Figure 1 Murine and human multipotent BMSCs express FAP and are recognized by T cells expressing FAP-reactive CARs.

Passage-5 in vitro–expanded murine BMSCs were stained with antibodies specific for Sca-1, PDGFR-α, and FAp< and assessed by flow cytometry (A). "Q" represents quadrant. Solid lines are isotype controls and filled histograms are FAP stained. UnTd or FAP5-CAR Td T cells were cultured overnight with murine BMSCs and supernatants were assessed for IFN-γ by ELISA (B), and cells were further analyzed for expression of CD107a and production of IFN-γ and TNF by ICS (C). Mean ± SD. Data are representative of two independent experiments. Flow cytometric phenotype of in vitro–expanded human BMSCs derived from three different donors (D). BMSCs from D were stained with the FAP-specific monoclonal antibodies Sibrotuzumab (E) and FAP5 (F) and assessed by flow cytometry. Solid lines are isotype or secondary antibody controls and filled histograms are FAP or Sibrotuzumab stained. UnTd or Sibro-CAR Td T cells were cultured overnight with BMSCs and the supernatants assessed for IFN-γ by ELISA (G), and cells were further analyzed for CD107a expression and IFN-γ and TNF production by ICS (H). Mean ± SD. Similar results were seen with two additional T cell donors.

Tran, E., Chinnasamy, D., Yu, Z., Morgan, R. A., Lee, C. C. R., Restifo, N. P., & Rosenberg, S. A. (2013). Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia. Journal of Experimental Medicine, jem-20130110.

Figure 2 IHC staining for FAP in various human tumors, and design and in vitro activity of FAP-reactive CARs.

Representative IHC staining for FAP in human melanoma (A), colorectal (B), pancreatic (C), and breast (D) adenocarcinomas. Isotype stains were negative (not depicted). Bars: 400 µm (A); 200 µm (B–D). Schematic of the FAP-reactive CAR constructs FAP5-CAR (E) and Sibro-CAR (F). LS, GM-CSFR leader sequence; VH and VL, variable heavy and light chains; L, 218 linker; CD8, transmembrane domain; CD28, 4-1BB, and CD3-ζ, intracellular signaling domains; m, murine; h, human. Both constructs were cloned into the MSGV1 retroviral vector. Retrovirus containing FAP5-CAR or Sibro-CAR constructs were generated and used to transduce mouse and human T cells, respectively, and flow cytometry was used to assess transduction efficiency at day 2 after transduction for FAP5-CAR (G) and day 8–10 after transduction for Sibro-CAR (H). Solid line is isotype control and filled histogram is FAP5 or Sibrotuzumab stained. Day 5-stimulated untransduced (UnTd) and FAP5-CAR–transduced (Td) mouse T cells were assessed for reactivity against plate-bound BSA, α-CD3 mAb, and recombinant human FAP (r-huFAP), and against HEK293 cell lines expressing or not expressing FAP. After an overnight stimulation, supernatants were assessed for IFN-γ with an IFN-γ ELISA (I), and cells were further assessed for cell surface CD107a expression, and production of IFN-γ and TNF by ICS (J). For ICS, cells are gated on FAP5-CAR Td cells. Day ∼14-stimulated UnTd or Sibro-CAR Td human T cells were assessed for in vitro reactivity as described for mouse. IFN-γ ELISA (K), and ICS results gated on Sibro-CAR Td T cells (L) are shown. Mean ± SD. All results are representative of at least three independent experiments.

Tran, E., Chinnasamy, D., Yu, Z., Morgan, R. A., Lee, C. C. R., Restifo, N. P., & Rosenberg, S. A. (2013). Immune targeting of fibroblast activation protein triggers recognition of multipotent bone marrow stromal cells and cachexia. Journal of Experimental Medicine, jem-20130110.

Figure 1 Anti-Human FAP Antibody (TAB-211) in ELISA

ELISA analysis of TAB-211 was performed by coating with human FAP antigen.