Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Focusing and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb

doi: 10.1073/pnas.1506357112

Figure Lengend Snippet: Synergistic combination of adoptive T-cell therapy and anti-CD137 mAb requires CD137 expression on both transferred OT-1 and endogenous CD8+ T lymphocytes. Mice were s.c. inoculated with 5 × 105 B16F10-OVA melanoma cells on day 0. Graphs represent individual follow-up of tumor size and the fraction of tumor-free mice at the end of the experiment. (A) Mice were left untreated or treated on day +3 with rat IgG or cognate peptide-activated OT-1 lymphocytes. (B) Mice were adoptively transferred with 2 × 106 polyclonally activated T cells from non–TCR-transgenic CD45.1 congenic mice and anti-CD137 mAb (1D8) on day +3. (C) Mice were adoptively transferred with activated OT-1 CD8+ T lymphocytes and 100 μg of anti-CD137 mAb on day +3. (D) Mice were treated as in C but treatment was postponed to day +7 following tumor cell inoculation. (E) Mice were treated as in C but starting one day before treatment (day +2) mice received a depleting course of anti-CD4 mAb that was dosed every 5 d for up to four doses. (F) Mice were treated as in C, but OT-1 cells were harvested from OT-1 CD137-deficient mice. (G) B16F10-OVA–bearing CD137−/− mice were treated as in C with CD137-sufficient OT-1 lymphocytes. (H) Tumor-bearing recipient mice were RAG1−/− and were treated as in C. Experiments were repeated at least twice rendering comparable results with at least six animals per group.

Article Snippet: Adoptively T-cell transferred mice were dosed anti-CD137 agonist antibodies (1D8 or 2A) or control antibodies to subsequently follow up tumor size, retrieve intratumoral lymphocytes for flow cytometry evaluation or to perform imaging experiments by intravital microscopy of tumors implanted in dorsal skin-fold chambers, using a customized multiphoton microscope (TriMScope-II, LaVision BioTec).

Techniques: Expressing, Transgenic Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Focusing and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb

doi: 10.1073/pnas.1506357112

Figure Lengend Snippet: Combination with CD137 mAb does not result in increased numbers of transferred OT-1 lymphocytes in the tumor but enhances recruitment of endogenous T cells in a CD137-dependent manner. Absolute numbers of CD8+ T lymphocytes were counted in cell suspensions obtained from tumors (n = 6 per group) excised 7 d following treatment with OT-1 T lymphocytes and anti-CD137 on day 3 after tumor cell inoculation. Transferred CD45.1+ (A) and endogenous CD45.2+CD8+ T (B) cells were differentially counted using perfect count beads as internal standards. Recipient mice and OT-1 lymphocytes were WT or CD137−/− as indicated in the figure. Treatment with antibodies (Ab): 1D8 or control rat antibody (Ab) as indicated. Two similar independent experiments were performed rendering similar results. Statistical differences were assessed by Student’s t tests. (C) Representative dot plots are shown to indicate the relative abundance of endogenous (CD45.2) and transferrect (CD45.1) CD8+ T lymphocyte in the tumor infiltrates. *P ≤ 0.01.

Article Snippet: Adoptively T-cell transferred mice were dosed anti-CD137 agonist antibodies (1D8 or 2A) or control antibodies to subsequently follow up tumor size, retrieve intratumoral lymphocytes for flow cytometry evaluation or to perform imaging experiments by intravital microscopy of tumors implanted in dorsal skin-fold chambers, using a customized multiphoton microscope (TriMScope-II, LaVision BioTec).

Techniques: Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Focusing and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb

doi: 10.1073/pnas.1506357112

Figure Lengend Snippet: Combined treatment enhances the effector phenotype in both transferred and endogenous CD8+ TILs. Results from FACS analyses of cell suspensions retrieved on day 10 from tumors as those in Fig. 2 treated with OT-1 cells and anti-CD137 mAb (1D8). When indicated, recipient mice or transferred OT-1 lymphocytes were CD137−/−. Transferred and endogenous lymphocytes were differentially gated as CD45.1+ or CD45.1− CD8+ lymphocytes and analyzed for surface CD107a (A), KLRG1 (B), and intracellular IFNγ (C). Results are presented as the mean intensity of fluorescence for the indicated marker from individual mice including three pooled experiments. p represents Student’s t test P values. Lines represent the median values. n.s., not significant; *P ≤ 0.01; **P ≤ 0.001; ***P ≤ 0.0001. CD137KO, CD137−/−; WT, wild type.

Article Snippet: Adoptively T-cell transferred mice were dosed anti-CD137 agonist antibodies (1D8 or 2A) or control antibodies to subsequently follow up tumor size, retrieve intratumoral lymphocytes for flow cytometry evaluation or to perform imaging experiments by intravital microscopy of tumors implanted in dorsal skin-fold chambers, using a customized multiphoton microscope (TriMScope-II, LaVision BioTec).

Techniques: Fluorescence, Marker

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Focusing and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb

doi: 10.1073/pnas.1506357112

Figure Lengend Snippet: EOMES is induced in transferred and endogenous intratumor CTLs by anti-CD137 mAb treatment. FACS analyses experiments as in Fig. 3 with intracellular staining for the transcription factors EOMES (A) and T-bet (B) in CD8+ TILS from the indicated experimental groups. In each graph, the adoptively transferred cells, the recipient mouse, and the antibody treatment are provided in the horizontal axis [WT: wild type; CD137KO: CD137−/−]. Results in A are from two pooled experiments performed identically. Statistical differences were assessed with Mann–Whitney u test. n.s., not significant, *P ≤ 0.01.

Article Snippet: Adoptively T-cell transferred mice were dosed anti-CD137 agonist antibodies (1D8 or 2A) or control antibodies to subsequently follow up tumor size, retrieve intratumoral lymphocytes for flow cytometry evaluation or to perform imaging experiments by intravital microscopy of tumors implanted in dorsal skin-fold chambers, using a customized multiphoton microscope (TriMScope-II, LaVision BioTec).

Techniques: Staining, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Focusing and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb

doi: 10.1073/pnas.1506357112

Figure Lengend Snippet: Intravital microscopy shows focused effector dynamics of adoptively transferred OT1 CTL. (A) OT1 CTL migration dynamics and pattern. Heat map of CTL dwell time shows confined CTL migration tracks (arrow heads) in CD137 cotreated tumors. (B) OT1 CTL migration speed in parental, OVA expressing and anti-CD137 mAb treated tumors. (C) The mean square displacement relates to the area covered by the migrating cells at increasing time intervals. For each time interval ∆t, the mean squared displacement is plotted for tracks of OT1 CTL with and without anti-CD137 treatment. Significance test, two-way ANOVA. (D) Cumulative OT1 CTL–tumor cell interactions. Quantifications are based on at least three independent experiments with at least 180 CTL tracks of 75 min length analyzed per condition. Black lines indicate the median. Statistical differences were assessed using the Mann–Whitney u test; ***P ≤ 0.0001. (Scale bars, 50 µm.)

Article Snippet: Adoptively T-cell transferred mice were dosed anti-CD137 agonist antibodies (1D8 or 2A) or control antibodies to subsequently follow up tumor size, retrieve intratumoral lymphocytes for flow cytometry evaluation or to perform imaging experiments by intravital microscopy of tumors implanted in dorsal skin-fold chambers, using a customized multiphoton microscope (TriMScope-II, LaVision BioTec).

Techniques: Intravital Microscopy, Migration, Expressing, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Immunomodulatory Monoclonal Antibodies in Combined Immunotherapy Trials for Cutaneous Melanoma

doi: 10.3389/fimmu.2017.01024

Figure Lengend Snippet: Clinical trials combining immune checkpoint blockade with immunostimulatory strategies.

Article Snippet: NCT02554812 Phase II (ongoing, 2015) , • Avelumab (antagonist PD-L1 mAb) • utomilumab (agonist 41BB mAb) • PF-04518600 (agonist OX-40 mAb) • PD 0360324 (neutralizing M-CSF mAb) , Metastatic tumors including MM , Pfizer , A Phase 1b/2 open-label study to evaluate safety, clinical activity, pharmacokinetics and pharmacodynamics of avelumab (msb0010718c) in combination with other cancer immunotherapies in patients with advanced malignancies , • Arm A: avelumab + utomilumab at three different dose levels. • Arm B: dose escalation PF-04518600 + avelumab. • Arm C: dose escalation PD 0360324 + avelumab. • Arm D: dose escalation utomilumab + PF-04518600 + avelumab. Afterward, dose expansion utomilumab + PF-04518600 + avelumab in selected tumor types.

Techniques: Clinical Proteomics, Immunopeptidomics, Activity Assay, Adjuvant, Vaccines, Injection, Modification, Virus, Expressing, Biomarker Discovery, Ex Vivo, Comparison, Drug discovery, In Situ