mouse recombinant il12  (R&D Systems)

Journal: Molecular Cancer Therapeutics

Article Title: XTX301, a Tumor-Activated Interleukin-12 Has the Potential to Widen the Therapeutic Index of IL12 Treatment for Solid Tumors as Evidenced by Preclinical Studies

doi: 10.1158/1535-7163.MCT-23-0336

Figure Lengend Snippet: Masking of XTX301 activity and reactivation by MMPs in vitro (A) SPR was used to measure the binding kinetics of XTX301, proteolytically cleaved XTX301 (XTX301 + MMP), and rhIL12 to IL12Rβ2. IL12Rβ2 was immobilized to a sensor chip. Then XTX301, proteolytically cleaved XTX301, and rhIL12 were flowed over at concentrations ranging from 3 nmol/L to 400 nmol/L. The concentrations of each analyte decrease from top to bottom within each panel. XTX301 activity was measured in a reporter cell line and primary human cells. B, IL12 HEK-Blue reporter gene cells were incubated with either rhIL12, unmasked control, or XTX301 at varying doses, and the reporter activity was measured. The data represents one of three independent experiments, data points represent the mean of 2 replicate wells and the error bars represent SD. C, Pre-activated primary human PBMCs were incubated with either rhIL12, unmasked control or XTX301 at varying doses, and STAT4 phosphorylation was assessed in CD8 + T cells via flow cytometry. The data points represent the mean of 2 replicate wells and the error bars represent SD. The data represents one of two independent experiments, each conducted with 2 different PBMC donors.

Article Snippet: Murine splenocytes were isolated via mechanical dissociation and pre-activated with PMA/Ionomycin (BioLegend, catalog #423301) for 2 days at 37°C before incubating with varying doses of rm IL12 (R&D Systems, catalog no. 419-ML/CF), murine surrogate mXTX301 or proteolytically activated mXTX301 for 24 hours.

Techniques: Activity Assay, In Vitro, Binding Assay, Incubation, Control, Phospho-proteomics, Flow Cytometry

Journal: Cancer immunology research

Article Title: P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy.

doi: 10.1158/2326-6066.CIR-21-0691

Figure Lengend Snippet: Fig. 1: P2RX7 is required for IL12-primed CD8+ T cells to effectively control tumors. a) C57BL/6 or B6.SJL mice were injected with 3×105 B16.gp33 melanoma cells subcutaneously. and once tumors became palpable (~7 days post-injection) 5×105 WT or P2rx7−/− P14 cells were transferred i.v. after 72 hours activation with anti-CD3/-CD28 and 2.5 IU/mL IL2, with (b-c) or without (d) 5 ng/mL IL12. b) Survival curve and c) tumor growth curves for individual tumor-bearing mice that received no cell transfer (n=27), WT P14 cells (n=23), or P2rx7−/− P14 cells (n=18) activated with IL12 priming. d) Survival curve for tumor-bearing mice that received WT (n=15) or P2rx7−/− (n=9) P14 cells activated

Article Snippet: A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript well plates with anti-CD3 (10 ug/mL) (BioXCell, #BE0001-1), anti-CD28 (20 ug/mL) (BioXCell, #BE0015-1), human IL2 (2.5 IU/mL) (Peprotech, #200-02), and with or without murine IL12 (5 ng/mL) (R&D Systems #419-ML-050/CF) for 72 hours in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin/streptomycin, 2mM Lglutamine at 37°C/5% CO2.

Techniques: Control, Injection, Activation Assay

Journal: Cancer immunology research

Article Title: P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy.

doi: 10.1158/2326-6066.CIR-21-0691

Figure Lengend Snippet: Fig. 2: Tumor-specific P2rx7−/− CD8+ T cells within dLN have increased expression of exhaustion markers. WT or P2rx7−/− P14 cells (activated in the presence of IL12) were transferred into C57BL/6 or B6.SJL with palpable B16.gp33 tumors. Tumors, tumor dLNs, and spleens were harvested from recipient mice 7 days after T-cell transfer. a) Number of live P14 cells per gram tumor and within dLN and spleen. b) Percentage of Tim3+ P14 cells within tumor (left) and dLN (right). c) PD1 gMFI of P14 cells within tumor (left) and dLN (right). d) Percentage of GzmB+ P14 cells post-ex vivo restimulation with PMA/Ionomycin. Data are from 3–4 independent experiments with 25 mice per group. Graphical data shown as means with error bars indicating SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Statistical significance determined by a two-tailed, unpaired t-test.

Article Snippet: A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript well plates with anti-CD3 (10 ug/mL) (BioXCell, #BE0001-1), anti-CD28 (20 ug/mL) (BioXCell, #BE0015-1), human IL2 (2.5 IU/mL) (Peprotech, #200-02), and with or without murine IL12 (5 ng/mL) (R&D Systems #419-ML-050/CF) for 72 hours in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin/streptomycin, 2mM Lglutamine at 37°C/5% CO2.

Techniques: Expressing, Ex Vivo, Two Tailed Test

Journal: Cancer immunology research

Article Title: P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy.

doi: 10.1158/2326-6066.CIR-21-0691

Figure Lengend Snippet: Fig. 3: P2rx7−/− CD8+ T cells show signs of mitochondrial dysfunction. Activated WT or P2rx7−/− P14 cells were cultured with IL12 then transferred into C57BL/6 or B6.SJL bearing palpable B16.gp33 tumors. Tumors, tumor dLNs and spleens were harvested from recipient mice 7 days after T-cell transfer. Mitochondrial mass and membrane potential were measured in WT and P2rx7−/− P14 cells within tumors of B16.gp33-engrafted mice by MTG and TMRE gMFI, respectively. Fold changes of a) MTG gMFI, b) TMRE gMFI, c) relative TMRE gMFI (normalized to MTG gMFI). (a-c) For each independent experiment, fold change in gMFI for the indicated molecular probe was calculated relative to average gMFI of donor WT P14 cells within the spleen. d) Frequency of P14 cells with depolarized (MTGhiTMRElo) mitochondria. Data are from 3 independent experiments with 25 mice per group. Graphical data shown as means with error bars indicating SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Statistical significance determined by a two-tailed, unpaired t-test.

Article Snippet: A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript well plates with anti-CD3 (10 ug/mL) (BioXCell, #BE0001-1), anti-CD28 (20 ug/mL) (BioXCell, #BE0015-1), human IL2 (2.5 IU/mL) (Peprotech, #200-02), and with or without murine IL12 (5 ng/mL) (R&D Systems #419-ML-050/CF) for 72 hours in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin/streptomycin, 2mM Lglutamine at 37°C/5% CO2.

Techniques: Cell Culture, Membrane, Two Tailed Test

Journal: Cancer immunology research

Article Title: P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy.

doi: 10.1158/2326-6066.CIR-21-0691

Figure Lengend Snippet: Fig. 4: Cell-intrisic defects in P2rx7−/− CD8+ T cells responding to tumor growth. Equal numbers of activated WT and P2rx7−/− OT-I cells (activated with IL12) were co- transferred into C57BL/6 or B6.SJL with palpable B16.OVA tumors. Tumors, tumor dLNs, and spleens were harvested from recipient mice 7 and 18 days after T-cell transfer. a) Weight of tumors from B16.OVA engrafted mice at indicated time of harvest. b) Ratio P2rx7−/ /WT OT-I donor cells within tumor at days 7 and 18 after T-cell transfer. c) Frequency of donor WT or P2rx7−/− OT-I cells that express (from left) CD39, Tim3, PD1, and Lag3 at each time point was determined and ratio P2rx7−/−/WT OT-I cells within each population

Article Snippet: A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript well plates with anti-CD3 (10 ug/mL) (BioXCell, #BE0001-1), anti-CD28 (20 ug/mL) (BioXCell, #BE0015-1), human IL2 (2.5 IU/mL) (Peprotech, #200-02), and with or without murine IL12 (5 ng/mL) (R&D Systems #419-ML-050/CF) for 72 hours in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin/streptomycin, 2mM Lglutamine at 37°C/5% CO2.

Techniques:

Journal: Cancer immunology research

Article Title: P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy.

doi: 10.1158/2326-6066.CIR-21-0691

Figure Lengend Snippet: Fig. 5: Impaired mitochondrial fitness of P2rx7−/− CD8+ T cells is cell-intrinsic. Equal numbers of WT and P2rx7−/− OT-I CD8+ T cells were activated in the presence of IL12 and equal numbers of cells were co-transferred into mice with palpable B16.OVA tumors. Tumors, tumor dLNs, and spleens were harvested from recipient mice 7 and 18 days after T-cell transfer. a) Frequency of OT-I donor cells with depolarized (MTGhiTMRElo) mitochondria within the tumors of mice harvested at days 7 or 18. Fold changes of b) MTG gMFI, c) TMRE gMFI, d) relative TMRE gMFI (normalized to MTG gMFI) in OT-I donor cells from tumors and dLN at indicated time points.(b-d) For each independent experiment,

Article Snippet: A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript well plates with anti-CD3 (10 ug/mL) (BioXCell, #BE0001-1), anti-CD28 (20 ug/mL) (BioXCell, #BE0015-1), human IL2 (2.5 IU/mL) (Peprotech, #200-02), and with or without murine IL12 (5 ng/mL) (R&D Systems #419-ML-050/CF) for 72 hours in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin/streptomycin, 2mM Lglutamine at 37°C/5% CO2.

Techniques:

Journal: Cancer immunology research

Article Title: P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy.

doi: 10.1158/2326-6066.CIR-21-0691

Figure Lengend Snippet: Fig. 6: Metabolic defects in P2rx7−/− CD8+ T cells arise during in vitro activation. WT and P2rx7−/− P14 cells were activated for 72 hours in vitro with anti-CD3, anti-CD28, IL2, and +/− IL12. (a-d) Oxygen consumption rate (OCR) of indicated groups after 72 hour activation was evaluated using Seahorse Extracellular Flux assay. a) OCRs were determined for the indicated groups after sequential addition of the listed inhibitors. b) Baseline OCR of indicated groups (determined prior to Oligomycin addition). c) Maximum OCR of indicated groups (determined after FCCP addition). d) Spare respiratory capacity (SRC) of indicated groups (calculated based on difference between Max OCR and baseline

Article Snippet: A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript well plates with anti-CD3 (10 ug/mL) (BioXCell, #BE0001-1), anti-CD28 (20 ug/mL) (BioXCell, #BE0015-1), human IL2 (2.5 IU/mL) (Peprotech, #200-02), and with or without murine IL12 (5 ng/mL) (R&D Systems #419-ML-050/CF) for 72 hours in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin/streptomycin, 2mM Lglutamine at 37°C/5% CO2.

Techniques: In Vitro, Activation Assay, XF Assay