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: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhancement of mycobacterial pathogenesis by host interferon-γ

doi: 10.1007/s00018-024-05425-7

Figure Lengend Snippet: Direct interaction of M. bovis BCG and M. tuberculosis with interferon-γ. ( A ) Specific binding of IFNγ to M. bovis BCG in a dose-dependent manner. Mycobacteria were fixed, incubated with different concentrations of IFNγ, heat inactivated IFNγ (HI-IFNγ), BSA, IL12, IL1β, and IL18, and analyzed by ELISA. ( B ) Immunofluorescence demonstrated IFNγ binding to M. bovis BCG. Mycobacteria were fixed, blocked, incubated with 10 µg/ml BSA or cytokines, immunostained, and analyzed by fluorescence microscopy. Scale bar, 1 μm. Arrow indicates protein colocalization. ( C ) Immunoblotting analysis demonstrated IFNγ binding to M . bovis BCG and M. tuberculosis . Bacteria were fixed, blocked, incubated with 10 µg/ml BSA or IFNγ, washed and analyzed by immunoblotting with antibodies against IFNγ and mycobacterial RpsO. Recombinant mouse IFNγ (10 ng) was used as a positive control. Data information: Statistical analysis in Fig. 1A was performed with two-way ANOVA followed by multiple comparisons among groups. The results are the mean values ± standard deviations of three biological replicates each with three technical replicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns , not significant

Article Snippet: Ten days after the final injection, serum samples were obtained, and ELISA was performed to determine the antibody titer; recombinant mouse IFNγ protein (#ab259378), recombinant mouse IL12 protein (#ab259419), recombinant mouse IL1β protein (#ab259421), recombinant mouse TNFα protein (#ab259411), and TMB ELISA substrate (#ab171523) from Abcam; mouse monoclonal anti-β-actin antibody (#3700S) from Cell Signaling; recombinant mouse IFNγ protein (#IF005) and glass bead (#G8772) from Sigma; recombinant mouse IL1β protein (#BMS332), recombinant mouse IL18 protein (#PMC0184), IFNγ rabbit monoclonal antibody (#701121), IFNγ monoclonal antibody (clone XMG1.2), eBioscience (#14-7311-81), rat anti-mouse IL12 p70 (clone 9A5) (#ENMM120), rabbit anti-mouse IL1β (#500-P51), rabbit anti-mouse IL18 (#210-401-323 S), rat IgG1 kappa isotype control (eBRG1), eBioscience (#14-4301-82), goat anti-rabbit IgG-HRP (#31460), goat anti-rat IgG-HRP (#31470), geneticin selective antibiotic (G418 Sulfate) (#10131035), and fluoromount-G mounting medium (#00-4958-02) from Thermo Fisher Scientific; FITC anti-mouse IFNγ antibody (#505806), FITC goat anti-rat IgG antibody (#405404), and Alexa fluor 647 anti-mouse IgG1 antibody (#406618) from BioLegend; rat anti-mouse IFNγ receptor 1 (CD119) (clone GR-20) and rat IgG2a isotype control (clone 2A3) from BioXCell; and heat shock protein 65 (mycobacterial) monoclonal antibody (clone 4H11) (#ADI-SPA-882-E) from Enzo Life Science.

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Fluorescence, Microscopy, Western Blot, Bacteria, Recombinant, Positive Control

Journal: Cancer Immunology Research

Article Title: CLN-617 Retains IL2 and IL12 in Injected Tumors to Drive Robust and Systemic Immune-Mediated Antitumor Activity

doi: 10.1158/2326-6066.CIR-23-0636

Figure Lengend Snippet: Bioactivity and in vivo pharmacokinetics of mCLN-617. A, Schematic of mCLN-617 design. B, ELISA-based binding assay on rat collagen I-coated plates, representative of three independent experiments. C, CTLL-2 or ( D ) 2D6 proliferation assay with normal or collagen-coated tissue culture plates, representative of four and two independent experiments, respectively. E,F, 50 pmol mCLN-617 was intratumorally injected in MC38-bearing mice and IL2 and IL12 levels detected in tumor and in serum at the indicated timepoints using MSD analysis.

Article Snippet: ELISA was utilized in studies measuring only IL2 (mouse IL2 Quantikine ELISA, R&D Systems) and IL12 (mouse IL12p70 Valukine ELISA, R&D Systems).

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Binding Assay, Proliferation Assay, Injection

Journal: Cancer Immunology Research

Article Title: CLN-617 Retains IL2 and IL12 in Injected Tumors to Drive Robust and Systemic Immune-Mediated Antitumor Activity

doi: 10.1158/2326-6066.CIR-23-0636

Figure Lengend Snippet: In vivo bioactivity of mCLN-617 in the injected tumor. A and B, Mice bearing ∼100 mm 3 B16F10 tumors were intratumorally treated with the indicated test articles on days 0 and 6 ( n = 10 mice/group). Shown are ( A ) survival curves and ( B ) body weight changes. C, Mice bearing B16F10 tumors were intratumorally or intravenously treated with the indicated dose levels of PBS or mCLN-617 on days 0, 6, and 12, and tumor growth kinetics are shown ( n = 10 mice/group). D, Mice bearing ∼100 mm 3 MC38 tumors were intratumorally treated with the indicated dose of mCLN-617 or PBS on days 0 and 6 or IP-treated with 10 mg/kg anti-PD1 antibody or isotype control BIW. Survival curves are shown ( n = 10 mice/group). E, Mice were treated as in D but with 400 pmol mCLN-617 on the indicated days, and survival curves are shown ( n = 10 mice/group). F, Mice were treated as in D but with 400 pmol mCLN-617 ( n = 10 mice/group). Shown are body weight changes. G and H, mCLN-617-treated survivors from the 400 pmol group in D and age-matched naïve controls were rechallenged with MC38 tumors on the contralateral flank and Pan02 tumors on the ipsilateral flank. Shown are growth curves for MC38 ( G ) and Pan02 ( H ; n = 10 mice/group). In A , statistical significance was evaluated using the Mantel–Cox log-rank test. n.s. indicates that group comparisons LAIR1-MSA-IL2 + IL12-MSA-LAIR1 (10 pmol each) vs. CLN-617 (10 pmpl) and LAIR1-MSA-IL2 + IL12-MSA-LAIR1 (100 pmol each) vs CLN-617 (100 pmol) were not statistically significant. In B , body weight differences were compared using two-way ANOVA with multiple comparison’s test at each timepoint. For both A and B , LAIR1-MSA-2 (10 pmol) + 12-MSA-LAIR1 (10 pmol) was compared against mCLN-617 (10 pmol), and LAIR1-MSA-2 (100 pmol) + 12-MSA-LAIR1 (100 pmol) was compared against mCLN-617 (100 pmol).

Article Snippet: ELISA was utilized in studies measuring only IL2 (mouse IL2 Quantikine ELISA, R&D Systems) and IL12 (mouse IL12p70 Valukine ELISA, R&D Systems).

Techniques: In Vivo, Injection, Control

Journal: Cancer Immunology Research

Article Title: CLN-617 Retains IL2 and IL12 in Injected Tumors to Drive Robust and Systemic Immune-Mediated Antitumor Activity

doi: 10.1158/2326-6066.CIR-23-0636

Figure Lengend Snippet: Characterization of CLN-617. A, CLN-617 was incubated in the indicated matrix for the indicated duration at 37°C and a Western blot performed detecting IL2 or IL12. B–I, Human PBMCs were CD3 stimulated overnight and then treated with the indicated test articles for 10 min ( C and D ), 2 h ( B ), 48 h ( E–G and I ), or 96 h ( H ). PBMC lysate was analyzed by signaling microarray ( B ) or CD8 + T cells assessed by flow cytometry ( C–I ). Data from one of the three representative donors is shown. In B , correlation analysis was assessed using Pearson correlation. Results from C–I are representative of two independent experiments.

Article Snippet: ELISA was utilized in studies measuring only IL2 (mouse IL2 Quantikine ELISA, R&D Systems) and IL12 (mouse IL12p70 Valukine ELISA, R&D Systems).

Techniques: Incubation, Western Blot, Microarray, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: USP28 protects development of inflammation in mouse intestine by regulating STAT5 phosphorylation and IL22 production in T lymphocytes

doi: 10.3389/fimmu.2024.1401949

Figure Lengend Snippet: Luminex – plasma.

Article Snippet: CD8+ T cells were cultured with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) in the presence of recombinant mouse IL12 (20 ng/ml), and mouse anti-IL4 (500-P54, 1 μg/ml, both from PeproTech).

Techniques: Luminex, Clinical Proteomics

Journal: Cancer Immunology Research

Article Title: Unleashing Natural IL18 Activity Using an Anti-IL18BP Blocker Induces Potent Immune Stimulation and Antitumor Effects

doi: 10.1158/2326-6066.CIR-23-0706

Figure Lengend Snippet: Mouse and human IL18 pathway share similar biological properties. A, IL18 and IL18BP levels were measured in sera and TDS taken from patients with cancer and tumor-bearing mice using ELISA kits (mouse sample information is indicated in Supplementary Table S9). B, Mouse T cells were purified from mouse tumors and matched spleens ( n = 16) and stained for IL18Rα expression by flow cytometry. Each dot represents one mouse. C, Affinity of mouse IL18 to mouse IL18BP measured by KinExA. Two curves with different mouse IL18BP concentrations were run and analyzed using n -curve analysis to determine the K d . Y -axis represents the free IL18BP. Free fraction of mouse IL18BP is measured pre-equilibrium, and the signal is a function of time and concentration of the tittered IL18. D , Affinity of anti-mouse IL18BP Ab to mouse IL18BP measured by surface plasmon resonance (SPR). Different colors represent the different concentrations of mouse IL18BP (0.125–256 nmol/L). E and F , Mouse cell lines (E) and mouse splenocytes, purified from mouse spleens ( F ), were stained with anti-mouse IL18BP Ab. Staining was analyzed by flow cytometry. G, Mouse CD3 + T cells were isolated from mouse splenocytes, activated with anti-CD3 and anti-CD28 and incubated with IL12 (2 ng/mL), and IL18:IL18BP (0.5 ng/mL:2 μg/mL) preformed complexes before addition of anti-mouse IL18BP Ab (10 μg/mL). Following the 24-hour culture, supernatant was collected for IFNγ secretion analysis. The median is depicted by a short black line in the violin plots. Bar graph shows the mean ± SEM; ***, P < 0.001 and ****, P < 0.0001 by two-tailed t test or by one-way ANOVA followed by two-tailed t test.

Article Snippet: Human IL18 (R&D), human IL18BPa (R&D), human IL12 (R&D), human IL18BPa mIgG1 His monomer (produced by GenScript Biotech), mouse IL18 (R&D), mouse IL18BPd (R&D), and mouse IL12 (R&D) proteins were used for biochemical, affinity measurements, and in vitro assays.

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Staining, Expressing, Flow Cytometry, Concentration Assay, SPR Assay, Isolation, Incubation, Two Tailed Test