The CD155/TIGIT axis promotes and maintains immune evasion in neoantigen-expressing pancreatic cancer

Abstract

The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. Pancreatic adenocarcinoma (PDAC) is a highly lethal malignancy, and immune-based strategies to combat this disease have been largely unsuccessful to date. We corroborate prior reports that a substantial portion of PDAC harbors predicted high-affinity MHC class I-restricted neoepitopes and extend these findings to advanced/metastatic disease. Using multiple preclinical models of neoantigen-expressing PDAC, we demonstrate that intratumoral neoantigen-specific CD8⁺ T cells adopt multiple states of dysfunction, resembling those in tumor-infiltrating lymphocytes of PDAC patients. Mechanistically, genetic and/or pharmacologic modulation of the CD155/TIGIT axis was sufficient to promote immune evasion in autochthonous neoantigen-expressing PDAC. Finally, we demonstrate that the CD155/TIGIT axis is critical in maintaining immune evasion in PDAC and uncover a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in preclinical models, now poised for clinical evaluation.

Keywords: CD155; TIGIT; immune evasion; immunotherapy; pancreatic cancer.

Figure 1.. A subset of pancreatic cancer harbors predicted MHC class I-restricted neoepitopes

(A–B) Neoepitope landscape in TCGA_PAAD (n=148) by (A) predicted affinity for MHC class I; or (B) predicted non-binder [NB] to binder [B] or strong binder [SB; <50 nM] neoepitopes. (C–D) Neoepitope landscape in DFCI-PancSeq (n=57) by (C) predicted affinity for MHC class I; or (D) predicted NB to B or NB to SB neoepitopes. See also Figure S1 and Table S1.

Figure 2.. Neoantigen-expressing pancreatic organoids model immune clearance and immune evasion in the same tissue and antigenic context

(A) ‘H11-SIIN’ (top) and ‘H11-mLAMA4’ or ‘H11-mALG8’ (bottom) genomic loci. (B) Brightfield (left) and fluorescent (right) images of KP;SIIN pancreatic organoids. (C–D) Brightfield (left) and fluorescence stereomicroscopic (right) images of 8-week [wk] tumors following orthotopic transplantation of neoantigen-expressing pancreatic organoids into (C) immune-deficient or (D) immune-competent animals. (E) Proportion of outcomes 5 wks post-orthotopic transplantation (KP [n=15]; KP;SIIN [n=45]; KP;mLAMA4 [n-25]; KP;mALG8 [n=25]). (F) Tumor/pancreas weights 8–10 wks post-orthotopic transplantation of KP;SIIN pancreatic organoids (n=5 ‘immune-deficient’; n=24 ‘N’; n=6 ‘I’, n=30 ‘P’; bar=median). (G) Flow cytometry of mScarlet (left) or surface MHC-I [H-2K^b] (right) on tumor-derived organoids from progressor (n=7) or immune-deficient (n=5) animals −/+ interferon-γ (mean +/− SD). (H) Representative images of organoid/CD8 T cell co-culture with indicated E:T ratios. Statistical analyses: (F, G) two-sided Mann-Whitney U test (n.s. P=non-significant, ** P<0.01, *** P<0.001). See also Figure S2.

Figure 3.. T cell exhaustion typifies the neoantigen-specific TIL response in immune-evasive PDAC

(A) Flow cytometry of neoantigen-specific (CD44^hiTetramer⁺) CD8⁺ T cells in tumor/pancreata at 9–10 wks post-initiation, (mean +/− SD). (B) Ki67⁺ of CD44^hiTetramer⁺CD8⁺ TILs from progressor tumors (mean +/− SD). (C) Inhibitory receptor (PD-1, TIGIT, TIM-3, LAG-3) co-positivity as indicated by color in CD44^hiTetramer⁺CD8⁺ TILs from KP;SIIN tumors/pancreata; (mean +/− SD). (D) Flow cytometric characterization of neoantigen-specific (CD44^hiTetramer⁺) TILs at 5 wks post-initiation, (bar=median). (E) UMAP projection of scRNA-seq of neoantigen-specific (CD8⁺CD44^hiTetramer⁺) TILs from immune-evasive tumors. (F) Heatmap of differentially expressed genes between clusters with selected genes highlighted. (G–H) UMAP projections overlaid with (G) gene module expression for “LCMV T cell exhaustion” (CM1) and “LCMV T cell chronic effector” (CM2) or (H) indicated PAGODA gene expression programs. Statistical analyses: (A,B,D) two-sided Mann-Whitney U test (n.s. P=non-significant, * P<0.05). See also Figure S3 and Table S2.

Figure 4.. Human PDAC harbors exhausted CD8⁺ TILs

(A–E) Flow cytometric profiling of human PDAC CD8⁺ TILs for: (A) CD45RO; (B) Inhibitory receptor (TIGIT, PD-1, TIM-3, LAG-3) co-expression; (C) TIM3⁺TCF1^lo; (D) PD-1⁺TCF1^lo and PD-1⁺TCF1^hi; or (E) HLA-DR⁺Ki67⁺CD57^neg. (F) UMAP projection of human PDAC scRNA-seq data [n=24 patients] (Peng et al., 2019). (G) Computationally sorted cell subsets and UMAP projections overlaid with indicated genes/signatures. (A–E: bar=median). See also Figure S4.

Figure 5.. Elevated CD155 expression within the malignant compartment in murine and human PDAC

(A–B) Immunohistochemical analysis of CD155 on (A) murine and (B) human PDAC TMAs; quantified by H-score (right). (C–D) Flow cytometry of surface (C) CD155 or (D) PD-L1 on GDO (WT, K, P, KP) or tumor-derived organoids (progressor, immune-deficient). (E–F) ECDF analysis of CD155 (PVR) expression in (E) TCGA_PAAD or (F) TCGA_COAD within indicated genetic cohorts. (G) CD155 (PVR) expression in TCGA_PAAD stratified by total neoepitope burden (high: top 25%, low: bottom 25% from Figure 1A), (bar=median). Statistical analyses: A–B: two-sided Mann-Whitney U test, C–D: Welch’s t-test, E-G: Kolmogorov-Smirnov (n.s. P=non-significant, * P<0.05); A–D,G: bar=median. See also Figure S5.

Figure 6.. TIGIT/PD-1/CD40a combination immunotherapy elicits anti-tumor responses in immune-evasive PDAC

(A) Waterfall plot of evaluable tumors at 4 wks of treatment. (B) Spider plots of treatment response to PD-1/CD40a (top) and TIGIT/PD-1/CD40a (bottom). (C) Representative staining of CD8α, cytokeratin 19 (CK19), CD155, and PD-L1 of responders following 4 wks of TIGIT/PD-1/CD40a. (D) Representative mfIHC with Nanostring GeoMx DSP AOIs of a responder tumor (mPR) following 4 wks of TIGIT/PD-1/CD40a. (E–F) Flow cytometric analysis of (E) CD8⁺ T cells and (F) G-MDSCs [CD45⁺CD11b⁺F4/80^lowLy6C^lowLy6G^high], (mean +/− SD). (G) Differential protein expression in ‘CD8 high’ versus ‘CD8 low’ AOIs in non-responder tumors following 4 wks of TIGIT/PD-1/CD40a. Red: FDR<0.05. Statistical analyses: (A) two-sided Mann-Whitney U test of percent change at 4 wks of therapy, (E-F) two-sided Mann-Whitney U test, (G) linear mixed effect model with Benjamini-Hochberg FDR (n.s. P=non-significant, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001). See also Figure S6.

Figure 7.. Elevated CD155/TIGIT signaling is sufficient to promote immune evasion in autochthonous PDAC

(A) Lentiviral vectors to generate autochthonous neoantigen-expressing PDAC or control. (B) Retrograde pancreatic ductal instillation of lentivirus. (C–D) Brightfield (left) and fluorescence stereomicroscopic (right) images of representative 9-wk autochthonous tumors generated using mScarletSIIN in (C) CD8α-depleted or (D) immune-competent animals. (E) Proportion of animals with mScarlet-positivity as assessed by fluorescence stereomicroscopy, 9 wks post-initiation. (F) Lentiviral vectors and R26-dCas9-VPR knock-in allele used to modulate CD155 (Pvr) expression in autochthonous PDAC. (G) Flow cytometric assessment of surface CD155 on pancreatic organoids following transduction with indicated lentiviruses. (H) Proportion of animals with mScarlet-positivity by fluorescence stereomicroscopy at 9–12 wks post-initiation following indicated genetic or pharmacologic modulation. See also Figure S7.