Journal: Frontiers in Oncology

Article Title: Chimeric antigen receptor-T cells are effective against CEACAM5 expressing non-small cell lung cancer cells resistant to antibody-drug conjugates

doi: 10.3389/fonc.2023.1124039

Figure Lengend Snippet: CEACAM5 expressions in different normal/tumor tissues and DM4 sensitivity in NSCLCs and PDACs. (A) CEACAM5 immunohistochemistry (IHC) of 14 tumor tissues and 14 correspondent normal tissues in tissue microarray (left panel) and quantification values of IHC images (right panel). (B) DM4 response curves in NSCLCs (H1975, A549, H1299, H2030, and H2009 cells) and PDACs (HPAC and HPAF-II cells). Normalized % cell viability (ATP level) was calculated by normalizing luminescence values using buffer (DMSO)-treated respective cells. Results are shown as the mean ±SD for representative data from three independent experiments.

Article Snippet: For immunohistochemistry, the tissue slide was blocked with 10% normal horse serum for 1 h at 25°C and incubated with mouse anti-human CEACAM5 antibody (Novus biologicals, NB11058734, 1:100) for overnight at 4°C in humidified chamber.

Techniques: Immunohistochemistry, Microarray

Journal: Frontiers in Oncology

Article Title: Chimeric antigen receptor-T cells are effective against CEACAM5 expressing non-small cell lung cancer cells resistant to antibody-drug conjugates

doi: 10.3389/fonc.2023.1124039

Figure Lengend Snippet: in vitro cell killing activities of the ADC SAR408701 analog and CAR-T cells targeting CEACAM5 in DM4 S cells. (A) Cell surface CEACAM5 expression levels in DM4 S NSCLCs (H1299, H1299-CEACAM5, A549, and A549-CEACAM5 cells) and PDACs (HPAC and HPAF-II cells). (B, D) Cell killing activities with ADC SAR408701 analog against DM4 S NSCLCs (H1299, H1299-CEACAM5, and A549, A549-CEACAM5 cells) (B) and DM4 S PDACs (HPAC, and HPAF-II cells) (D) . Normalized % cell viability (ATP level) was calculated by normalizing luminescence values using vehicle (buffer)-treated respective cells. The IC 50 were then determined by nonlinear regression plot of percent specific cytotoxicity versus Log10 concentration of ADC SAR408701 analog using GraphPad Prism software. (C, E) Cytotoxic activities (%) of anti-CEACAM5 CAR-T cells against DM4 S NSCLCs (C) and PDACs (E) . Significance was tested using one-way ANOVA, followed by the tukey’s multiple post hoc test. ****, P <0.0001; ***, P <0.001; **, P <0.01; *, P <0.05; versus control T at each E:T ratio. (B–E) Results are shown as the mean ±SD for representative data from three independent experiments.

Article Snippet: For immunohistochemistry, the tissue slide was blocked with 10% normal horse serum for 1 h at 25°C and incubated with mouse anti-human CEACAM5 antibody (Novus biologicals, NB11058734, 1:100) for overnight at 4°C in humidified chamber.

Techniques: In Vitro, Expressing, Concentration Assay, Software, Control

Journal: Frontiers in Oncology

Article Title: Chimeric antigen receptor-T cells are effective against CEACAM5 expressing non-small cell lung cancer cells resistant to antibody-drug conjugates

doi: 10.3389/fonc.2023.1124039

Figure Lengend Snippet: in vitro cell cytotoxicity assay of the ADC SAR408701 analog and CAR-T cells targeting CEACAM5 in DM4 R cells. (A) Cell surface CEACAM5 expression levels in DM4 R NSCLCs (H1975, H1975-CEACAM5, H2009, and H2009-CEACAM5 cells). (B) Cell killing activities with ADC SAR408701 analog against CEACAM5-positive DM4 R cell lines (H1975-CEACAM5 and H2009-CEACAM5 cells) and CEACAM5-negative DM4 R cell lines (H1975 and H2009 cells). Normalized % cell viability (ATP level) was calculated by normalizing luminescence values for vehicle (buffer)-treated respective cells. (C) Cytotoxic activities (%) of anti-CEACAM5 CAR-T cells against CEACAM5-positive and CEACAM5-negative DM4 R NSCLC cells. Significance was tested using one-way ANOVA, followed by the tukey’s multiple post hoc test. ****, P <0.0001; ***, P <0.001; versus control T at each E:T ratio. (B, C) Results are shown as the mean ±SD for representative data from three independent experiments.

Article Snippet: For immunohistochemistry, the tissue slide was blocked with 10% normal horse serum for 1 h at 25°C and incubated with mouse anti-human CEACAM5 antibody (Novus biologicals, NB11058734, 1:100) for overnight at 4°C in humidified chamber.

Techniques: In Vitro, Cytotoxicity Assay, Expressing, Control

Journal: Frontiers in Oncology

Article Title: Chimeric antigen receptor-T cells are effective against CEACAM5 expressing non-small cell lung cancer cells resistant to antibody-drug conjugates

doi: 10.3389/fonc.2023.1124039

Figure Lengend Snippet: in vivo anti-tumor activities of the ADC SAR408701 analog and CAR-T cells targeting CEACAM5 in DM4 S and DM4 R NSCLC tumors. (A) Schematic representation of experimental design and treatment schedule for mice studies. (B, D, F) Tumor growth curve (left panels) and individual mice curves (right panels) of DM4 S A549-CEACAM5 (B) , DM4 R H1975-CEACAM5 (D) , and CEACAM5-negative DM4 S A549 (F) tumors. Significance was analyzed by comparing the tumor volume (mm 3 ) ±SD at endpoint of 1.0 cm 3 (B) or end day ( D, F ) and determined using one-way ANOVA, followed by the tukey’s multiple post hoc test. ****, P <0.0001; ***, P <0.001. (C, E, G) Survival curve showing the efficacy of the ADC SAR408701 analog and CAR-T cells in NSG mice. Tumor volume (mm 3 ) are shown as mean ±SD for n=6 or 7 per group. Survival was presented by Kaplan-Meier plot of percentage of mice with tumor volume ≥ 1-1.5 cm 3 . Significance was determined by log-rank (Mantel-Cox) test. ***, P <0.001; **, P <0.01.

Article Snippet: For immunohistochemistry, the tissue slide was blocked with 10% normal horse serum for 1 h at 25°C and incubated with mouse anti-human CEACAM5 antibody (Novus biologicals, NB11058734, 1:100) for overnight at 4°C in humidified chamber.

Techniques: In Vivo

Journal: Journal of Hematology & Oncology

Article Title: Accurate control of dual-receptor-engineered T cell activity through a bifunctional anti-angiogenic peptide

doi: 10.1186/s13045-018-0591-7

Figure Lengend Snippet: Construction and identification of effector cells, target cells, and bifunctional molecules. a Characterization of CD4 + T cells or CD8 + T cells enriched with the magnetic microbeads by flow cytometry. With anti-CD4-PE and anti-CD8-APC antibodies, the sorting efficiency of CD4 + T cells and CD8 + T cells was determined to be 92.4 and 90.7%, respectively (shown on the upper). At the single-cell level, our flow cytometry data indicated the size and morphology of cells attached to antibodies (shown on the lower). (Ch02 bright channel, Ch03 PE fluorescent channel, Ch11 APC fluorescent channel) b sdCAR-engineered T cells were successfully constructed by electroporation. We evaluated the transduction efficiency by flow cytometry with endogenous BFP expression to quantify fractions of sdCAR-CD4 + T cells and sdCAR-CD8 + T cells. c To assess sdCAR-T cell activity, K562 cells were lentivirally transduced to stably express human tumor antigens MSLN or CEA, respectively. Western blotting results confirmed the expression of cognate antigen (MSLN) on MSLN + K562 cells (shown on the upper), non-cognate antigen (CEA) on CEA + K562 cells (shown on the middle), and cognate antigen (MSLN) on MSLN + HT29 cells (shown on the lower). We used glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control. d Western blot results showed that the integrin αvβ3 was highly expressed on the surface of K562 cells (shown on the left). In contrast, HT29 cells did not express integrin αvβ3 (shown on the right). GAPDH was used as an endogenous control. e Analysis of site-specific FHBM conjugates after isolation and purification. Chromatography of obtained FHBM demonstrated that the final product was of high purity (> 98%) and can be used to regulate CAR-T activity (shown on the left). Characteristic ion peaks from mass spectrometry, such as [M+4H] 4+ , [M+3H] 3+ , and [M+2H] 2+ , showed that the molecular weight of FHBM was approximately 2565 and the ratio of FITC to HM-3 in FHBM conjugate was about 1.97 (shown on the right)

Article Snippet: Expression of tumor antigens was detected with Western blot in which target proteins were probed with an anti-MSLN antibody (R&D Systems) or an anti-CEA antibody (R&D Systems).

Techniques: Flow Cytometry, Construct, Electroporation, Transduction, Expressing, Activity Assay, Stable Transfection, Western Blot, Control, Isolation, Purification, Chromatography, Mass Spectrometry, Molecular Weight

Journal: Journal of Hematology & Oncology

Article Title: Accurate control of dual-receptor-engineered T cell activity through a bifunctional anti-angiogenic peptide

doi: 10.1186/s13045-018-0591-7

Figure Lengend Snippet: Switch molecule-controlled activation of CD4 + T cells engineered with sdCAR. a We engineered primary human CD4 + T cells derived from fresh human peripheral blood with sdCARs by electroporation and evaluated the cells by activation assays with an “AND logic gate” strategy. b , c We quantified production of IL-2 and IFNγ by enzyme-linked immunosorbent assay. The cytokines were produced only when sdCAR-T cells were exposed to cognate tumor cells (MSLN + K562) in the presence of FHBM but not when exposed to non-cognate tumor cells (K562 or CEA + K562). For MSLN + HT29 cells, high levels of cytokines were found only in MζBB CAR-T cells. ( n = 3, error bars denote standard deviation.) d Monitoring T cell activation by CD69 expression. CD69 was expressed on sdCAR-T cells in the presence of both MSLN + K562 cells and a switch molecule (FHBM) and also was expressed on MζBB CAR-T cells. For MSLN + HT29 cells, only MζBB CAR-T cells had significant CD69 expression. ( n = 3, error bars denote standard deviation.) e – h FHBM and cognate tumor cell-dependent T cell proliferation. As sdCAR-T cells express a fluorescence reporter protein (BFP), we quantified the number of activated cells by flow cytometry after 3, 4, or 5 days of incubation. For cognate tumor cells (K562 or CEA + K562), sdCAR-T cells and MSLN-specific CAR-T cells had no significant proliferation. sdCAR-T cell proliferation was regulated by FHBM in the presence of MSLN + K562 cells. A similar degree of T cell proliferation was found in MζBB CAR-T cells. For HT29 tumor cells, only MSLN-specific CAR-T cells had strong proliferation. ( n = 3, error bars denote standard deviation)

Article Snippet: Expression of tumor antigens was detected with Western blot in which target proteins were probed with an anti-MSLN antibody (R&D Systems) or an anti-CEA antibody (R&D Systems).

Techniques: Activation Assay, Derivative Assay, Electroporation, Enzyme-linked Immunosorbent Assay, Produced, Standard Deviation, Expressing, Fluorescence, Flow Cytometry, Incubation

Journal: Journal of Hematology & Oncology

Article Title: Accurate control of dual-receptor-engineered T cell activity through a bifunctional anti-angiogenic peptide

doi: 10.1186/s13045-018-0591-7

Figure Lengend Snippet: sdCAR-engineered CD8 + T cells yield antigen-specific and titratable killing of cognate target cells in vitro. a sdCAR-engineered CD8 + T cells eradicated cognate target cells expressing MSLN antigen and integrin αvβ3 with an “AND logic gate” strategy. b Specific cytotoxicity of sdCAR-engineered T cells. sdCAR-T cells combined with FHBM have significant cytotoxicity for MSLN + K562 cells. c Representative flow cytometry data for cytotoxicity of engineered-CD8 + T cells. T cells were incubated with a mixture of cognate target cells (GFP + ) and non-cognate target cells (mCherry + ). After a 22-h incubation, we quantified the abundance of both surviving cognate and non-cognate tumor cells. The normalized percentage of surviving cognate target cells is expressed as the percentage of MSLN + K562 cells (Q1) divided by that of CEA + K562 cells (Q4). d Cytotoxicity mediated by sdCAR-CD8 + T cells in a 22-h experiment. The lower surviving percentage of MSLN + K562 cells indicated a significant degree of cognate target cell killing by sdCAR T cells only upon the addition of FHBM. ( n = 3, error bars denote standard deviation.) e Representative fluorescence images of target cells. After 22 h of interaction, we observed mixtures of cognate and non-cognate target cells incubated with engineered-CD8 + T cells by fluorescence microscopy. f Time course of cognate target cell killing by sdCAR-T cells. The sdCAR-T cell cytotoxicity was not monitored in the absence of switch molecules for the first 4 h (−). However, sdCAR-CD8 + T cells exerted high cytotoxicity for cognate target cells only in the presence of FHBM (+). ( n = 3, error bars denote standard deviation.) g Effect of the switch molecule dose on the cytolytic capacities of sdCAR-T cells. The level of cognate target cell killing was correlated with concentrations of FHBM. When the concentration of the switch molecule was increased to more than 100 pM, there was no further increase of T cell cytotoxicity. ( n = 3, error bars denote standard deviation)

Article Snippet: Expression of tumor antigens was detected with Western blot in which target proteins were probed with an anti-MSLN antibody (R&D Systems) or an anti-CEA antibody (R&D Systems).

Techniques: In Vitro, Expressing, Flow Cytometry, Incubation, Standard Deviation, Fluorescence, Microscopy, Concentration Assay

Journal: Journal of Hematology & Oncology

Article Title: Accurate control of dual-receptor-engineered T cell activity through a bifunctional anti-angiogenic peptide

doi: 10.1186/s13045-018-0591-7

Figure Lengend Snippet: In vivo cytotoxic effect of sdCAR-T cells toward the cognate tumor cells. a Schematic of the mouse treatment strategy used in the in vivo experiment. Matched MSLN + /CEA + K562 cells described previously were injected into nude mice intraperitoneally (i.p.). Effector cells, switch molecules, or PBS were injected i.p. at the indicated time point. After 48 h, both target cells were recovered from peritoneal lavage and quantified by flow cytometry. b Various concentrations of FHBM were added to mice. The degree of cytotoxicity of sdCAR-T cells together with saturated FHBM (0.5 mg/kg) matched the level observed with the MζBB CAR-T cells that triggered significant cytotoxicity for cognate tumor cells. c At the end of the experiment, we quantitatively analyzed the remaining cognate and non-cognate tumor cells by flow cytometry, respectively. sdCAR-T cells eliminated the cognate tumor cells in mice treated with FHBM, comparable to the cytotoxicity of MζBB CAR-T cells. d Ratios of surviving MSLN + K562:CEA + K562 in each sample. Cognate target cells were killed by sdCAR-T cells only in the presence of FHBM. We observed similar results for MζBB CAR-T cells. ( n = 5, error bars denote standard deviation.) e – h The sdCAR-T cells produced lower levels of cytokines. During the experiment, sdCAR-T cells released IL-2 (~ 1300 pg/mL), IFNγ (~ 30 ng/mL), IL-6 (~ 1160 ng/mL), and TNFα (~ 380 pg/mL) only in the presence of FHBM. MζBB CAR-T cells always released high levels of cytokines, including ~ 2600 pg/mL of IL-2, ~ 60 ng/mL of IFNγ, ~ 2216 ng/mL of IL-6, and ~ 647 pg/mL of TNFα. ( n = 5, error bars denote standard deviation)

Article Snippet: Expression of tumor antigens was detected with Western blot in which target proteins were probed with an anti-MSLN antibody (R&D Systems) or an anti-CEA antibody (R&D Systems).

Techniques: In Vivo, Injection, Flow Cytometry, Standard Deviation, Produced

Journal: The Journal of Clinical Investigation

Article Title: Functional monocytic myeloid-derived suppressor cells increase in blood but not airways and predict COVID-19 severity

doi: 10.1172/JCI144734

Figure Lengend Snippet: (A) Gating strategy to identify M-MDSCs by flow cytometry. From live, single CD45+ leukocytes, cells expressing lineage markers (CD3, CD19, CD20, CD56, CD66abce) and HLA-DR were excluded and CD14+ M-MDSCs identified. (B) M-MDSC frequency per live CD45+ cells in blood and NPAs. HCs (blue): n = 12 (blood), n = 7 (NPAs). Patients with influenza (open circles): n = 19 (blood), n = 9 (NPAs). COVID-19 patients (solid circles): n = 140 (blood), n = 28 (NPAs). The dots are color-coded according to peak disease severity. (C) Peak frequency of blood M-MDSCs per live CD45+ cells across disease severity. HCs (blue): n = 12. Patients with COVID-19 (color-coded by peak disease severity): mild, n = 19; moderate, n = 53; severe, n = 56; fatal, n = 12. (D) Blood M-MDSC frequencies over time in patients with COVID-19: mild, n = 17; moderate, n = 53; severe, n = 56; fatal, n = 12. Line shows the locally estimated scatterplot smoothing (LOESS) with shaded 95% CI (fatal group wide CI, not presented). (E) Frequency of blood M-MDSCs in paired acute and convalescent samples from patients with COVID-19 (n = 6). (F) M-MDSC frequency in blood, NPA, and ETA samples from patients with severe (red, n = 16) and fatal (gray, n = 4) COVID-19. (G–I) Surface expression of (G) CD62L, (H) CD86, and (I) CCR2 on M-MDSCs in blood, NPAs, and ETAs from HCs (blue, NPAs n = 7, PBMCs n = 11) and COVID-19 patients (black, NPAs n = 25, ETAs n = 19, PBMCs n = 69). (J) Frequency of PMN-MDSCs of live CD45+ cells in blood from patients with COVID-19. HCs: n = 12. Patients with COVID-19: mild, n = 11; moderate, n = 47; severe, n = 42; and fatal, n = 8. (K) Frequency of blood PMN-MDSCs in paired acute and convalescent samples from patients with COVID-19 (n = 6). (B, C, and F–J) Comparisons of M-MDSC frequencies were performed using the nonparametric Kruskal-Wallis test with Dunn’s post hoc multiple-comparison test. In the strip charts, group medians are presented as horizontal lines and individual patients as jitter points.

Article Snippet: If a sufficient number of cells were available, a second staining was performed using antibodies against CD3 (SP34-2; BD), CD4 (L200; BD), CD11c (B-ly6; BD), CD14 (M5E2; BD), CD16 (3G8; BD), CD19 (SJ25-C1; Thermo Fisher Scientific), CD45 (HI30; BD), CD56 (HCD56; BioLegend), CD66abce (TET2; Miltenyi Biotec), CD123 (7G3; BD), LOX-1 (15C4; BioLegend), and HLA-DR (L243; BioLegend).

Techniques: Flow Cytometry, Expressing, Comparison, Stripping Membranes

Journal: iScience

Article Title: CEA cell adhesion molecule 5 enriches functional human hematopoietic stem cells capable of long-term multi-lineage engraftment

doi: 10.1016/j.isci.2023.108561

Figure Lengend Snippet:

Article Snippet: Anti-CEACAM5 Antibody (PE), Mouse Monoclonal , Sino Biological , Cat# 11077-MM02-P; RRID: AB_2860305.

Techniques: Recombinant, Lysis, Staining, Isolation, Sequencing, Software