Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Design considerations for single and dual-receptor engineered T cells (A) Schematic representation of a DAP12-associated synthetic antigen receptor (DAP12-SAR) composed of an antigen binding domain fused to the hinge, transmembrane (TM) and intracellular (ICD) domains of a DAP12-associated activating receptor (created using BioRender). (B) Schematic diagram of cDNA encoding DAP12 and the SAR separated by a Thoseasigna virus 2A (T2A) sequence for co-expression. (C) SAR surface expression was determined by binding of a myc-tag specific mAb or HER2-Fc to T cells engineered with the IL13Rα2-KIR and HER2-KIR, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + . Presented is SAR expression on the CD4 + NGFR + population (unfilled black = non-transduced; gray = SAR). (D) Representative cytotoxicity of IL13Rα2-KIR, HER2-KIR and non-specific SAR T cell products after 96-h co-culture with U-251 tumor cells in an Incucyte assay (E:T = 8:1). The experiment was performed in technical triplicates and error bars are standard error mean (SEM). (E) Schematic representation of dual-SAR constructs. (F) SAR surface expression on T cells engineered with single and dual SAR constructs, using anti-Myc tag mAb and HER2-Fc to detect binding of the IL13Rα2-KIR and HER2-KIR, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + .

Article Snippet: All proliferation assay samples were incubated for 3 days at 37°C and stained with Live/Dead Fixable Near-IR stain (Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (eBioscience) and VioBright FITC-conjugated mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Binding Assay, Virus, Sequencing, Expressing, Co-Culture Assay, Construct

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Dual targeting of IL13Rα2 and HER2 can be achieved by expression of different synthetic DAP12-associated receptors (A) Schematic representation of various HER2-SAR constructs evaluated. (B) Receptor surface expression and transduction efficiency of various HER2-SAR constructs, determined by binding to HER2-Fc and tNGFR expression, respectively. (C) Cytotoxicity of HER2-SAR engineered T cells after 120 h co-culture with HCT-116 and U-251 tumor cells in an Incucyte assay. (D) Proliferation of respective HER2-SAR constructs after 72 h co-culture with HCT-116 or U-251 tumor cells at a 1:1 ratio. Absolute cell count was determined by flow cytometry using 123count eBeads. (E) Schematic diagram of cDNA encoding single and dual SAR constructs. (F) SAR surface expression of single and dual IL13Rα2/HER2 SAR T cells determined by binding of a myc-tag specific mAb or HER2-Fc to detect the IL13Rα2-KIR and HER2-TREM1 SARs, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + . Presented is SAR expression on the CD4 + NGFR + population. (G) Mean fluorescence intensity (MFI) of respective SARs on single and dual-receptor engineered T cells. Data are from 4 experiments with 4 PBMC donors (each donor is represented by a unique symbol). (H) Cytotoxicity of single, dual, and non-transduced T cell products after 120-h co-culture with U-251 tumor cells in an Incucyte assay. All conditions were normalized to growth of tumor cells alone. Data are from 4 individual experiments with 3 PBMC donors. Error bars represent SEM of technical replicates. (I) Cytotoxicity of single, dual, and non-transduced T cell products after 120-h co-culture with U-251 IL13Rα2 KO and U-251 HER2 KO cells in an IncuCyte assay. Data are representative of 3 experiments with 3 PBMC donors. Error bars represent SEM. Statistical analysis for (G) and (H) were performed using a paired t test and two-way ANOVA with correction for multiple comparison (Tukey test), respectively (∗ p = ≤ 0.05, ∗∗ p = ≤ 0.01, ∗∗∗ p = ≤ 0.001, ∗∗∗∗ p = ≤ 0.0001).

Article Snippet: All proliferation assay samples were incubated for 3 days at 37°C and stained with Live/Dead Fixable Near-IR stain (Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (eBioscience) and VioBright FITC-conjugated mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Expressing, Construct, Transduction, Binding Assay, Co-Culture Assay, Cell Counting, Flow Cytometry, Fluorescence, Comparison

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Combinatorial targeting of CD133 and HER2 can be achieved using the dual-SAR approach (A) Schematic diagram of cDNA encoding CD133-SAR constructs. (B) SAR surface expression on primary human T cells as determined by binding of a myc-tag specific mAb or HER2-Fc to detect the IL13Rα2-KIR and HER2-TREM1 SARs, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + . (C) T cells were incubated with firefly luciferase-expressing HCT-116 tumor cells for 20 h at indicated effector:target ratios. Luminescence was read with an open filter upon addition of 0.15 mg/mL D-luciferin substrate and converted to % cytotoxicity. Error bars display standard deviation for technical replicates. (D) T cells were CTV-labeled and incubated for 72-h with HCT-116 tumor cells at a 1:1 ratio. T cell proliferation was measured by flow cytometry with live > CD3 + > CD4 + > NGFR+ cells presented. (E) Schematic diagram of cDNA encoding single and dual CD133/HER2 SAR constructs. (F) SAR surface expression of single and dual CD133/HER2 SAR T cells determined by binding of a FLAG tag specific mAb or HER2-Fc to detect the CD133-NKp44 and HER2-TREM1 SARs, respectively. Cells were gated as follows: lymphocytes > single cells > CD4/CD8 > NGFR + . Presented is SAR expression on the CD4 + NGFR + population. (G) Mean fluorescence intensity (MFI) of respective SARs on single and dual-receptor engineered T cells. Data are from 4 experiments with 4 PBMC donors (each donor is represented by a unique symbol). (H) Cytotoxicity of single, dual, and non-transduced T cell products after 72-h co-culture with HCT-116 tumor cells in an Incucyte assay. All conditions in were normalized to growth of tumor cells alone. Data are from 4 individual experiments with 3 PBMC donors. Error bars represent SEM. (I) Cytotoxicity of single, dual and non-transduced T cell products after 120-h co-culture with HCT-116 CD133 KO and HCT-116 HER2 KO cells in an IncuCyte assay. Data are representative of 3 experiments with 3 PBMC donors. Error bars represent SEM of technical replicates. Statistical analysis for (G) and (H) were performed using a paired t test and two-way ANOVA with correction for multiple comparison (Tukey test), respectively (∗ p = ≤ 0.05, ∗∗ p = ≤ 0.01, ∗∗∗ p = ≤ 0.001, ∗∗∗∗ p = ≤ 0.0001).

Article Snippet: All proliferation assay samples were incubated for 3 days at 37°C and stained with Live/Dead Fixable Near-IR stain (Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (eBioscience) and VioBright FITC-conjugated mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Construct, Expressing, Binding Assay, Incubation, Luciferase, Standard Deviation, Labeling, Flow Cytometry, FLAG-tag, Fluorescence, Co-Culture Assay, Comparison

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Expression of multiple receptors attenuates the production of inflammatory cytokines but not proliferation (A and D) Schematic representation of stimulation conditions for single- vs. dual-SAR engagement against (A) U-251 tumor cells or (D) HCT-116 tumor cells. (B and E) Intracellular cytokine production by (B) U-251 or (E) HCT-116 stimulated engineered T cells was measured by flow cytometry. Data are presented as percent NGFR+ CD4 and CD8 T cells producing respective cytokines. Data are from four independent experiments with 3 PBMC donors. Each donor is represented by a unique symbol. For the gating strategy see Figure S6 A. (C and F) Engineered T cells were labeled with CellTrace Violet (CTV) and stimulated with (C) U-251 or (F) HCT-116 tumor cells at a 1:1 ratio for 72 h. T cell proliferation and absolute cell count using 123count eBeads were measured by flow cytometry. Data are from three independent experiments with 2 T cell donors. Each donor is represented by a unique symbol. For the gating strategy see Figure S6 . Statistical analysis for (B), (C), (E), and (F) were performed using two-way ANOVA with correction for multiple comparison (Tukey test) (∗ p = ≤ 0.05, ∗∗ p = ≤ 0.01, ∗∗∗ p = ≤ 0.001, ∗∗∗∗ p = ≤ 0.0001, ns = not significant.

Article Snippet: All proliferation assay samples were incubated for 3 days at 37°C and stained with Live/Dead Fixable Near-IR stain (Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (eBioscience) and VioBright FITC-conjugated mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Expressing, Flow Cytometry, Labeling, Cell Counting, Comparison

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Expression of multiple receptors attenuates the early signal strength of individual receptors (A and B) Intracellular phospho-specific staining of ERK. Cells were stimulated for 30 min with respective WT or KO tumor lines, fixed, methanol-permeabilized and stained for phosphorylated ERK 1/2 (pT202/pY204). (A) Representative plots from 1 of 4 independent experiments and (B) %pERK+ of single and dual-SAR T cells following stimulation with a single target antigen. Data are from 4 independent experiments and 3 T cell donors. (C) Cells were stimulated with HCT-116 CD133 KO or U251 IL13Rα2 KO tumor cells for 1–4 h. The cells were surface stained for viability, CD4, CD8, NGFR and CD69, fixed/permeabilized, and then stained intracellularly/intranuclearly for Nur77. Cells were gated as follows: lymphocytes > single cells > live > CD4/CD8 > NGFR + > Nur77/CD69. Presented is Nur77 and CD69 expression on the CD8 + NGFR + population. Data are generated with T cells from one donor. For the gating strategy, see Figure S6 C.

Article Snippet: All proliferation assay samples were incubated for 3 days at 37°C and stained with Live/Dead Fixable Near-IR stain (Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (eBioscience) and VioBright FITC-conjugated mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Expressing, Staining, Generated

Journal: iScience

Article Title: DAP12-associated synthetic antigen receptors enable multi-targeting of T cells with independent chimeric receptors in a small genetic payload

doi: 10.1016/j.isci.2025.112142

Figure Lengend Snippet: Co-transduction improves dual-SAR receptor expression and rescues attenuated cytokine production (A and B) Schematic representation of vectors used to achieve dual-SAR expression through co-transduction. (C) CD133-and HER2-SAR surface expression on single vs. co-transduced T cells. FACS was performed on day 9 for co-expression of FLAG tag (CD133-SAR) and HER2-Fc (HER2-SAR). (D) IL13Rα2-and HER2-SAR surface expression on single vs. co-transduced T cells. FACS was performed on day 9 for co-expression of Myc-tag (IL13Rα2-SAR) and HER2-Fc (HER2-SAR). Cells were gated as follows: lymphocytes > single cells > CD4/CD8. Presented is representative SAR expression on the CD4 + population from 1 of 3 donors. (E and F) Intracellular cytokine production by (E) HCT-116 stimulated CD133/HER2-SAR T cells was measured by flow cytometry and (F) U251-stimulated IL13Rα2/HER2-SAR T cells. Data are presented as percent of CD4 + DAP12 + T cells producing respective cytokines ( n = 3) using cells from one donor. Comparable results were obtained with CD8 + T cells (data not shown).

Article Snippet: All proliferation assay samples were incubated for 3 days at 37°C and stained with Live/Dead Fixable Near-IR stain (Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (eBioscience) and VioBright FITC-conjugated mouse anti-human NGFR (Miltenyi Biotec).

Techniques: Transduction, Expressing, FLAG-tag, Flow Cytometry

Journal: Molecular Therapy Oncology

Article Title: Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signaling

doi: 10.1016/j.omton.2024.200842

Figure Lengend Snippet: Anti-DNP SAR αβ T cell production and CIR interaction (A) General structure and components of CIR (upper left) and a generic acceptor (lower right). (B) Steady-state interaction between the donor moiety and an acceptor leads to a balance of bound/unbound CIR/(N)CIR. In the case of the CIR, a subsequent covalent labeling step occurs when the covalent reactive chemistry is placed in close proximity to a nucleophilic amino acid. Covalent attachment of the CIR to the acceptor molecule leads to a state where the tumor binding domain is irreversibly bound to the acceptor molecule. (C) Structure of the DNP-acyl imidazole-glutamate-urea-lysine CIR (PSMA-CIR) and the DNP-Sufex-uPAR peptide CIR (uPAR-CIR). (D) An illustration of the receptor design and selection. All receptors employ the same anti-DNP as the acceptor. (E) Expression of the anti-DNP scFv containing TAC, KIR-CAR, and CAR on αβ T cells. Cells were gated on singlets > CD4 + /CD8 + > Myc + . Both CD4 and CD8 T cell population showed the same results, CD4-positive T cells are shown in this figure (see Figure S1 for gating and repeats). (F) Analyzed intracellular cytokine levels of TNF- α and IFN- γ of each universal SAR-engineered T cell when cultured in media alone to assess tonic signaling of each receptor.

Article Snippet: Cell surfaces were stained with Pacific Blue-conjugated mouse anti-human CD4 (cat. no. 558116, BD Pharmingen) and AlexaFluor700-conjugated mouse anti-human CD8α (cat. no. 56-0086-82, Invitrogen), VioBright FITC-conjugated mouse anti-human NGFR (cat. no. 130-113-423, Miltenyi Biotec), and anti-huCD69 (cat. no. 563835, BD Horizon), fixed, and permeabilized then fixed and permeabilized using the FoxP3 Transcription Factor Staining kit (cat. no. 00-5523-00, ThermoFisher), and stained intracellularly with PE-conjugated mouse anti-mouse Nur77 (cat. no. 12-5965-82, Invitrogen).

Techniques: Labeling, Binding Assay, Selection, Expressing, Cell Culture

Journal: Molecular Therapy Oncology

Article Title: Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signaling

doi: 10.1016/j.omton.2024.200842

Figure Lengend Snippet: CIRs produce selective antigen-specific activation of universal SARs (A) Structure of the DNP-acyl imidazole-desthiobiotin (AI-DTB CIR) and DNP-desthiobiotin (DTB (N)CIR). (B) DNP-TAC-T cells were labeled with AI-DTB CIR or AI-DTB CIR followed by streptavidin-PE. Non-anti-DNP scFv-containing cells (Control TAC-T cell) and non-engineered T cells were also labeled with AI-DTB and streptavidin-PE. As negative controls, DNP-TAC T cells were incubated with streptavidin-PE without prior labeling with CIR (No CIR) or the DNP-TAC T cells were labeled with AI-DTB CIR in the presence of 200-fold excess DNP (CIR +200X DNP). Both CD4 and CD8 T cell population showed the same results, CD8 T cell population used for this figure (see Figure S2 for gating and repeats). (C and D) Analysis of AI-DTB CIR rate of labeling over 3 h at a range of concentrations with TAC-, KIR-CAR-, and CAR-engineered T cells. DTB (N)CIR control is also included to show importance of covalent binding. Cells were gated on singlets > CD4 + /CD8 + > streptavidin-PE + , y axis corresponds to % of T cells labeled with CIR or (N)CIR. CD4 and CD8 labeling results were averaged and graphed (see Figure S3 for gating and repeats). (E) Structure of the DNP-acyl-imidazole-glutamate-urea-lysine CIR (PSMA-CIR) and DNP-glutamate-urea-lysine (N)CIR (PSMA-(N)CIR). (F) Anti-DNP SAR-engineered αβ T cells with 100 nM PSMA-CIR, or media alone, were incubated with either wild-type or PSMA-engineered HEK-293 cells and CD69 and Nur77 were measured by flow cytometry. Data were gated on live > singlets > CD4 + /CD8 + . The fraction of T cells expressing only CD69 or CD69 and Nur77 is shown in each flow plot (see Figure S8 for gating). Both the CD4 and CD8 T cell population revealed the same results. The CD4 T cell population is shown in this figure.

Article Snippet: Cell surfaces were stained with Pacific Blue-conjugated mouse anti-human CD4 (cat. no. 558116, BD Pharmingen) and AlexaFluor700-conjugated mouse anti-human CD8α (cat. no. 56-0086-82, Invitrogen), VioBright FITC-conjugated mouse anti-human NGFR (cat. no. 130-113-423, Miltenyi Biotec), and anti-huCD69 (cat. no. 563835, BD Horizon), fixed, and permeabilized then fixed and permeabilized using the FoxP3 Transcription Factor Staining kit (cat. no. 00-5523-00, ThermoFisher), and stained intracellularly with PE-conjugated mouse anti-mouse Nur77 (cat. no. 12-5965-82, Invitrogen).

Techniques: Activation Assay, Labeling, Control, Incubation, Binding Assay, Flow Cytometry, Expressing

Journal: Molecular Therapy Oncology

Article Title: Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signaling

doi: 10.1016/j.omton.2024.200842

Figure Lengend Snippet: Functional testing of CIR programmed T cells (A) T cells were incubated with PSMA-CIR (1 nM, 10 nM, 100 nM), PSMA-(N)CIR (1 nM, 10 nM, 100 nM) in the presence of 293-PSMA cells and stained intracellularly for both interferon gamma (IFN γ ) and TNF alpha (TNF α ); increasing CIR/(N)CIR concentration is shown as a blue or red triangle, respectively. As negative controls, T cells were incubated in medium alone (Media), wild-type 293 and 100 nM PSMA-CIR (CIR +293-WT), or PSMA-expressing 293 in the absence of CIR (293-PSMA + T cell). The data were gated on singlets > CD4 + /CD8 + > cells producing either IFN γ or TNF α . Both CD4 and CD8 T cell populations revealed the same results and the data in the figure reflect CD8 + T cells (see Figure S9 for gating). (B) LNCaP tumor cells expressing Nuclight Red were co-cultured with anti-DNP SAR-engineered αβ T cells (E:T of 8:1) and various concentrations of PSMA-CIR (blue symbols) or PSMA-(N)CIR (red symbols) and tumor growth was monitored using live cell imaging. The area under the curve of the LNCaP tumor growth curves was used to determine % cytotoxicity. (C) Anti-DNP SAR-engineered αβ T cells were labeled with either 1 μM of CIR (blue bar), (N)CIR (red bar), or media alone (gray bar) for 1 h followed by three washes to remove any unbound molecule. The labeled T cells were co-cultured with LNCaP tumor cells expressing Nuclight Red (E:T of 8:1) for cytotoxicity analysis as in (B). (D) Anti-DNP SAR-engineered αβ T cells were stained with CellTrace Violet (CTV) and co-cultured with PSMA-CIR (1 nM, 10 nM, 100 nM), PSMA-(N)CIR (1 nM, 10 nM, 100 nM), and 293-PSMA cells; increasing CIR/(N)CIR concentration is shown as a blue or red triangle, respectively. As negative controls, T cells were incubated in medium alone (Media), wild-type 293 and 100 nM PSMA-CIR (CIR +293-WT), or PSMA-expressing 293 in the absence of CIR (293-PSMA + T cell). Flow cytometry data were gated on live > singlets > CD3 + > CD4 + /CD8 + . FCS Express analysis software was used to determine the proliferation statistics (see Figure S10 for gating). The % of CD8+ T cells that divided over the course of the experiment is shown. The results for CD4 + T cells were similar but not shown. (E) CTV histograms for 100 nM conditions in (D). Statistical analysis for (A) and (C) was performed using ordinary one-way ANOVA, while (D) was performed with a two-way ANOVA, all with correction for multiple comparison (Tukey test) (∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001, ns = not significant).

Article Snippet: Cell surfaces were stained with Pacific Blue-conjugated mouse anti-human CD4 (cat. no. 558116, BD Pharmingen) and AlexaFluor700-conjugated mouse anti-human CD8α (cat. no. 56-0086-82, Invitrogen), VioBright FITC-conjugated mouse anti-human NGFR (cat. no. 130-113-423, Miltenyi Biotec), and anti-huCD69 (cat. no. 563835, BD Horizon), fixed, and permeabilized then fixed and permeabilized using the FoxP3 Transcription Factor Staining kit (cat. no. 00-5523-00, ThermoFisher), and stained intracellularly with PE-conjugated mouse anti-mouse Nur77 (cat. no. 12-5965-82, Invitrogen).

Techniques: Functional Assay, Incubation, Staining, Concentration Assay, Expressing, Cell Culture, Live Cell Imaging, Labeling, Flow Cytometry, Software, Comparison

Journal: Molecular Therapy Oncology

Article Title: Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signaling

doi: 10.1016/j.omton.2024.200842

Figure Lengend Snippet: Testing SuFEX CIR labeling and targeting of uPAR (A) Structures of the DNP-SuFEX-Desthiobiotin (SuFEX-DTB CIR), DNP-Sufex-uPAR peptide CIR (uPAR-CIR), and DNP-uPAR peptide NCIR (uPAR-(N)CIR). (B) DNP-TAC-T cells were labeled with SuFEX-DTB CIR or AI-DTB CIR followed by streptavidin-PE. Non-engineered T cells were also labeled with SuFEX-DTB and streptavidin-PE. As negative controls, DNP-TAC T cells were incubated with streptavidin-PE without prior labeling with CIR (No CIR) or the DNP-TAC T cells were labeled with SuFEX-DTB CIR in the presence of 200-fold excess DNP (CIR +200X DNP). Both CD4 and CD8 T cell populations showed the same results, CD4 T cell population used for this figure. See Figure S2 for gating strategy. (C) SAR-engineered T cells were incubated with uPAR-CIR (100 nM; blue bars), uPAR-(N)CIR (100 nM; red bars) in the presence of A172 cells and stained intracellularly for both interferon gamma (IFN γ ) and TNF alpha (TNF α ). As negative controls, T cells were incubated in medium and uPAR-CIR without A172 cells (Media+CIR; dark gray bars) or A172 cells without uPAR-CIR (A172 control; light gray bars). The data were gated on singlets > CD4 + /CD8 + > cells producing either IFN γ or TNF α . Both CD4 and CD8 T cell populations revealed the same results and the data in the figure reflect CD8 + T cells (see Figure S9 for gating). (D) A172 cells expressing eGFP were co-cultured with anti-DNP SAR engineered αβ T cells (E:T of 8:1) and various concentrations of uPAR-CIR (blue circles) and uPAR-(N)CIR (red circles) and tumor growth was monitored by live cell imaging. The area under the curve of the A172 growth curves was used to determine % cytotoxicity. Statistical analysis for (C) was performed using ordinary one-way ANOVA with correction for multiple comparison (Tukey test) (∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001, ns = not significant).

Article Snippet: Cell surfaces were stained with Pacific Blue-conjugated mouse anti-human CD4 (cat. no. 558116, BD Pharmingen) and AlexaFluor700-conjugated mouse anti-human CD8α (cat. no. 56-0086-82, Invitrogen), VioBright FITC-conjugated mouse anti-human NGFR (cat. no. 130-113-423, Miltenyi Biotec), and anti-huCD69 (cat. no. 563835, BD Horizon), fixed, and permeabilized then fixed and permeabilized using the FoxP3 Transcription Factor Staining kit (cat. no. 00-5523-00, ThermoFisher), and stained intracellularly with PE-conjugated mouse anti-mouse Nur77 (cat. no. 12-5965-82, Invitrogen).

Techniques: Labeling, Incubation, Staining, Control, Expressing, Cell Culture, Live Cell Imaging, Comparison

Journal: Molecular Therapy Oncology

Article Title: Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signaling

doi: 10.1016/j.omton.2024.200842

Figure Lengend Snippet: CIR-mediated anti-DNP SAR T-cell activation and tonic signaling (A) Brief overview of signaling cascade in T cells. (B) Anti-DNP SAR-engineered αβ T cells were co-incubated with 293-PSMA cells in the presence 100 nM (dark colors; upper panels) or 10 nM (light colors; lower panels) of PSMA-CIR (blue) or PSMA-(N)CIR (red). Cells were fixed, permeabilized, and stained for phosphorylated ERK. The percentage of pERK-positive cells was plotted over a 45-min time course (see Figure S11 for gating). (C) Anti-DNP SAR engineered αβ T cells were co-cultured with PSMA-CIR (1 nM, 10 nM, 100 nM), PSMA-(N)CIR (1 nM, 10 nM, 100 nM) and 293-PSMA cells; increasing CIR/(N)CIR concentration is shown as a blue or red triangle, respectively. As negative controls, T cells were incubated in medium alone (Media Only), PSMA-expressing 293 in the absence of CIR (293-PSMA Control) or wild-type 293 and 100 nM PSMA-CIR (CIR +293-WT). CD69 and Nur77 were measured by flow cytometry. The y axis reflects the fraction of cells expressing both CD69 and Nur77. Three independent donors were tested—each donor is shown as an individual symbol (triangle, square, circle). Both CD4 and CD8 T cell population revealed the same results. Data for CD8+ T cells is shown here (see Figure S8 for gating). Statistical analysis for (C) was performed using two-way ANOVA with correction for multiple comparison (Tukey test) (∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001, ns = not significant).

Article Snippet: Cell surfaces were stained with Pacific Blue-conjugated mouse anti-human CD4 (cat. no. 558116, BD Pharmingen) and AlexaFluor700-conjugated mouse anti-human CD8α (cat. no. 56-0086-82, Invitrogen), VioBright FITC-conjugated mouse anti-human NGFR (cat. no. 130-113-423, Miltenyi Biotec), and anti-huCD69 (cat. no. 563835, BD Horizon), fixed, and permeabilized then fixed and permeabilized using the FoxP3 Transcription Factor Staining kit (cat. no. 00-5523-00, ThermoFisher), and stained intracellularly with PE-conjugated mouse anti-mouse Nur77 (cat. no. 12-5965-82, Invitrogen).

Techniques: Activation Assay, Incubation, Staining, Cell Culture, Concentration Assay, Expressing, Control, Flow Cytometry, Comparison

Journal: Molecular Therapy Oncology

Article Title: Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signaling

doi: 10.1016/j.omton.2024.200842

Figure Lengend Snippet: Anti-DNP SAR αβ T cell production and CIR interaction (A) General structure and components of CIR (upper left) and a generic acceptor (lower right). (B) Steady-state interaction between the donor moiety and an acceptor leads to a balance of bound/unbound CIR/(N)CIR. In the case of the CIR, a subsequent covalent labeling step occurs when the covalent reactive chemistry is placed in close proximity to a nucleophilic amino acid. Covalent attachment of the CIR to the acceptor molecule leads to a state where the tumor binding domain is irreversibly bound to the acceptor molecule. (C) Structure of the DNP-acyl imidazole-glutamate-urea-lysine CIR (PSMA-CIR) and the DNP-Sufex-uPAR peptide CIR (uPAR-CIR). (D) An illustration of the receptor design and selection. All receptors employ the same anti-DNP as the acceptor. (E) Expression of the anti-DNP scFv containing TAC, KIR-CAR, and CAR on αβ T cells. Cells were gated on singlets > CD4 + /CD8 + > Myc + . Both CD4 and CD8 T cell population showed the same results, CD4-positive T cells are shown in this figure (see Figure S1 for gating and repeats). (F) Analyzed intracellular cytokine levels of TNF- α and IFN- γ of each universal SAR-engineered T cell when cultured in media alone to assess tonic signaling of each receptor.

Article Snippet: Cells were then stained with live/dead fixable near-IR stain (cat. no. L10119, Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (cat. no. 45-0088-42, eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (cat. no. 56-0048-82, eBioscience), VioBright FITC-conjugated mouse anti-human NGFR (cat. no. 130-113-423, Miltenyi Biotec), and BV605-conjugated mouse anti-human CD3 (cat. no. 300460 BioLegend).

Techniques: Labeling, Binding Assay, Selection, Expressing, Cell Culture

Journal: Molecular Therapy Oncology

Article Title: Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signaling

doi: 10.1016/j.omton.2024.200842

Figure Lengend Snippet: CIRs produce selective antigen-specific activation of universal SARs (A) Structure of the DNP-acyl imidazole-desthiobiotin (AI-DTB CIR) and DNP-desthiobiotin (DTB (N)CIR). (B) DNP-TAC-T cells were labeled with AI-DTB CIR or AI-DTB CIR followed by streptavidin-PE. Non-anti-DNP scFv-containing cells (Control TAC-T cell) and non-engineered T cells were also labeled with AI-DTB and streptavidin-PE. As negative controls, DNP-TAC T cells were incubated with streptavidin-PE without prior labeling with CIR (No CIR) or the DNP-TAC T cells were labeled with AI-DTB CIR in the presence of 200-fold excess DNP (CIR +200X DNP). Both CD4 and CD8 T cell population showed the same results, CD8 T cell population used for this figure (see Figure S2 for gating and repeats). (C and D) Analysis of AI-DTB CIR rate of labeling over 3 h at a range of concentrations with TAC-, KIR-CAR-, and CAR-engineered T cells. DTB (N)CIR control is also included to show importance of covalent binding. Cells were gated on singlets > CD4 + /CD8 + > streptavidin-PE + , y axis corresponds to % of T cells labeled with CIR or (N)CIR. CD4 and CD8 labeling results were averaged and graphed (see Figure S3 for gating and repeats). (E) Structure of the DNP-acyl-imidazole-glutamate-urea-lysine CIR (PSMA-CIR) and DNP-glutamate-urea-lysine (N)CIR (PSMA-(N)CIR). (F) Anti-DNP SAR-engineered αβ T cells with 100 nM PSMA-CIR, or media alone, were incubated with either wild-type or PSMA-engineered HEK-293 cells and CD69 and Nur77 were measured by flow cytometry. Data were gated on live > singlets > CD4 + /CD8 + . The fraction of T cells expressing only CD69 or CD69 and Nur77 is shown in each flow plot (see Figure S8 for gating). Both the CD4 and CD8 T cell population revealed the same results. The CD4 T cell population is shown in this figure.

Article Snippet: Cells were then stained with live/dead fixable near-IR stain (cat. no. L10119, Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (cat. no. 45-0088-42, eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (cat. no. 56-0048-82, eBioscience), VioBright FITC-conjugated mouse anti-human NGFR (cat. no. 130-113-423, Miltenyi Biotec), and BV605-conjugated mouse anti-human CD3 (cat. no. 300460 BioLegend).

Techniques: Activation Assay, Labeling, Control, Incubation, Binding Assay, Flow Cytometry, Expressing

Journal: Molecular Therapy Oncology

Article Title: Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signaling

doi: 10.1016/j.omton.2024.200842

Figure Lengend Snippet: Functional testing of CIR programmed T cells (A) T cells were incubated with PSMA-CIR (1 nM, 10 nM, 100 nM), PSMA-(N)CIR (1 nM, 10 nM, 100 nM) in the presence of 293-PSMA cells and stained intracellularly for both interferon gamma (IFN γ ) and TNF alpha (TNF α ); increasing CIR/(N)CIR concentration is shown as a blue or red triangle, respectively. As negative controls, T cells were incubated in medium alone (Media), wild-type 293 and 100 nM PSMA-CIR (CIR +293-WT), or PSMA-expressing 293 in the absence of CIR (293-PSMA + T cell). The data were gated on singlets > CD4 + /CD8 + > cells producing either IFN γ or TNF α . Both CD4 and CD8 T cell populations revealed the same results and the data in the figure reflect CD8 + T cells (see Figure S9 for gating). (B) LNCaP tumor cells expressing Nuclight Red were co-cultured with anti-DNP SAR-engineered αβ T cells (E:T of 8:1) and various concentrations of PSMA-CIR (blue symbols) or PSMA-(N)CIR (red symbols) and tumor growth was monitored using live cell imaging. The area under the curve of the LNCaP tumor growth curves was used to determine % cytotoxicity. (C) Anti-DNP SAR-engineered αβ T cells were labeled with either 1 μM of CIR (blue bar), (N)CIR (red bar), or media alone (gray bar) for 1 h followed by three washes to remove any unbound molecule. The labeled T cells were co-cultured with LNCaP tumor cells expressing Nuclight Red (E:T of 8:1) for cytotoxicity analysis as in (B). (D) Anti-DNP SAR-engineered αβ T cells were stained with CellTrace Violet (CTV) and co-cultured with PSMA-CIR (1 nM, 10 nM, 100 nM), PSMA-(N)CIR (1 nM, 10 nM, 100 nM), and 293-PSMA cells; increasing CIR/(N)CIR concentration is shown as a blue or red triangle, respectively. As negative controls, T cells were incubated in medium alone (Media), wild-type 293 and 100 nM PSMA-CIR (CIR +293-WT), or PSMA-expressing 293 in the absence of CIR (293-PSMA + T cell). Flow cytometry data were gated on live > singlets > CD3 + > CD4 + /CD8 + . FCS Express analysis software was used to determine the proliferation statistics (see Figure S10 for gating). The % of CD8+ T cells that divided over the course of the experiment is shown. The results for CD4 + T cells were similar but not shown. (E) CTV histograms for 100 nM conditions in (D). Statistical analysis for (A) and (C) was performed using ordinary one-way ANOVA, while (D) was performed with a two-way ANOVA, all with correction for multiple comparison (Tukey test) (∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001, ns = not significant).

Article Snippet: Cells were then stained with live/dead fixable near-IR stain (cat. no. L10119, Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (cat. no. 45-0088-42, eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (cat. no. 56-0048-82, eBioscience), VioBright FITC-conjugated mouse anti-human NGFR (cat. no. 130-113-423, Miltenyi Biotec), and BV605-conjugated mouse anti-human CD3 (cat. no. 300460 BioLegend).

Techniques: Functional Assay, Incubation, Staining, Concentration Assay, Expressing, Cell Culture, Live Cell Imaging, Labeling, Flow Cytometry, Software, Comparison

Journal: Molecular Therapy Oncology

Article Title: Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signaling

doi: 10.1016/j.omton.2024.200842

Figure Lengend Snippet: Testing SuFEX CIR labeling and targeting of uPAR (A) Structures of the DNP-SuFEX-Desthiobiotin (SuFEX-DTB CIR), DNP-Sufex-uPAR peptide CIR (uPAR-CIR), and DNP-uPAR peptide NCIR (uPAR-(N)CIR). (B) DNP-TAC-T cells were labeled with SuFEX-DTB CIR or AI-DTB CIR followed by streptavidin-PE. Non-engineered T cells were also labeled with SuFEX-DTB and streptavidin-PE. As negative controls, DNP-TAC T cells were incubated with streptavidin-PE without prior labeling with CIR (No CIR) or the DNP-TAC T cells were labeled with SuFEX-DTB CIR in the presence of 200-fold excess DNP (CIR +200X DNP). Both CD4 and CD8 T cell populations showed the same results, CD4 T cell population used for this figure. See Figure S2 for gating strategy. (C) SAR-engineered T cells were incubated with uPAR-CIR (100 nM; blue bars), uPAR-(N)CIR (100 nM; red bars) in the presence of A172 cells and stained intracellularly for both interferon gamma (IFN γ ) and TNF alpha (TNF α ). As negative controls, T cells were incubated in medium and uPAR-CIR without A172 cells (Media+CIR; dark gray bars) or A172 cells without uPAR-CIR (A172 control; light gray bars). The data were gated on singlets > CD4 + /CD8 + > cells producing either IFN γ or TNF α . Both CD4 and CD8 T cell populations revealed the same results and the data in the figure reflect CD8 + T cells (see Figure S9 for gating). (D) A172 cells expressing eGFP were co-cultured with anti-DNP SAR engineered αβ T cells (E:T of 8:1) and various concentrations of uPAR-CIR (blue circles) and uPAR-(N)CIR (red circles) and tumor growth was monitored by live cell imaging. The area under the curve of the A172 growth curves was used to determine % cytotoxicity. Statistical analysis for (C) was performed using ordinary one-way ANOVA with correction for multiple comparison (Tukey test) (∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001, ns = not significant).

Article Snippet: Cells were then stained with live/dead fixable near-IR stain (cat. no. L10119, Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (cat. no. 45-0088-42, eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (cat. no. 56-0048-82, eBioscience), VioBright FITC-conjugated mouse anti-human NGFR (cat. no. 130-113-423, Miltenyi Biotec), and BV605-conjugated mouse anti-human CD3 (cat. no. 300460 BioLegend).

Techniques: Labeling, Incubation, Staining, Control, Expressing, Cell Culture, Live Cell Imaging, Comparison

Journal: Molecular Therapy Oncology

Article Title: Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signaling

doi: 10.1016/j.omton.2024.200842

Figure Lengend Snippet: CIR-mediated anti-DNP SAR T-cell activation and tonic signaling (A) Brief overview of signaling cascade in T cells. (B) Anti-DNP SAR-engineered αβ T cells were co-incubated with 293-PSMA cells in the presence 100 nM (dark colors; upper panels) or 10 nM (light colors; lower panels) of PSMA-CIR (blue) or PSMA-(N)CIR (red). Cells were fixed, permeabilized, and stained for phosphorylated ERK. The percentage of pERK-positive cells was plotted over a 45-min time course (see Figure S11 for gating). (C) Anti-DNP SAR engineered αβ T cells were co-cultured with PSMA-CIR (1 nM, 10 nM, 100 nM), PSMA-(N)CIR (1 nM, 10 nM, 100 nM) and 293-PSMA cells; increasing CIR/(N)CIR concentration is shown as a blue or red triangle, respectively. As negative controls, T cells were incubated in medium alone (Media Only), PSMA-expressing 293 in the absence of CIR (293-PSMA Control) or wild-type 293 and 100 nM PSMA-CIR (CIR +293-WT). CD69 and Nur77 were measured by flow cytometry. The y axis reflects the fraction of cells expressing both CD69 and Nur77. Three independent donors were tested—each donor is shown as an individual symbol (triangle, square, circle). Both CD4 and CD8 T cell population revealed the same results. Data for CD8+ T cells is shown here (see Figure S8 for gating). Statistical analysis for (C) was performed using two-way ANOVA with correction for multiple comparison (Tukey test) (∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001, ns = not significant).

Article Snippet: Cells were then stained with live/dead fixable near-IR stain (cat. no. L10119, Invitrogen), PerCP-Cy5.5-conjugated mouse anti-human CD8α (cat. no. 45-0088-42, eBioscience), Alexa Fluor 700-conjugated mouse anti-human CD4 (cat. no. 56-0048-82, eBioscience), VioBright FITC-conjugated mouse anti-human NGFR (cat. no. 130-113-423, Miltenyi Biotec), and BV605-conjugated mouse anti-human CD3 (cat. no. 300460 BioLegend).

Techniques: Activation Assay, Incubation, Staining, Cell Culture, Concentration Assay, Expressing, Control, Flow Cytometry, Comparison

Journal: Nature medicine

Article Title: Pembrolizumab and cabozantinib in recurrent metastatic head and neck squamous cell carcinoma: a phase 2 trial

doi: 10.1038/s41591-023-02275-x

Figure Lengend Snippet: a, Univariate Cox proportional-hazards association with OS by patient and clinical characteristics at baseline. Data are presented as the HR and 95% CI by potential confounders. HR < 1 represents a lower hazard; HR > 1 represents a higher hazard. OP, oropharynx carcinoma. b, OS of 36 patients treated with PD-L1-CPS. CPS < 20, median OS of 14.6 months (95% CI = 8.2–NE); 1-year OS of 54.9% (95% CI = 24.5%–31.4%). CPS ≥ 20, median OS of 32.9 months (95% CI = 6.9–32.9); 1-year OS of 83.6% (95% CI = 48.0%–95.7%). Association of OS with CPS was assessed using the Kaplan–Meier method (log-rank test); the significance level was set at P < 0.05 (two-tailed). c, Preexisting CD8+ T cell tumor infiltration (25 patients) with representative IHC. Data are presented as mean values ± s.d. The length of the error bars is the s.d. Mean number of CD8+ positive cells per field in the PD + SD and PR groups is 52.57 and 137.8, respectively. An unpaired two-tailed t-test was used. WCI, Winship Cancer Institute.

Article Snippet: Sections were placed in 10 mM sodium citrate buffer (pH 6.0) at subboiling temperatures for 10 min and incubated with 10% normal goat serum, followed by incubation with a primary monoclonal antibody against human CD8α (clone C8/144B, 1:300, catalog no. 90257, Cell Signaling Technology).

Techniques: Two Tailed Test