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Chen, Limo

Diao, Lixia

Fradette, Jared

Gibbons, Don

Gibson, Laura

Konen, Jessica Marie

Li, Yanli

Miao, Shucheng

Ochieng, Joshua K

Padhye, Aparna

Peng, David H

Rodriguez, B. Leticia

Wang, Jing

Zhou, Xiaofei, PhD

English

OpenWorks @ MD Anderson

Targeting immunosuppressive classical monocytes prevents immunotherapy resistanceB. Leticia Rodriguez1, Limo Chen1, Yanli Li1,2, Shucheng Miao1, David H. Peng1, Jared J. Fradette1, Lixia Diao3, Jessica M. Konen1, Aparna Padhye1,6, Laura A. Gibson1, Joshua K.Ochieng1, Xiaofei Zhou4, Jing Wang3, Don L. Gibbons1,51Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 2Department of Obstetrics and Gynecology, Shanghai General Hospital, Shanghai JiaoTong University, School of Medicine, Shanghai, Shanghai 200080, China. 3Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 4Department ofImmunology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 5Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 6UTHealth Graduate School of Biomedical Sciences, Houston, Texas 77030, USA.Fig 3. 129/Sv mice were treated weekly with IgG control, anti-PD-1_CTLA-4, or anti-PD-1_CTLA-4_Ly6C (A) mice were subcutaneouslyimplanted with 344P cells, (B) 412P cell, and (C) 393P cells.IntroductionEmail: blrodriguez@mdanderson.orgImmune checkpoint inhibitors have revolutionized the treatment of lung cancer. However, most patients fail to respond or acquire resistance overtime. Understanding resistance mechanism to immune checkpoint blockade in NSCLC is critical to developing new therapeutic strategies andimproving patient survival. In this study, we evaluated the immune cell infiltration in anti-PD-1+anti-CTLA-4 combination therapy resistanttumors. Previously derived murine lung cancer cells lines from primary lung lesions of the KrasLA1/+/p53R172H∆g/+ genetically engineered mousemodel were used (3). The mesenchymal 344SQ tumors were found to have lower CD8 T-cell infiltration and an increase in exhaustion markers tocompared to epithelial tumors (4). Here we found several immunosuppressive cell types including T-regs, MDSC, neutrophils, and monocytes inthe IO resistant 344SQ tumors. We then assessed the role of the immune cells in contributing to resistance to combination anti-PD-1+anti-CTLA-4therapy by in vivo depletion. We found that addition of anti-Ly6C to the combination of anti-PD-1+anti-CTLA-4 was capable of complete tumoreradication. We confirmed this triple combinatorial approach in multiple models and found similar results. Increased infiltration ofCD115+CD14+Ly6C+ monocytes were found in the anti-PD-1+anti-CTLA-4 combination therapy resistant tumors. Two distinct monocytespopulations were characterized in immunotherapy resistant tumors based upon Ly6C staining. The Ly6C- monocytes differentiate into dendriticcells whereas the Ly6C+ differentiate into M2 macrophages. Dendritic cells derived from tumor-infiltrating monocytes had high levels of antigenpresentation and robust B7 signaling. Triple combination of anti-PD1+anti_CTLA+anti-LyC tumors show high levels of Ly6C- monocytes as wellas monocyte-derived dendritic cells. Additional clinical data from several lung cancer patient datasets shows a strong correlation betweenmonocytes and dendritic cells. This study suggests that immunotherapy resistance is associated with progenitor monocytes and if we can controlthe differentiation process of monocytes, we can have a lasting impact on the ability of the immune system to control tumor growth.ResultsDual PD-1/CTLA4 blockade leads to long term acquired resistance due to infiltrating immunosuppressive cellsFig 2. (A) anti-PD-1, anti-CTLA-4, combination or IgG control was IP injected into 129/Sv mice bearing KP 344SQ subcutaneously implantedtumors for 4 weeks. (B) Combination anti-PD-1_CTLA-4 or IgG control was IP injected in 129/Sv mice bearing KP 344SQ tumors for 8 weeks.(C) Flow cytometry tSNE CD45 plots from 344SQ tumors treated with combination anti-PD-1_CTLA-4 or IgG control for 8 weeks. (D)Enrichment of CD45+CD4+CD25+FoxP3+ T-regulatory cells, CD45+CD3-CD11b+Ly6G+ neutrophils, and CD45+CD3-CD14+Ly6C+monocytes from combination therapy resistant tumors. p values were calculated with t-test, n.s., no significant difference; *p<0.05; **p< 0.01;***p<0.001; ****p<0.0001.Conclusions1) Hellmann et al. “Nivolumab plus Ipilimumab in Advanced Non-Small-Cell Lung Cancer”, NEJM. 2019.2) Larkin et al. “Five-Year Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma”, NEJM. 2019.3) Gibbons et al. “Contextual extracellular cues promote tumor cell EMT and metastasis by regulating miR-200 family expression”, Genes Dev. 2009.4) Chen et al. “Metastasis is regulated via microRNA-200/ZEB1 axis control of tumor cell PD-L1 expression and intratumoralimmunosuppression”, Nat. Commun. 2014. References Funding- NIH P50CA70907-16A1- NIH P50CA70907-18- Lung cancer discovery award LCD-615867- CPRIT RP150405, RP200235, RP160652- MDACC Lung Cancer Moon Shot ProgramAnti-PD-1_CTLA-4 treatment resistance is eradicated by addition of anti-Ly6C antibody treatmentTumor-infiltrating monocytes trans-differentiation into dendritic cells via Ly6C blockadeFig 5. (A) Tumor-infiltrating monocytes sorted from 344SQ tumors have Ly6C+ and Ly6C- expression. (B) Differentiation assay scheme.Ly6C+ and Ly6C- monocytes are incubated with IL-4 to generate M2 macrophage, GM-CSF+IL4+TNFa to generate dendritic cells, GM-CSFto generate MDSC, and M-CSF + IFNg to generate M1 macrophages over 6 days at 37C +5% CO2. Flow cytometry was used to accessdifferentiation percentage. (C) Differentiation assay results; percentage of M1 macrophages, iNOS+MHCIIhigh,CD86+ from tumor treatedwith IgG CTL or combo. Percentage of M2 macrophages Arg1+, MHCII low , percentages of dendritic cells CD11c+, MHCII+, CD209+,percentage of MDSC CD11b++GR1++. Monocytes were Ly6C sorted -/+.Monocyte-derived dendritic cells enhance antitumor immunity of anti-PD-1_CTLA-4 treatment via antigen-presentationFig 6. (A) Left:C57BL/6 mice were treated weekly with combined anti-PD-1_CTLA-4 or their IgG control beginning on day 7 after subcutaneousmelanoma B16 cell injection for 8 weeks. B16 tumor growth plotted over time. (right) B16 tumors were processed for single cell FACS analysis.Percentage of CD45+CD14+ monocytes from tumors treated at week 8. (B) left; B16-OVA melanoma cells subcutaneously injected in C57BL/6mice. Day 7 mice were treated with IgG control, anti-PD-1_CTLA-4, or BSA pulsed DC and anti-PD-1_CTLA-4 or OVA-pulsed DC and anti-PD-1_CTLA-4. Pulsed OVA or BSA dendritic cell were administered via tail via on week 1 and week2. (middle) at week 4 mice were euthanized andtumor volume weighed. (C) B16-OVA tumors were processe d for FACS analysis. Percentage of CD209a+CD11c+CD11b- mature dendritic cellsshown. (D) Dendritic cells from CD11c-DTR mice were transferred to C57BL/6 to generate chimerical mice. Mice were treated with diphtheriatoxin weekly starting on week1. Mice were also treated with anti-PD-1_CTLA-4_Ly6C.Fig 8. (A) 344SQ tumor bearing 129/Sv mice were treated weekly with IgG control, anti-PD-1_CTLA-4, anti-Ly6C, or anti-PD-1_CTLA-4_Ly6Cbeginning on week2 after subcutaneous cell injection for 4 weeks. At week 6 mice were euthanized and to measure tumor weight (middle) and #of lung metastatic lesion (right). Tumors from (A) were harvested to prepare single cell suspensions for FACS analysis. (B) FACS analysis revealpercentage of CD45+CD3-CD14+Ly6C- monocytes (left), percentage of CD45+CD3-CD209+MHCII+CD11b+ monocyte derived dendritic cells(middle), percentage of CD45+CD36+CD64+CD209+MHCII+CD11b+ cells (right). The results were analyzed using ANOVA test. n.s. nosignificant difference; *p<0.05; **p< 0.01; ***p<0.001; ****p<0.0001.BackgroundFig 1. Overall survival in advanced non-small-cell lung cancer patients in open-label, phase 3 trial with patients treated with nivolumab plusipilimumab or chemotherapy. First line treatment of nivolumab plus ipilimumab resulted in a longer duration of overall survival thanchemotherapy independent of PD-L1 expression level. Hellmann et al. The New England Journal of Medicine. 2019.Nivolumab plus ipilimumab show improvement in NSCLC patientsTumor-derived dendritic cells play a vital role in maximizing the antitumor immunity of anti-PD-1_CTLA-4 treatmentLoss of Flt3/PU.1 in classical Ly6C+ monocytes in the anti-PD-1/CTLA4 resistant tumors prevents differentiationinto antigen presenting cellsFig 7. (A) Ly6C+ monocytes heterogeneity based upon CD11c and Flt3 staining of R1 (CD11c-Flt3-), R2 (CD11c-Flt3+), and R3 (CD11c+Flt3+)monocytes. (B) RT-QCR of Sfpi1 (PU.1) expression of Ly6C+ monocytes from IgG CTL or combo treated tumors. (C) RT-QCR of Flt3 expressionof Ly6C+ monocytes from IgG CTL or combo treated tumors. (D) Percentage of PU.1 on R1 monocytes from (A) from IgG CTL or combo treatedtumors.Monocyte infiltration in anti-PD-1/CTLA-4 resistant tumor are mediated by IFN-g and the CCL2/CCR2 axisFig 4. (A) 344SQ tumors were treated with IgG control or anti-PD-1_CTLA4 after week 8 tumors were processing for single cells FACSanalysis. Percentage of CD115+CD14+Ly6C+ and CD115+CD14+Ly6C- monocytes was compared and found to be much higher incombination resistant tumors. (B) Classical monocyte and non-classical monocytes expression in melanoma patients stratified based onresponse of progressive disease/stable disease (PD/SD) or progressive response/complete response (PR/CR). (C) IFN-g concentration fromtumors treated with IgG control or combination therapy resistant tumors. (D) Monocytes sorted from 344SQ tumors were treated with IFNgfor 12 hours. Expression of Ly6C was evaluated over time.(E) Tumors from 344SQ tumors were treated with IgG control or combination therapy over week 8. Total levels of CLL2 (F) and CCR2expression on the monocytes were determined via FACS analysis. (G) Transwell migration assays were completed on sorted monocytes from344SQ tumors. Sorted monocytes were added to top chamber and transmigration was determined. (H) Heat map of mRNA levels betweenmonocyte gene signature and IFNg pathway in TCGA lung adenocarcinoma. (I) Correlation between CD14/CD16a/CD16b and CCL2 orCCR2 TCGA-lung adenocarcinoma.

Targeting immunosuppressive classical monocytes prevents immunotherapy resistance

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1053&context=leading-edge

Targeting immunosuppressive classical monocytes prevents immunotherapy resistance

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