Targeting T cell checkpoints 41BB and LAG3 and myeloid cell CXCR1/CXCR2 results in antitumor immunity and durable response in pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is considered non-immunogenic, with trials showing its recalcitrance to PD1 and CTLA4 immune checkpoint therapies (ICTs). Here, we sought to systematically characterize the mechanisms underlying de novo ICT resistance and to identify effective therapeutic options for PDAC. We report that agonist 41BB and antagonist LAG3 ICT alone and in combination, increased survival and antitumor immunity, characterized by modulating T cell subsets with antitumor activity, increased T cell clonality and diversification, decreased immunosuppressive myeloid cells and increased antigen presentation/decreased immunosuppressive capability of myeloid cells. Translational analyses confirmed the expression of 41BB and LAG3 in human PDAC. Since single and dual ICTs were not curative, T cell-activating ICTs were combined with a CXCR1/2 inhibitor targeting immunosuppressive myeloid cells. Triple therapy resulted in durable complete responses. Given similar profiles in human PDAC and the availability of these agents for clinical testing, our findings provide a testable hypothesis for this lethal disease.

Extended Data Fig. 1. Prominent infiltration of myeloid immunosuppressive cells in iKRAS tumors.

A. PDAC tumor development in syngeneic mouse model with representative images of tumor detected by bioluminescence, PET/CT and MRI at indicated timepoints. B. Tumor volume measured by MRI at indicated timepoints (top) and Kaplan-Meier curve depicting overall survival (bottom) for untreated iKRAS tumor bearing mice (n=10 mice). C. Representative images of normal pancreas, orthotopic and autochthonous (GEMM) iKRAS tumors with H&E, Masson Trichrome, smooth muscle actin (SMA) and vimentin staining. Scale bars: 100μm. D. Representative H&E images of iKRAS tumors invading into adjacent lymph nodes (left=4x magnification, right=20x magnification). E. Representative coronal and axial MRI images of iKRAS tumor invading into duodenum. F. Representative images of normal pancreas and human PDAC tumors with SMA, Vimentin and CD45 staining. Scale bars: 100μm. Red arrow indicates positively stained cells. G. Percentage of granulocytic (CD45⁺CD11b⁺Ly6G⁺Ly6C^-) and monocytic MDSCs (CD45⁺CD11b⁺Ly6G^-Ly6C⁺) within syngeneic iKRAS tumors (n=10 tumors) assessed by CyTOF at 4 weeks after initial tumor detection. Two-sided Student’s t-test. H. Representative images (bottom) of normal pancreas, orthotopic and autochthonous (GEMM) iKRAS tumors with indicated staining. n=6 biological replicates. Scale bars: 100μm. The bar graph (top) shows quantification of each cell type as analyzed by IHC. Two-sided Student’s t-test. I. Representative images of normal pancreas and orthotopic iKRAS tumors with indicated staining. Scale bars: 100μm. J. Percentage of Treg (CD45⁺CD3⁺TCRβ⁺CD4⁺FoxP3⁺) among CD4⁺ T cells within syngeneic iKRAS tumors (n=10 tumors) assessed by CyTOF at 4 weeks after initial tumor detection. Two-sided Student’s t-test. K. Representative images of normal pancreas and human PDAC tumors with indicated staining. Scale bars: 100μm. Red arrow indicates positively stained cells. Data in G, H and J are presented as mean ± s.e.m.

Extended Data Fig. 2. Prominent infiltration of myeloid immunosuppressive cells in human PDAC tumors.

A. Representative images of normal pancreas and human PDAC tumors with indicated staining. Scale bars: 100μm. Red arrow indicates positively stained cells. B. Clustering of human TCGA PDAC samples (n=178 patients) into MDSC-high, MDSC-low, and MDSC-medium groups using a 39-gene MDSC signature [16]. C. CIBERSORTx quantification of monocyte/macrophage subset fraction in human PDAC samples; TCGA (n=178 patients) and ICGC-AU (n=92 patients). D. Representative images of normal pancreas and human PDAC tumors with indicated staining. Scale bars: 100μm. Red arrow indicates positively stained cells. E. Kaplan-Meier plot depicting overall survival of TCGA PDAC patients (n=178 patients) grouped by the gene expression signatures of C1q⁺ TAM (top) and Spp1⁺ TAM (bottom).

Extended Data Fig. 3. Heterogeneity of myeloid cells in iKRAS PDAC tumors identified by single cell gene expression profiling.

A. UMAP of all live CD45⁺ cells used for scRNA-seq analysis of untreated iKRAS tumors (n=4,080 cells). B. Representative genes and functional markers used for identification of immune cell clusters. C. Heatmap of six immune cell clusters with unique signature genes. D. Representative genes and functional markers used for identification of myeloid cell clusters. E. Heatmap of myeloid cell clusters with unique signature genes. F. Representative genes and functional markers used for identification of dendritic cell clusters.

Extended Data Fig. 4. Dysfunctional phenotype of T cells in iKRAS PDAC tumors identified by single cell gene expression profiling.

A. Representative genes and functional markers used for identification of T cell clusters. B. Heatmap of two CD4⁺ and four CD8⁺ T cell clusters with unique signature genes. C. Cell cycle scoring for two CD4⁺ and four CD8⁺ T cell clusters. D. Relative expression of select genes in CD8⁺ T cells as a function of pseudotime from Monocle2 inferred trajectory. Each point corresponds to a single cell, colored by CD8⁺ T cell cluster. Lines represent average expression at that location in the trajectory. E. Quantification of immune checkpoint expression on infiltrating CD4⁺ and CD8⁺ T cells in iKRAS tumors (n=3 biological replicates), assessed by flow cytometry and analyzed by FlowJo.

Extended Data Fig. 5. Efficacy of immune checkpoint therapy (ICT) and treatment effects on immune microenvironment.

A. Treatment schedule and monitoring procedures for preclinical trials to evaluate effect of ICT on iKRAS PDAC bearing mice. B. Heatmap of immune checkpoint expression on T cells after 4-week treatment with control, anti-PD1 or anti-CTLA4 antibody (n=3 mice/ group). C. UMAP demonstrating cell types in single-cell RNA sequencing of human PDAC samples from Peng et al. [27] and Steele et al. [10] (left), and expression of LAG3 and 41BB (TNFRSF9) on T cells (right). D. UMAP of all live CD45⁺ cells used for scRNA-seq analysis of iKRAS tumors treated with control, anti-PD1, anti-CTLA4, anti-41BB, anti-LAG3, SX-682 or combination (anti-LAG3+anti-41BB+SX-682) treatment (n=3 mice/group). E. UMAP projection of immune cell clusters (top) and cells with TCR detected (bottom). F. Violin plots displaying relative expression of representative genes and functional markers used for identification of immune cell clusters.

Extended Data Fig. 6. Efficacy of immune checkpoint therapy (ICT) and treatment effects on immune microenvironment.

A. Heatmap of six immune cell clusters with unique signature genes. B. UMAP projection of T cell clusters (top) and violin plots displaying relative expression of representative genes and functional markers used for identification of T cell clusters (bottom). C. Heatmap of ten T cell clusters with unique signature genes. D. UMAP projection of neutrophil/granulocyte clusters (top) and violin plots displaying relative expression of representative genes and functional markers used for identification of neutrophil/granulocyte clusters (bottom).

Extended Data Fig. 7. Efficacy of immune checkpoint therapy (ICT) and treatment effects on immune microenvironment.

A. Heatmap of five neutrophil/granulocyte clusters with unique signature genes. B. UMAP projection of monocyte/macrophage clusters (top) and violin plots displaying relative expression of representative genes and functional markers used for identification of monocyte/macrophage clusters (bottom). C. Heatmap of five monocyte/macrophage clusters with unique signature genes. D. Proportion of immune cell subtypes in single-cell sequencing analysis of established iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control, anti-PD1, anti-CTLA4, anti-41BB or anti-LAG3 antibody for 4 weeks (n=3 tumors/group).

Extended Data Fig. 8. Effects of immune checkpoint therapy (ICT) treatment on immune microenvironment.

A Proportion of T cell subtypes in scRNA-seq analysis of established iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control, anti-PD1, anti-CTLA4, anti-41BB or anti-LAG3 antibody for 4 weeks (n=3 tumors/group). B. Top gene ontologies from GSEA of differential expression in T cells from anti-41BB and control antibody treated mice (n=3 mice/group). C. Circos plots of T cell receptor clonotype frequencies and expression states of CD8⁺ T cells in iKRAS tumors after treatment with control (left) and anti-41BB antibody (right) for 4 weeks. Outer histogram is the frequency of each clonotype. Inner bars show the fraction of cells of particular clonotype in each expression state (colors correspond to the clusters in Extended Data Fig 8a). Inner dendrograms are the hierarchical clustering of gene expression centroids for each clonotype. D. Multiple-testing corrected 95% binomial confidence intervals on the probability of a cell in each treatment group containing a TCR CD3R sequence which overlaps that of another cluster. (*p<0.05) E. Violin plots showing CCR7 expression in CD4⁺, CD8⁺ and CD4^-CD8^- T cells. (*p<0.05 two-sided unpaired Wilcox test) F. Proportion of CD4⁺, CD8⁺ and CD4^-CD8^- T cells with expression of CCR7 (left) and IL2RB (right). G. Expression of genes and functional markers on CD3⁺CD4^-CD8^- T cells. H. Violin plots showing expression of Stat6, Socs3 and Il1b among myeloid cells from control and anti-LAG3 antibody-treated tumors (n=3 mice/group). (*p<0.05 two-sided unpaired Wilcox test) I. Violin plots showing expression of Cxcl10, Stat1, Il10, Mrc1 and Socs3 among myeloid cells from control and anti-41BB antibody-treated tumors (n=3 mice/group). (*p<0.05 two-sided unpaired Wilcox test)

Extended Data Fig. 9. Efficacy of targeted therapy directed against Cxcr1/2 and treatment effects on immune microenvironment.

A. Kaplan-Meier plot depicting overall survival differences between patients with MDSC-high vs. MDSC-low signatures based on clustering of human TCGA PDAC samples (n=178 patients) shown in Extended Data Fig 2b. B. Representative images (left) of established iKRAS tumors treated with control and anti-Gr1 neutralizing antibody for 4 weeks with indicated staining. Scale bars: 100μm. The bar graph (right) shows quantification of each cell type as analyzed by IHC. n=6 biological replicates. Two-sided Student’s t-test. C. Tumor volume after 4 weeks of treatment with control or anti-Gr1 neutralizing antibody in mice bearing established (tumor volume ~250mm³ prior to treatment initiation) orthotopic iKRAS tumors (n=10 mice/group). Two-sided Student’s t-test. D. Expression of Cxcr2 on granulocytic MDSCs in untreated iKRAS tumors, assessed by flow cytometry and analyzed by FlowJo (n=3 tumors). E. Representative images of human PDAC tumors with indicated staining. Scale bars: 100μm. Red arrow indicates positively stained cells in the same area of a core specimen. F. UMAP demonstrating cell types in single-cell RNA sequencing of human PDAC samples from Steele et al. [10] with expression of CXCR1 and CXCR2 on granulocytes/neutrophils, and expression of CSF1R, CCR2 and TREM2 on monocytes/macrophages. G. Migration of MDSCs toward conditioned medium from iKRAS tumor cells treated with control or SX-682 (n=3 biological replicates). Student’s t-test. H. Tumor volume after 4 weeks of treatment with control or SX-682 in mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) (n=10 mice/group). Two-sided Student’s t-test. I. Stratification of infiltrating CD4⁺ and CD8⁺ T cells as naive (CD44^lowCD62L^high), central memory (CD44^highCD62L^high), and effector memory (CD44^highCD62L^low), in established iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control, SX-682 or combination (anti-LAG3+anti-41BB+SX-682) for 4 weeks assessed by flow cytometry and analyzed by FlowJo (n=3 biological replicates). Two-sided Student’s t-test. J. Quantification of total tumor associated macrophages (TAM) and dendritic cells (DC) in established iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control, SX-682 or combination (anti-LAG3+anti-41BB+SX-682) for 4 weeks assessed by flow cytometry and analyzed by FlowJo (n=3 biological replicates). Two-sided Student’s t-test. K. Expression of Cxcr2 on myeloid cells in established iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control, SX-682 or combination (anti-LAG3+anti-41BB+SX-682) for 4 weeks assessed by flow cytometry and analyzed by FlowJo (n=3 biological replicates). L. Representative images (left) of control, SX-682 or combination (anti-LAG3+anti-41BB+SX-682) treated iKRAS tumors with indicated staining. Scale bars: 100μm. The bar graphs (right) show quantification of each cell type as analyzed by IHC. n=6 biological replicates. Two-sided Student’s t-test. M. Quantification of change in the proportion of cells in cluster M_c2 as a proportion of total monocyte/macrophage cells in scRNA-seq analysis of iKRAS tumors following treatment with control, SX-682 or combination (anti-LAG3+anti-41BB+SX-682) for 4 weeks (n=3 mice/group). (*p<0.05 mixed effect model) N. Tumor volume of mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control, SX-682 or SX-682 with CD8 T cell depleting antibody (n=10 mice/group). Two-sided Student’s t-test. Data in D, G, I, J and M are presented as mean ± s.e.m.

Extended Data Fig. 10. Efficacy of ICT in combination with targeted therapy directed against Cxcr1/2 and treatment effects on immune microenvironment.

A. Tumor volume of mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control or anti-LAG3+anti-41BB antibodies or combination (anti-LAG3+anti-41BB+SX-682) for 4 weeks (n=10 mice/group). Two-sided Student’s t-test. B. Tumor volume of mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control or anti-PD1+anti-CTLA4 antibodies or SX-682 or anti-PD1+anti-CTLA4+SX-682 for 4 weeks (n=10 mice/group). Two-sided Student’s t-test. C. Body weight of mice (top left), before (pre-treatment), during (2 weeks) and after (4 weeks) treatment with control, SX-682 or combination (anti-LAG3+anti-41BB+SX-682) for 4 weeks (n=4 biological replicates). Mouse toxicity tests including creatinine, blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, and alkaline phosphatase in the indicated treatment groups (n=4 biological replicates). Representative images of H&E staining (middle) of the lung, heart, liver, kidney and spleen in the indicated treatment groups (n=4 mice/group). Inset (bottom) shows representative H&E staining of liver tissues in the indicated treatment groups at higher magnification. D. CyTOF analysis of tumors from syngeneic iKRAS 2 and iKRAS 3 tumor bearing mice with equivalent tumor volume (~1000mm³) (n=10 tumors/group). E. Quantification of tumor infiltrating CD45⁺ cells in syngeneic iKRAS 2 and iKRAS 3 tumors with equivalent tumor volume (~1000mm³) assessed by CyTOF (n=10 tumors/group). F. Overall survival of mice bearing established orthotopic iKRAS 2 and iKRAS 3 tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control or anti-LAG3+anti-41BB+SX-682 for 4 weeks (n=10 mice/group). Statistical differences were identified by Kaplan-Meier with log-rank test. G. Treatment schedule and monitoring procedures for preclinical trial to evaluate overall survival of mice bearing established autochthonous iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control or anti-PD1+anti-CTLA4 antibodies or anti-LAG3+anti-41BB antibodies or SX-682 or combination (anti-LAG3+anti-41BB+SX-682) (n=10 mice/group). Animals in the “extended” treatment group received treatment with the combination regimen for 6 months or until death. Data in E are presented as mean ± s.e.m.

Figure 1.

Prominent infiltration of myeloid immunosuppressive cells in iKRAS tumors. A. Quantification of tumor infiltrating CD45⁺ cells in syngeneic iKRAS tumors assessed by CyTOF at 4 weeks after initial tumor detection (n=10 samples/group). Two-sided Student’s t-test. B. SPADE tree derived from CyTOF analysis of whole-tumor cell population from syngeneic iKRAS PDAC tumors (n=10 tumors). Live single cells were used to construct the tree. Cell populations were identified as pancreatic ductal adenocarcinoma (PDAC) cells (EpCAM⁺CD45^-), non-immune TME cells (EpCAM^-CD45^-), CD4 or CD8 T cells (CD45⁺CD3⁺TCRβ⁺), B cells (CD45⁺B220⁺CD19⁺), Natural killer (NK) cells (CD45⁺NK1.1⁺), dendritic cells (CD45⁺CD11c⁺), MDSCs (CD45⁺CD11b⁺Gr1⁺) and macrophages (CD45⁺CD11b⁺Gr1^-F4/80⁺). C. CyTOF analysis of tumors from syngeneic and autochthonous iKRAS PDAC tumors with equivalent tumor volume (~1000mm³) (n=10 samples/group). D. Representative CFSE flow-cytometry histograms (left) showing the effect on in vitro T cell proliferation by MDSCs isolated from iKRAS tumors, and summarized result (right). Unstimulated T cells were used as negative control. Position of CFSE peaks can be used to denote the T cell division times. High and low proliferation were defined as T cell division ≥2 and ≤1, respectively (n=3 biological replicates). E. Effect on IFN-γ secretion from CD8⁺ T cells by MDSCs isolated from iKRAS tumors, measured by ELISA (n=3 biological replicates). Two-sided Student’s t-test. F. Quantification of tumor infiltrating CD4⁺ and CD8⁺ T cells in iKRAS tumors (n=3 biological replicates), assessed by flow cytometry and analyzed by FlowJo. Cell populations were identified as naive (CD44^lowCD62L^high), central memory (CD44^highCD62L^high), and effector memory (CD44^highCD62L^low). Data in A, E and F are presented as mean ± s.e.m.

Figure 2.

Prominent infiltration of myeloid immunosuppressive cells in human PDAC tumors. A. Representatives multiplex immunofluorescence images of human PDAC tissues on FFPE slides stained with the indicated proteins. Each experiment was replicated twice with similar results. B. Representatives multiplex immunofluorescence images of human PDAC tissues on FFPE slides stained with the indicated proteins. Each experiment was replicated twice with similar results. C. CIBERSORTx quantification of immune cell subsets in human PDAC samples; TCGA (n=178 patients) and ICGC-AU (n=92 patients). D. SPADE tree derived from CyTOF analysis of whole-tumor cell population from human PDAC samples (n=5 patients). Live single cells were used to construct the tree. See Supplementary Table 1 for clinicopathologic and demographic information about patients; Supplementary Table 2 for antibodies. Cell populations were identified as pancreatic ductal adenocarcinoma (PDAC) cells (EpCAM⁺CD45^-), non-immune TME cells (EpCAM^-CD45^-), CD4 (CD45⁺CD3⁺CD4⁺) or CD8 T cells (CD45⁺CD3⁺CD8⁺), B cells (CD45⁺CD19⁺), Natural killer (NK) cells (CD45⁺CD161⁺CD56⁺), Dendritic cells (CD45⁺CD33⁺HLA-DR⁺CD14^-CD15^-CD16^-CD11c⁺), MDSCs (CD45⁺CD33⁺HLA-DR^-CD11b⁺CD14^-CD15⁺ [neutrophilic/granulocytic] or CD45⁺CD33⁺HLA-DR^-CD11b⁺CD14⁺CD15^- [monocytic]) and macrophages (CD45⁺CD33⁺HLA-DR⁺CD14⁺CD15^-CD16^-CD11c⁺). E. CyTOF analysis of human PDAC tumors (n=5 patients). See Supplementary Table 1 for clinicopathologic and demographic information about patients; Supplementary Table 2 for antibodies. Red indicates high level of indicated marker expression; blue indicates no marker expression.

Figure 3.

Heterogeneity of myeloid cells in iKRAS PDAC tumors identified by single cell gene expression profiling (n=3 tumors). A. UMAP projection of immune cell clusters and B. proportion of immune cell subtypes. C. UMAP projection of myeloid cell clusters and D. proportion of myeloid cell subtypes. E. Cell cycle scoring for five myeloid cell clusters. F. UMAP projection of dendritic cell clusters and G. proportion of dendritic cell subtypes.

Figure 4.

Dysfunctional phenotype of T cells in iKRAS PDAC tumors identified by single cell gene expression profiling (n=3 tumors). A. UMAP projection of T cell clusters and B. proportion of T cell subtypes. C. Ordering of CD8⁺ T cells along pseudotime in a two-dimensional state-space defined by Monocle2. Each point corresponds to a single cell, and each color represents a CD8⁺ T cell cluster. D. Heatmap of immune checkpoint expression on various clusters of CD4⁺ and CD8⁺ T cells. E. Volcano plot showing differentially expressed genes between naïve/central memory CD8⁺ T cells and exhausted CD8⁺ T cells (left), CD8⁺ T cells and exhausted CD8⁺ T cells (middle), and naïve/central memory CD4⁺ T cells and Tregs (right).

Figure 5.

Efficacy of immune checkpoint therapy (ICT) and treatment effects on immune microenvironment. A. Tumor volume after 4 weeks of treatment with control or anti-PD1 or anti-CTLA4 or anti-41BB or anti-TIM3 or anti-OX40 or anti-LAG3 antibody in mice bearing established (tumor volume ~250mm³ prior to treatment initiation) orthotopic iKRAS tumors (n=13–14 mice/group). Two-sided Student’s t-test. B. Overall survival of mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control or anti-PD1 or anti-CTLA4 or anti-41BB or anti-TIM3 or anti-OX40 or anti-LAG3 antibody (n=13–14 mice/group). C. Tumor volume after 4 weeks of treatment with control or anti-41BB or anti-LAG3 or anti-41BB+anti-LAG3 antibodies in mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) (n=13 mice/group). Two-sided Student’s t-test. D. Overall survival of mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control or anti-41BB or anti-LAG3 or anti-41BB+anti-LAG3 antibodies (n=13 mice/group). E. Representative spectral composite image of immunofluorescence staining in human PDAC samples with the indicated proteins (left). Each experiment was replicated twice with similar results. Quantification of proportion of human PDAC samples with positive and negative staining for indicated proteins (right; n=54 patients). F. Quantification of change in proportion of immune cell subtypes in single-cell sequencing analysis of established iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) after treatment with anti-PD1, anti-CTLA4, anti-41BB or anti-LAG3 antibody for 4 weeks (n=3 tumors/group). Mixed effect model. Statistical differences in B and D were identified by Kaplan-Meier with log-rank test. Data in A, C and F are presented as mean ± s.e.m.

Figure 6.

Effects of immune checkpoint therapy (ICT) treatment on immune microenvironment. A. Fraction of top clonotypes in each T cell cluster among control and anti-41BB antibody-treated mice (n=3 mice/group). B. Relative expression levels of Granzyme K (Gzmk) and Granzyme B (Gzmb) among T cells in control and anti-41BB antibody-treated mice (n=3 mice/group). (*p<0.05 two-sided unpaired Wilcox test) C. Relative expression of gene signatures of T cell inhibition [43], terminal differentiation [25], progenitor exhaustion [33] and terminal exhaustion [28] in T cells from clusters T_c1 and T_c3. (n=1762 cells [T_c1], 1809 cells [T_c3]) (*p<0.05 two-sided unpaired Wilcox test) D. Fraction of overlapping T cell receptor CDR3 sequences between mice after 4 weeks of treatment with control or anti-PD1 or anti-CTLA4 or anti-41BB or anti-LAG3 antibody in mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) (n=3 mice/group). E. Fraction of CD3⁺CD4^-CD8^-NK1.1^- T cells among CD3⁺ T cells after 4 weeks of treatment with control or anti-PD1 or anti-CTLA4 or anti-41BB or anti-LAG3 antibody in mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) (n=3 mice/group). Mixed effect model. F. Relative expression of IL17a among subtypes of CD3⁺ T cells. G. Violin plots showing expression of β2m, H2-Aa, Cd74 and H2-Ab1 among myeloid cells from control, anti-PD1, anti-CTLA4, anti-41BB and anti-LAG3 antibody-treated tumors (n=3 tumors/group) (*p<0.05 two-sided unpaired Wilcox test). Data in E are presented as mean ± s.e.m.

Figure 7.

Efficacy of targeted therapy directed against Cxcr1/2 and treatment effects on immune microenvironment. A. Overall survival of mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control or anti-Gr1 neutralizing antibody for 4 weeks (n=10 mice/group). B. Overall survival of mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control or SX-682 for 4 weeks (n=10 mice/group). C. Quantification of total CD45⁺ immune cells, CD4⁺ and CD8⁺ T cells, granulocytic- and monocytic-MDSCs, in established iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control, SX-682 or combination (anti-LAG3+anti-41BB+SX-682) for 4 weeks assessed by flow cytometry and analyzed by FlowJo (n=3 biological replicates). Two-sided Student’s t-test. D. Relative expression of Ifng and Tnf on T cells in scRNA-seq analysis of iKRAS tumors following treatment with control, SX-682 or combination (anti-LAG3+anti-41BB+SX-682) (n=3 tumors/group). (*p<0.05 two-sided unpaired Wilcox test) E. Multiple-testing corrected 95% binomial confidence intervals on the probability of a cell in each treatment group containing a TCR CD3R sequence which overlaps that of another cluster. (*p<0.05) F. Overall survival of mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control, SX-682 or SX-682 with CD8 T cell depleting antibody (n=10 mice/group). Statistical differences in A, B and F were identified by Kaplan-Meier with log-rank test. Data in C and D are presented as mean ± s.e.m.

Figure 8.

Efficacy of ICT in combination with targeted therapy directed against Cxcr1/2 and treatment effects on immune microenvironment. A. Overall survival of mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control or anti-LAG3+anti-41BB or anti-LAG3+anti-41BB+SX-682 for 4 weeks (n=10 mice/group). B. Changes in the fraction of cells in clusters T_c2, T_c3, T_c4 and T_c5 as a proportion of total T cells in scRNA-seq analysis of iKRAS tumors following treatment with control or combination (anti-LAG3+anti-41BB+SX-682) for 4 weeks (n=3 tumors/group). Mixed effect model. C. Relative expression of effector, memory, naïve and exhausted signatures [62] in T cells from scRNA-seq analysis of iKRAS tumors after combination treatment (anti-LAG3+anti-41BB+SX-682) for 4 weeks compared to control (n=3 tumors/group). (*p<0.05 two-sided unpaired Wilcox test) D. Overall survival of mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control or anti-PD1+anti-CTLA4 antibodies or SX-682 or anti-PD1+anti-CTLA4+SX-682 for 4 weeks (n=10 mice/group). E. Overall survival of mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) that were cured (survival >6 months after treatment discontinuation) by the combination in A. (anti-LAG3+anti-41BB+SX-682) re-challenged with secondary tumors (n=5 mice/group). Treatment naïve mice were animals who had never been exposed to iKRAS cells previously. F. Overall survival of mice bearing established autochthonous iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control or anti-PD1+anti-CTLA4 antibodies or anti-LAG3+anti-41BB antibodies or SX-682 or anti-LAG3+anti-41BB+SX-682 for 4 weeks (n=10 mice/group). Animals in the “LAG3+41BB+SX-682 Extended” treatment group received extended treatment with the combination regimen for 6 months or until death. Statistical differences in A, D, E and F were identified by Kaplan-Meier with log-rank test. Data in B is presented as mean ± s.e.m.