Zhang T, Barber A, Sentman CL. CAR T cells have robust cellular cytotoxicity and IFN- secretion when co-cultured with B7H6+ tumor cells, and they exhibit little self-reactivity to immature dendritic cells (iDCs) or pro-inflammatory monocytes. mRNA is not expressed in 48 normal tissues under steady state conditions.11 This suggests that NKp30-based CARs may target multiple tumor types. However, BAG-6, another ligand for NKp30, is expressed on iDCs and can trigger NK cell killing of iDCs,14, 15 and NKp30-based CARs mediated self-reactivity against PBMCs and iDCs.8 A potential approach to overcome the self-reactivity of NKp30-based CARs to PBMCs and iDCs is to create CARs targeting B7H6. In this study, we show that B7H6-specific CAR T cells mediate robust and activity against B7H6 expressing tumor cells with little activity against PBMCs or iDCs. Thus, a B7H6-specific CAR T cell therapy may be beneficial for a variety of patients with hematologic or solid tumors. RESULTS Construction and expression of B7H6-specific CARs and NKp30-based CARs To generate a CAR specific to B7H6 but not other NKp30 ligands, a single chain variable fragment from an anti-B7H6 mAb (47.39) was constructed by linking heavy chain variable region and light chain variable region having a (Glycine4Serine3) linker. This anti-B7H6 scFv was fused with human being CD28 hinge (H), transmembrane (TM), and cytoplasmic (CYP) domains, followed by a human being CD3 CYP website to create a B7H6-specific CAR (anti-B7H6 CAR) (Number 1a). Wild Cd24a type (WT) NKp30 and a NKp30-centered CAR (NKp30 CAR) were used for assessment with the anti-B7H6 CAR.8 T cells communicate WT NKp30 poorly and no specific activity is expected from this CAR, so WT NKp30 transduced T cells were used like a transduction control. The NKp30 CAR consists of human being CD28 TM and CYP domains between the NKp30 extracellular (EC) and CD3 CYP domains (Number 1a). These CARs can be indicated efficiently within the T cell surface and confer main and CD28 costimulatory signals through CD3 CYP and CD28 CYP domains upon CAR binding to its ligand.8 In order to assess anti-B7H6 CAR expression and to facilitate sorting of CAR+ T cells, a retroviral vector with the anti-B7H6 CAR, a furin cleavage site containing T2A sequence, and a truncated human being CD19 gene was also constructed (Number 1a). Surface manifestation of anti-B7H6 CARs on transduced human being T cells were analyzed by circulation cytometry after staining T cells with soluble B7H6 or by using CD19 expression like a surrogate marker of the CAR expression (Number 1b). Although there is potential for donor to donor variability in CAR manifestation, the manifestation of anti-B7H6 CAR on T cells from different human being PBMC donors showed very similar patterns of manifestation (Number 1c). NKp30 CAR and anti-B7H6 CARs can be indicated efficiently on human being T cells, whereas WT NKp30 communicate poorly on T cells (Number 1b), as previously shown.8 Open in a separate window Number 1 Design and expression of NKp30-based CAR (NKp30 CAR) and B7H6-specific CARs (anti-B7H6 CARs)(a) WT NKp30 is the full length wild-type Bergaptol NKp30 gene. A NKp30 CAR was Bergaptol created by fusing NKp30 extracellular (EC) website with human being CD28 transmembrane (TM), and cytoplasmic (CYP) domains, followed by a human being CD3 CYP website. A B7H6-specific CAR was created by fusing anti-B7H6 scFv DNA with the human being CD28 hinge (H), transmembrane (TM), and cytoplasmic (CYP) domains, followed Bergaptol by a human being CD3 CYP website DNA. The anti-B7H6 CAR-T2A-tCD19 create was created by combining the anti-B7H6 CAR DNA having a T2A sequence comprising a furin cleavage site and a truncated (t) human being CD19 DNA sequence. (b) Human being PBMCs were transduced with WT NKp30, NKp30 CAR, or anti-B7H6 CAR-T2A-tCD19 constructs. Transduced T cells were stained with anti-CD4 mAbs, anti-NKp30 mAbs, soluble B7H6 (sB7H6), and/or anti-CD19 mAbs. CD4- T cells are CD8+ T cells. The data are representative of data from 3 different human being donors. (c) Anti-B7H6 CAR manifestation on T cells from different PBMC donors were analyzed. The ideals in the graph represent the mean fluorescent intensities of CD19 expression for each sample. (d) RMA/B7H6, B16F10/B7H6, and ID8/B7H6 were stained with anti-B7H6 mAbs followed by goat anti-msIgG Abdominal muscles (open histograms) or with goat anti-msIgG Abdominal muscles only (stuffed histograms). Data demonstrated are representative of 3 self-employed experiments. B7H6 manifestation on tumor cells To identify the potential focuses on for anti-B7H6 CAR T cell therapy, we screened a panel of human being tumor cell lines for B7H6 manifestation.8 B7H6 expression was found in several hematological malignancy cell lines, including lymphoma, leukemia, and multiple myeloma and in several solid tumor cell lines, including melanoma, breast cancer, and pancreatic cancer cell lines. A broader survey of B7H6 manifestation reported that it was found on.