Anti-PD-1/L1 lead-in before MAPK inhibitor combination maximizes antitumor immunity and efficacy

Abstract

Rationally sequencing and combining PD-1/L1-and MAPK-targeted therapies may overcome innate and acquired resistance. Since increased clinical benefit of MAPK inhibitors (MAPKi) is associated with previous immune checkpoint therapy, we compare the efficacies of sequential and/or combinatorial regimens in subcutaneous murine models of melanoma driven by Braf^V600, Nras, or Nf1 mutations as well as colorectal and pancreatic carcinoma driven by Kras^G12C. Anti-PD-1/L1 lead-in preceding MAPKi combination optimizes response durability by promoting pro-inflammatory polarization of macrophages and clonal expansion of interferon-γ^hi, and CD8⁺ cytotoxic and proliferative (versus CD4⁺ regulatory) T cells that highly express activation genes. Since therapeutic resistance of melanoma brain metastasis (MBM) limits patient survival, we demonstrate that sequencing anti-PD-1/L1 therapy before MAPKi combination suppresses MBM and improves mouse survival with robust T cell clonal expansion in both intracranial and extracranial metastatic sites. We propose clinically testing brief anti-PD-1/L1 (± anti-CTLA-4) dosing before MAPKi co-treatment to suppress therapeutic resistance.

Trial registration: ClinicalTrials.gov NCT02196181.

Keywords: BRAF/NRAS/KRAS/NF1; MAPK/BRAF/MEK inhibitor resistance; anti-CTLA-4; anti-PD-1/L1; brain metastasis; colorectal carcinoma; melanoma; pancreatic ductal adenocarcinoma; sequential-combination therapy; tumor immune microenvironment.

Figure 1.. Progression-free survival of patients with BRAFV600MUT melanoma treated with dabrafenib plus trametinib and stratified by prior exposure to ICT

Results from the S1320 SWOG trial and analyzed by the univariate Cox regression model. ICT, immune checkpoint therapy. See also Figure S1, Table S1, S2A and S2B.

Figure 2.. Two doses of anti-PD-1/L1 before MAPKi combination forestall therapy resistance and induce pro-inflammatory TAM polarization

(A) Schematic of timelines for subcutaneous tumor progression and for dosing anti-PD-L1 and/or MEKi therapies in (B to I) and for tumor sampling in (J and K). Anti-PD-L1 (200 μg/mouse) twice per week IP; MEKi (dosage variable depending on the tumor model) daily PO via chow. Gray circles indicate regimen and time points for CyTOF analysis in Figure 2 and scRNA-seq + scTCR-seq analysis in Figure 3. (B to H) Tumor volumes of YUMM1.7ER (B), NIL (C), NILER1–4 (D), mSK-Mel254 (E, H), CT26 (F), and KPC (G) treated with indicated regimens in (A). Trametinib at 1 (B, C, D), 2 (G), 3 (E) or 5 (F) mg/kg/d PO via chow. Anti-PD-L1 (200 μg/mouse) and anti-PD-1 (300 μg/mouse first week only and then 200 μg/mouse) twice per week IP. N = 8 tumors/group. Data are means ± SEMs (P-values, Student’s t test) and representative of two independent experiments. (I) Overall survival (cutoff tumor volume ≥ 1000 mm³) of mice bearing YUMM1.7ER tumors treated as indicated. PLX4032, 50 mg/kg/d PO; trametinib, 0.3 mg/kg/d PO. CTRL, historical control. All p-values (logrank test) are for pairwise comparisons relative to mice treated with anti-PD-L1 (d0 to d7) followed by the triplet (anti-PD-L1 + PLX4032 + trametinib) combination. (J) t-SNE maps (left) of tumor-infiltrating CD45⁺ cells analyzed by CyTOF in three indicated syngeneic subcutaneous tumor models at time points and in treatment regimens indicated in (A). Heatmaps (right) showing the expression values of immune phenotypic protein markers normalized to the maximum mean value across subsets. (K) Frequencies of iNOS⁺ TAMs in the CD45⁺ population of three syngeneic tumor models at time points and treatment regimens indicated in (A). Mean ± SEMs. Pairwise comparisons were performed in (i) vehicle vs. two doses of anti-PD-L1, (ii) MEKi d7 vs. MEKi d7, anti-PD-L1 d0 to d7, (iii) MEKi d7, anti-PD-L1 d7 vs. MEKi d7, anti-PD-L1 d0. P-value, Student’s t test. *p < 0.05, **p < 0.01 and ***p < 0.001. See (A). See also Figure S2.

Figure 3.. TAM and T-cell phenotypes associated with response to optimized therapy regimen

(A) Uniform manifold approximation and projection (UMAP) of tumor-infiltrating CD45⁺ cells (n = 53,841) analyzed by scRNA-seq. Dissociated live, CD45⁺ cells were pooled from 4 tumors (mSK-Mel254) per regimen and per time point. Inferred cell types are indicated by clusters denoted by distinct colors. (B) UMAP of tumor-infiltrating Mo/MΦ population (n = 28,857) analyzed by scRNA-seq. Clusters with differentially expressed genes are denoted by distinct colors. (C) Heatmap showing differentially expressed genes (rows) among different Mo/MΦ subpopulations (columns). Representative genes of each cluster are highlighted (right). (D) Ratios between the proportions of pro- and anti-inflammatory MΦ clusters across distinct regimens and time points. See Figure 2A. (E) UMAP of tumor-infiltrating T cells (n = 13,803) analyzed by scRNA-seq. Clusters with differentially expressed genes are denoted by distinct colors. (F) Heatmap showing differentially expressed genes (rows) among different T-cell clusters (columns) and highlighted genes of each cluster (right). (G) UMAP in (E) colored by clonality based on scTCR-seq. (H) Clonal expansion indices of T-cell subpopulations. P-value, Kruskal–Wallis test. (I) Developmental transition indices of CD8⁺ T_C cells with other CD8⁺ subpopulations. Pairwise comparisons were performed between Ki-67^hi CD8⁺ T cells and each of the other CD8⁺ subpopulations with the Wilcoxon test. P-values, ***p < 0.001. (J) Relative expansion ratios between T_C vs. T_REG (left) and Ki-67^hi CD8⁺ vs. T_REG (right) across different treatment regimens and time points. See Figure 2A. (K) Developmental transition indices between CD8⁺ T_C and Ki-67^hi CD8⁺ T cells across distinct regimens and time points. See Figure 2A. (L) Fold changes of top 5 (top) or 10 (bottom) clone sizes for CD8⁺ T_C or Ki-67^hi CD8⁺ T cells vs. CD4⁺ T_REG cells. See Figure 2A. (M) Violin plots of Ifng expression in distinct T-cell subpopulations. (N) Heatmap displaying the scaled mean expression levels of highlighted functional genes (rows) in T_C cells across treatment regimens and time points (see Figure 2A). Gene expression levels were row-scaled for only T_C cells. See also Figure S3 and Table S3.

Figure 4.. Functional roles of TAMs and CD8+ T cells in the development of acquired resistance

(A to C) Tumor volumes of mSK-Mel254 without active treatment or treated with the indicated regimens. Trametinib at 3 mg/kg/d PO; RP-832c (CD206 peptide) at 10 mg/kg/d subcutaneously; anti-PD-L1 (200 μg/mouse IP twice per week); and anti-CD8 antibody at 200 μg/mouse IP initiated one day before any treatment regimen and then twice a week. N = 8 tumors/group. Data are means ± SEMs (P-values, Student’s t test).

Figure 5.. Immune checkpoint blockade before MAPKi co-treatment prolongs MBM suppression and survival

(A) Schematic of timelines for metastatic tumor progression of Braf^V600MUT murine melanoma (YUMM1.7ER) and for dosing anti-PD-L1 and/or MEKi therapies in (B to F). Anti-PD-L1 (200 μg/mouse) IP on d-4 and d-2, when applicable, and, thereafter, twice per week; MEKi, 1 mg/kg/d PO. Gray circles indicate regimens and time points for TCR-seq analysis in Figure 6. (B) Representative ex vivo BLI of organs from necropsies at experimental endpoints (total ventral or dorsal BLI signal in the range of 1e9–1e10, moribund state of health, or death). Scale for radiance, photons/sec. (C, D) Temporal BLI quantification (radiance, photons/sec) based on the dosing timeline in (A) of dorsal extracranial (C) or intracranial (D) tumor burden. Data are mean ± SD based on the indicated numbers of mice in the untreated and treatment regimen groups. Pairwise comparisons (mixed model framework with Bonferronic correction for multiple testing) of the group MEKi d0, anti-PD-L1 d-4 vs. the group denoted by the specific color of symbol #. # p = 0.05–0.001, ## p < 0.001. *Indicates death of mouse or mice, resulting in drops in mean BLI values. True or confirmed complete responses or CRs defined in STAR Methods. (E) In vivo BLI of representative mice from the untreated group vs. all mice treated with MEKi d0 monotherapy at indicated timepoints. All images were adjusted to the same radiance scale. (F) Survival of untreated mice and mice on each treatment regimen. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (logrank test). Survival criteria indicated in (B). (G, H) Survival of untreated mice and mice on indicated treatment regimens. P values, logrank test. Day on which BRAFi + MEKi in (G) were started, defined as day 0, was the same as in (A) but pushed back by 2 days (day 10 after intracardiac injection) in (H). Low tumor burden model, MAPKi started on day 8 after IC injection; high tumor burden model, MAPKi started on day 10 after IC injection. (I, J) As in (C, D). (K) As in (F). (L, M) As in (C, D), except anti-CTLA-4 at 200 μg/mouse twice a week IP and # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001. Pairwise comparisons (mixed model framework with Bonferronic correction for multiple testing) of the group BRAFi+MEKi d0, anti-PD-L1 d-4 vs. the group denoted by the specific color of symbol #. (N) As in (F) except anti-CTLA-4 at 200 μg/mouse twice a week IP. (G to N) BRAFi, PLX4032, 50 mg/kg/d PO; MEKi, trametinib, 0.3 mg/kg/d PO. See also Figure S4.

Figure 6.. Anti-PD-L1 lead-in before MAPKi co-treatment augments clonal T-cell expansion

(A) Tumor cell-involved brain and ovarian tissues were collected from mice at time points and in groups as indicated in Figure 5A (brain, n = 2 mice per group, except the no treatment group; ovary, n = 1 mouse per group; two geographically distinct regions of each organ site were sampled for TCR-seq analysis). The total sizes of large TCR clones (≥ 5%) for the α or β chain in tumor-involved brain or ovarian tissues (red dots, average values). Pairwise comparisons were performed between the group of MEKi d0, anti-PD-L1 d-4 vs. each of the other groups with Student’s t-test. P-values, *p < 0.05, **p < 0.01, ***p < 0.001. (B) The fractions of overlapping TCR clones (α or β chain) between two distinct geographic regions of tumor-involved brain tissues from individual mice. (C) Total sizes of large clones (≥ 5%) shared by two distinct geographic regions of tumor-involved brain or ovarian tissues in each mouse. Pairwise comparisons were performed as in (A). See also Figure S4.