Proimmunogenic impact of MEK inhibition synergizes with agonist anti-CD40 immunostimulatory antibodies in tumor therapy

Cancer types with lower mutational load and a non-permissive tumor microenvironment are intrinsically resistant to immune checkpoint blockade. While the combination of cytostatic drugs and immunostimulatory antibodies constitutes an attractive concept for overcoming this refractoriness, suppression of immune cell function by cytostatic drugs may limit therapeutic efficacy. Here we show that targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) does not impair dendritic cell-mediated T cell priming and activation. Accordingly, combining MEK inhibitors (MEKi) with agonist antibodies (Abs) targeting the immunostimulatory CD40 receptor results in potent synergistic antitumor efficacy. Detailed analysis of the mechanism of action of MEKi shows that this drug exerts multiple pro-immunogenic effects, including the suppression of M2-type macrophages, myeloid derived suppressor cells and T-regulatory cells. The combination of MEK inhibition with agonist anti-CD40 Ab is therefore a promising therapeutic concept, especially for the treatment of mutant Kras-driven tumors such as pancreatic ductal adenocarcinoma.


Statistics
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n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

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October 2018

Data analysis
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-OT-I killing assay: GDC-0623: 3 experiments performed (2/3 successful with 3-4 mice per treatment group; cumulative data shown in Fig. 2). In 1/3 experiment no killing For experiments aimed at assessing efficacy of tumor treatment in conjuction with biomarker analyses, groups of at least 6 mice were used. Details on sample size and number of replicates experiments can be found in the individual figure legends.
Tumor treatment experiments: Mice were allocated to individual treatment grouped in a semi-randomized fashion by grouping according to initial tumor size so that all treatment groups had a comparable baseline mean tumor volume at treatment start. For all other experiments, e.g. in vivo T cell assays, animals were randomly assigned to different groups.
Control of other co-variates: Sex: In all experiments more Ly5.1 mice were used. Experimenter: In most experiments, the same experimenter performed the experiment of the same type. Age: All mice had an age of 8-12 weeks Facility: C57BL/6-Ly5.1 (CD45.1+, Ptprca) and NSG (NOD-Prkdcscid) mice were bred in animal facilities of the German Cancer Research Center. OT-I mice (C57BL/6-Ly5.2/CD45.2+; Tg(TcraTcrb)1100Mjb) were purchased from Charles River. Food & Environment: Food and water were provided ad libitum. Mice were maintained on a 12 hour light/dark cycle and environmental enrichment was provided; temperature was main-tained between 20-24°C. Drug cross-contamination: In order to avoid cross-contamination of animals with different small molecule inhibitors and chemotherapy, drugs were allocated to seperate cages.
In vitro: Blinding of in vitro experiments was not performed, also because drug solutions were colored differently and not necessary as the final data acquisition was performed by a machine automatically. Where suitable, automatic data analyses was performed, e.g. gating strategy for flow cytometry data and analysis templates for luciferase signal for cytotoxicity experiments. In addition, whenever possible, objective values were used, e.g. mean fluorescence intensity instead of frequency of gated populations. Lots: Different Lots of antibodies were used.
Triple block: Was performed because myeloid cells in the tumor microenvironment bind antibodies with different Fc receptors that cannot be blocked by one single clone. We used dissociated murine tumor specimens and evaluated single versus triple block for T cell gating. When using triple block, we observed less CD3+/CD90+ dim-med double positive cells. In addition, we used CD11b to gate on lineage negative cells in some experiments.