Stereoselective Construction of 2,6-<i>cis</i>-Disubstituted Tetrahydropyrans via Intramolecular Amide Enolate Alkylation: Total Synthesis of (−)-Centrolobine

A highly stereoselective construction of 2,6-<i>cis</i>-disubstituted tetrahydropyrans was achieved by using an intramolecular amide enolate alkylation with KHMDS. The efficiency and practicality of this methodology was successfully demonstrated in the total synthesis of (−)-centrolobine (<b>1</b>)

Folate receptor 1 (FOLR1) targeted chimeric antigen receptor (CAR) T cells for the treatment of gastric cancer

<div><p>Gastric cancer is a malignancy that has a high mortality rate. Although progress has been made in the treatment of gastric cancer, many patients experience cancer recurrence and metastasis. Folate receptor 1 (FOLR1) is overexpressed on the cell surface in over one-third of gastric cancer patients, but rarely is expressed in normal tissue. This makes FOLR1 a potential target for chimeric antigen receptor (CAR) T cell immunotherapy, although the function of FOLR1 has not been elucidated. CAR are engineered fusion receptor composed of an antigen recognition region and signaling domains. T cells expressing CAR have specific activation and cytotoxic effects against cancer cells containing the target antigen. In this study, we generated a CAR that targets FOLR1 composed of a single-chain variable fragment (scFv) of FOLR1 antibody and signaling domains consisting of CD28 and CD3ζ. Both FOLR1-CAR KHYG-1, a natural killer cell line, and FOLR1-CAR T cells recognized FOLR1-positive gastric cancer cells in a MHC-independent manner and induced secretion of various cytokines and caused cell death. Conclusively, this is the first study to demonstrate that CAR KHYG-1/T cells targeting FOLR1 are effective against FOLR1-positive gastric cancer cells.</p></div

Specific activity of FOLR1-CAR T cells via ZAP70 signaling pathway is similar to the TCR reaction and KB cell death is caused by granzyme B.

<p>To investigate FOLR1-CAR T cell activation and KB cell death signaling, mock or FOLR1-CAR T cells were co-cultured with KB cells at an E/T ratio of 10:1 for 4 h. (A) ZAP70, AKT, JNK, p38, and ERK associated with TCR signaling were measured by western blot analysis in mock or FOLR1-CAR T cells when incubated alone or with KB cells. The graphs presented ratio of phosphorylation by quantification of the western blot bands from three independent experiments. The data are represented as the mean ratio of densitometry values ± SD. Statistical analysis was performed using the two-sided unpaired t-test (*P<0.05, *P<0.01, and ***P<0.001). (B) Pro-apoptotic proteins PARP, cleaved caspase 8, caspase 9, caspase 3, and tBid were measured in KB cells by western blot analysis. Experiments were repeated three times with similar results.</p

Antitumor efficacy of FOLR1-CAR KHYG cells against xenografts derived from FOLR1-positive MKN1 cells in mice.

<p>Ten NOD-SCID IL2R γ null (NSG) mice were subcutaneously implanted with MKN1 cells for approximately 14 days until tumor volume reached approximately 100 mm³ and then randomly divided into two groups (n = 5). The mice were injected intratumorally with either mock or FOLR1-CAR KHYG-1 cells on days 0 and 7. Body weight (A) and tumor size (B) were measured simultaneously at intervals of 2 to 4 days. Line graphs represented an individual animal. Experiments were repeated two times with similar results. The data are represented as the mean body weight ± SD (g) and the mean tumor size ± SD (mm³). Statistical analysis was performed using the two-sided unpaired t-test (*P<0.05).</p

Specific activity of FOLR1-CAR Jurkat cells against FOLR1-positive GC cells.

<p>FACS was used to measure the surface expression of FOLR1 in a series of human GC cell lines, including MKN1, MKN7, SNU216, SNU484, SNU601, SNU638, SNU668, and GCIY. GC cell lines were separated into (A) FOLR1-positive GC cell lines (MKN1, MKN45, SNU484, and GCIY) and (B) FOLR1-negative GC cell lines (SNU216, SNU601, SNU638 and SNU668) based on the FACS results. (C) GC cell lines were co-cultured with FOLR1-CAR Jurkat cells at E/T ration of 10:1 for 24 h. After incubation, the levels of IL-2 were measured by ELISA. Experiments were repeated at least three times with similar results. The data are represented as the mean cytokine concentrations ± SD (pg/ml) from triplicate cultures. Statistical analysis was performed using the two-sided unpaired t-test and data were compared to FOLR1-CAR Jurkat cells cultured alone (***P<0.001).</p

Analysis of pro-apoptotic proteins in GC cells co-cultured with T cells.

<p>T cells were co-cultured with FOLR1-positive GC cells (MKN1, MKN7, SNU484, and GCIY) or FOLR1-negative GC cells (SNU216, SNU601, SNU638, and SNU668) at an E/T ratio of 10:1 for 4 h. Pro-apoptotic proteins PARP, cleaved caspase 8, caspase 9, caspase 3, and tBid in FOLR1-positive or -negative GC cells were measured by western blot analysis. Experiments were repeated three times with similar results.</p

Schematic representation of the FOLR1-CAR construct and the specific activity against target cells expressing FOLR1.

<p>(A) A FOLR1-CAR gene consisting of a FOLR1-specific scFv, CD28 and CD3ζ was constructed. (B) The FOLR1-CAR vector was transfected into Jurkat cells, and the Myc tagged FOLR1 gene was transfected into K562 cells. FOLR1-CAR and FOLR1-Myc tag vectors were measured by western blot using CD3ζ and myc antibody, respectively. (C) FOLR1-CAR Jurkat cells were co-cultured with transfected K562 cells at an effector-target (E/T) ratio of 10:1 for 24 h. The levels of IL-2 were measured by enzyme-linked immunosorbent assay (ELISA). (D−E) The specific activity of Jurkat cells transfected with the FOLR-CAR vector was analyzed by co-culture with FOLR1-positive KB cells. (D) Expression of FOLR1 in KB cells was measured using FACS. (E) The levels of IL-2 secreted by FOLR1-CAR Jurkat cells were measured by ELISA after co-cultured with KB cells at an E/T ratio of 10:1 for 24 h. Experiments were repeated at least three times with similar results. The data are represented as the mean cytokine concentrations ± SD (pg/ml) from triplicate cultures. Statistical analysis was performed using the two-sided unpaired Student’s t test (***P<0.001).</p

Generation and characterization of FOLR1-CAR T cells.

<p>T cells were obtained from human PBMCs of three healthy donors and the FOLR1-CAR gene was cloned into the pLVX-IRES-GFP vector and the lentivirus infection efficiency was determined. (A) The growth rate of infected or untransduced T cells was measured by CellTiter-Glo assay for 12 days. (B) Lentivirus infection rate was analyzed by GFP fluorescence on day 12 using FACS analysis. (C) Expression of FOLR1-CAR in T cells was measured by western blot analysis. (D-E) The population of T cells was evaluated by FACS analysis using CD3, CD4, and CD8 antibodies on day 12. Experiments were repeated three times with similar results. The data are represented as the mean of luminescence ± SD and positive rate ± SD (%) from triplicate cultures. Statistical analysis was performed using the two-sided unpaired t-test.</p

Specific cytotoxicity of FOLR1-CAR KHYG-1 cells against FOLR1-positive cells.

<p>KHYG-1 cells were infected with lentivirus encoding the FOLR1-CAR or mock vector and were selected with puromycin for 2 weeks. (A) FOLR1-CAR expression was measured by western blot using the CD3ζ antibody in KHYG-1 cells. (B) The cytotoxic effects of KHYG-1 cells on K562 cells transfected with FOLR1 or mock vector were measured by luciferase assay at an E/T ratio of 10:1 for 4 h. (C) The lysis percentages of mock KHYG-1 and FOLR1-CAR KHYG-1 cells against KB cells were determined in a dose-dependent manner for 4 h using the luciferase assay. (D−E) To determine the lysis percentages of FOLR1-CAR KHYG-1 cells against GC cells, mock KHYG-1 and FOLR1-CAR KHYG-1 cells were co-cultured with (D) FOLR1-positive GC cells or (E) FOLR1-negative GC cells at an indicated E/T ratio for 4 h. Experiments were repeated at least three times with similar results. The data are represented as the mean rate of lysis ± SD (%) from triplicate cultures. Statistical analysis was performed using the two-sided unpaired t-test (***P<0.001).</p

Specific activity of FOLR1-CAR T cells against KB cells and analysis of T cell-mediated cytotoxic factors.

<p>Mock or FOLR1-CAR T cells were co-cultured with KB cells at an E/T ratio of 10:1 for 4 h. (A) The levels of cytokines IFN-γ, TNF-α, GM-CSF, and granzyme B were measured by ELISA and (B) the cytotoxic effects were evaluated by luciferase assay. (C) To determine the factors that induced cell death, FOLR1-CAR T cells were co-cultured with KB cells in the presence of 100 μM of Z-AAD-CMK, 0.1 μg/ml of anti-FasL antibody, and anti-TNF-a antibody. Experiments were repeated at least three times with similar results. The data are represented as the mean cytokine concentrations ± SD (pg/ml) and rate of lysis ± SD (%) from triplicate cultures. Statistical analysis was performed using the two-sided unpaired t-test (***P<0.001).</p