preclinical development of a chimeric antigen receptor T cell therapy targeting FGFR4 in rhabdomyosarcoma

doi: 10.1016/j.xcrm.2023.101212

Figure Lengend Snippet: Clinical-grade 3A11 CAR T cells show specific cytotoxicity to FGFR4 + cells (A) Schematic of 3A11 CAR construct targeting FGFR4. HTM, hinge and transmembrane domain; hu tEGFR, human truncated EGFR. (B) Cytotoxicity assays of 3A11 CAR T cells show potent killing activity toward target RMS cells at an E:T ratio of 0.75:1 in a xCELLigence Real-Time Cell Analysis (RTCA). Vertical black arrows show the time point for adding CAR T cells into a plate seeded with target cells. Representative of n = 3 independent experiments with n = 3 individual donors for (B)–(D). Values represent mean ± SD (standard deviation, error bars). (C) Cytotoxic assay shows 3A11 CAR does not cause cytolysis to FGFR4-KO or FGFR4 − cells at an E:T ratio of 0.75:1. Values represent mean ± SD (error bars). (D) Cytokine release assay shows 3A11 CAR T cells only release high level of IFN-γ when cocultured with FGFR4-expressing RMS cells (RH30, RH4, or RMS559) rather than the respective FGFR4-KO cell lines or 7250. Values represent mean ± SEM (error bars). Two-way ANOVA is used to compare secreted IFN-γ by mock T or 3A11 CAR T cells cocultured with the target cells by calculating the p value. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001; ns: no significant difference.

Article Snippet: Recombinant mouse FGFR4 , Sino Biological , 50194-M08H.

Techniques: Construct, Activity Assay, Standard Deviation, Release Assay, Expressing

Journal: Cell Reports Medicine

Article Title: Preclinical development of a chimeric antigen receptor T cell therapy targeting FGFR4 in rhabdomyosarcoma

doi: 10.1016/j.xcrm.2023.101212

Figure Lengend Snippet: Low or absence of cell surface FGFR4 in primary human cells does not induce cytokine release when cocultured with 3A11 CAR T cells (A) Representative flow cytometry plots show low or absence of FGFR4 cell surface expression on human cardiomyocytes, renal epithelial cells, renal cortical epithelial cells, renal proximal epithelial cells, HEK293, cholangiocytes, and hepatocytes pooled from 10 individuals using 3A11 scFvFc. MFI of 3A11 scFvFc or huIgG1 isotype control staining on these primary cells is shown in the top right table. Relative FGFR4 expression levels on these primary cells compared with RH30 cells are calculated as R e l a t i v e e x p r e s s i o n = [ M F I ( s c F v F c ) − M F I ( h u I g G i s o t y p e ) ] P r i m a r y c e l l [ M F I ( s c F v F c ) − M F I ( h u I g G i s o t y p e ) ] R H 30 . (B) Log 2 ratio of cytokine (IFN-γ, IL-2, and TNF-α) release in the supernatant, by 3A11 CAR T cells cocultured with primary cells, as indicated, in their respective media, compared with the RMS cell RH30. Cell line 7250 serves as a FGFR4 − control. Values represent n = 3 independent experiments with n = 3 individual donors. Values represent mean ± SEM (error bars).

Article Snippet: Recombinant mouse FGFR4 , Sino Biological , 50194-M08H.

Techniques: Flow Cytometry, Expressing, Staining

Journal: Cell Reports Medicine

Article Title: Preclinical development of a chimeric antigen receptor T cell therapy targeting FGFR4 in rhabdomyosarcoma

doi: 10.1016/j.xcrm.2023.101212

Figure Lengend Snippet:

Article Snippet: Recombinant mouse FGFR4 , Sino Biological , 50194-M08H.

Techniques: Purification, Recombinant, Labeling, Virus, Microarray, Cytokine Assay, Transfection, Chromatin Immunoprecipitation, ChIP-sequencing, Plasmid Preparation, Software, Imaging