Fig 1: 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 TYRP1low 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.
Fig 2: 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 S4B and S4C. Scale bars represent 50 μm. For (B) and (C), means (SEMs) are plotted.
Fig 3: 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.
Fig 4: IHC for TYRP1 on mouse normal tissuesRepresentative 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.
Fig 5: 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 log2 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 (log2 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 (log2 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 log2 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.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for TYRP1 Antibody (TA99) [Alexa Fluor® 647]
Available conjugates: Alexa Fluor 647