Figure S2 shows the gating strategy for T panel in immune profiling analysis.

This figure shows the single cell RNA sequencing analysis of EGFR high tumor subcluster in the humanized LUSC PDX model.

Individual whole slide images of HNSCC PDX tumor used for TME analysis.

Figure S3 shows the gating strategy for M panel in immune profiling analysis.

This figure shows the selection of cell lines used for in vitro evaluation of the effects of amivantamab on the protein and surface levels EGFR, MET, LDHA and SLC16A3.

Abstract Background: The Aryl hydrocarbon receptor (AhR) is one of the most predominant regulators of cancer metabolism. AhR plays a crucial role in inhibiting the activation of immune cells and promoting the growth of tumor cells. Here, we propose that a best-in-class AhR inhibitor, DA-4505, improves anti-tumor efficacy in combination with anti-PD-1. Methods: To explore the anti-tumor effects, DA-4505 was administered at a daily dose of 10 mg/kg either alone or in combination with anti-PD-1 (10 mg/kg) in a syngeneic mouse model. Tumor volume, survival rates, and metastasis were measured, and immune profiles were evaluated using mIHC, flow cytometry, and scRNAseq. Results: The synergistic anti-tumor effects of the DA-4505 and aPD-1 combination therapy were observed across four mouse tumor models (CT26, 4T1, LLC, TC1). The combination group demonstrated a significant improvement in anti-tumor efficacy compared to the monotherapy groups. Despite the moderate response of aPD-1 in the CT26 colon tumor model, the combination group displayed heightened anti-tumor effects through increase in CD8 T cell activity (P<0.05). In the LLC and TC1 lung cancer models, the combination therapy demonstrated improved anti-tumor effects and survival rates (P<0.001). Notably, the LLC model exhibited a significant increase in M1-type macrophages, while the TC1 model showed a significant augmentation in functional markers of CD3+ T cells within the combination group (P<0.05). In both models, there was a significant reduction in the ratio of immunosuppressive M-MDSC and PMN-MDSC in the combination group. Immune depletion assays elucidated that DA-4505 mediated anti-tumor effects by increase in CD8+ T cells, NK cells, and macrophages in the CT26 model. In contrast, the LLC model exhibited the contribution of macrophages and CD4+ T cells to anti-tumor effects in the combination group (P<0.05). To assess the impact on metastasis inhibition, an orthotopic model of 4T1 mouse tumor was established, revealing a significantly increased anti-tumor effects and survival rate in the combination group, accompanied by a reduced lung metastasis ratio. H&E staining confirmed diminished migration of tumor tissue to adjacent areas. scRNAseq analysis of CT26 tumor samples indicated consistent changes at the gene expression level, reflecting a decrease in metastasis-related MMP and VEGF genes in the combination group and an increasing trend in cytotoxicity-related genes following DA-4505 treatment. Conclusion: The AhR inhibitor, DA-4505, has demonstrated enhanced anti-tumor efficacy when administered in combination with anti-PD-1. Furthermore, it not only promoted tumor elimination but also demonstrated significant efficacy in inhibiting metastasis. This study highlights the potential of combining AhR inhibitors with immunotherapies for clinical treatment. Citation Format: Dong Kwon Kim, Sujeong Baek, Seung Min Yang, Young Taek Kim, Joon Yeon Hwang, Seul Lee, Seong-san Kang, Mi hyun Kim, Heekyung Han, Kwangmin Na, Chai Young Lee, Yu Jin Han, Taedong Han, Hyounmie Doh, Jongho Cho, Dajeong Kim, Daewon Cha, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Mi Ran Yun, Jii Bum Lee, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho. Enhanced anti-cancer efficacy of the AhR Inhibitor DA-4505 in combination with anti-PD-1 treatment: Attenuation of lung metastasis and elimination of tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4039.

Natural killer (NK) cells have recently shown renewed promise as therapeutic cells for use in treating hematologic cancer indications. Despite this promise, NK cell manufacturing workflows remain largely manual, open, and disconnected, and depend on feeders, as well as outdated unit operations or processes, often utilizing research-grade reagents. Successful scale-up of NK cells critically depends on the availability and performance of nutrient-rich expansion media and cryopreservation conditions that are conducive to high cell viability and recovery post-thaw. In this paper we used Cytiva hardware and media to expand the NK92 cell line in a model process that is suitable for GMP and clinical manufacturing of NK cells. We tested a range of cryopreservation factors including cooling rate, a range of DMSO-containing and DMSO-free cryoprotectants, ice nucleation, and cell density. Higher post-thaw recovery was seen in cryobags over cryovials cooled in identical conditions, and cooling rates of 1°C/min or 2°C/min optimal for cryopreservation in DMSO-containing and DMSO-free cryoprotectants respectively. Higher cell densities of 5x107 cells/ml gave higher post-thaw viability than those cryopreserved at either 1x106 or 5x106 cells/ml. This enabled us to automate, close and connect unit operations within the workflow while demonstrating superior expansion and cryopreservation of NK92 cells. Cellular outputs and performance were conducive to clinical dosing regimens, serving as a proof-of-concept for future clinical and commercial manufacturing.

Upregulation of EGFR and MET in HNSCC PDX (YHIM-3003) tumor induced increased expression of immune checkpoints regulators in the EGFRHIGH/METHIGH subcluster (EMHIGH). A, Volcano plot of top 50 genes in EGFRHIGH/METHIGH against EGFRLOW/METLOW tumor subcluster analyzed by log2 fold change (FC) against P-values. Red dots indicating transcripts with significantly increased fold changes including MET, PD-L1, and MET-regulated genes. B, Expression of STAT-4/PD-L1 (MET, STAT4, CD274), MET-regulated (BACE2, STK40, PRSS23, DPYD, CAV1, S100A4, PYGL), and MET-related immune checkpoints (HAVCR2, CD276) generally increased in the EGFRHIGH/METHIGH tumor subcluster compared to the EGFRLOW/METLOW subcluster. C, Expression of MET-related markers in different treatment groups.

Antitumor effects of amivantamab w/wo pembrolizumab in HNSCC and LUSC tumor–bearing humanized PDX preclinical models. A, The intensity of EGFR and MET in YHIM-3003 (HNSCC) and YHIM-2010 (LUSC) tumors. B, The tumor progression of YHIM-3003 model over 19 days showing significant tumor regression by the combination treatment of amivantamab (30 mpk) and pembrolizumab (10 mpk) compared to single treatment of amivantamab and pembrolizumab (P < 0.001, n = 10 in each group). C, Tumor growth inhibition represented in a waterfall plot of HNSCC PDX model at day 19. D, Survival curve of HNSCC PDX demonstrating improved survival by the combination treatment (n = 10 in each group). E, Treatment of LUSC PDX using amivantamab at 30 mpk in combination with pembrolizumab (10 mpk, n = 10 in each group). No rebound of tumor growth was observed after the termination of amivantamab and combination treatment. F, Tumor regression in LUSC PDX showing significant tumor reduction by combination treatment of amivantamab (10 mpk) and pembrolizumab (10 mpk) compared to single treatment of amivantamab (P < 0.05) and pembrolizumab (P < 0.001, n = 10 in each group). G, Survival curve of LUSC PDX demonstrating improved survival by the combination treatment.

Flow cytometry analysis of memory subsets of T cells in the different treatment groups of humanized HNSCC (YHIM-3003) and LUSC (YHIM-2010) PDX tumor of internally sacrificed mice (n = 5 in each treatment group). A, Heatmap of memory T cell subsets (central memory, effector memory, and effector T cells) and activation markers in the tumor samples of HNSCC PDX. B, Heatmap of memory T-cell subsets (central memory, effector memory, and effector T cells) and activation markers in the tumor samples of YHIM-2010. C, Factors that combination of amivantamab and pembrolizumab positively affected in each humanized PDX model and both models shared enhancement of CD8+ T central memory subset by combination therapy. D, Tumor reactive (CEA-stained) CD8 T cells in HNSCC PDX tumor were abundant in the combination treatment group and were significantly higher in proportion compared to the control group (8.28 ± 2.67 and 3.02 ± 0.75, respectively, P < 0.05).

Single-cell RNA sequencing analysis of EGFRHIGH/METHIGH and EGFRLOW/METLOW subclusters in the tumor of humanized HNSCC (YHIM-3003) PDX mice showing relative increase in EGFR and MET expressing subcluster after treatment of pembrolizumab (n = 5 in each treatment group). Top 50 genes were analyzed by log2 fold change (FC) against P-values and immune related genes were divided into three categories as following: immunomodulation, metastasis potential and cancer progression (drug resistance/cancer stemness). A, Heatmap of tumor indicating a region of tumor subcluster with elevated expression of both EGFR and MET. B, Density plot illustrating EGFRHIGH/METHIGH and EGFRLOW/METLOW subclusters in treatment groups. Expression of EGFR and MET was relatively higher in the pembrolizumab treated group compared to the other treatment groups (C). D, Tumor subcluster with elevated dual expression of EGFR and MET (EMHIGH) was define and analyzed for DEGs. E, EGFR and MET in EMHIGH and EMLOW, showing increased expression of both markers in EMHIGH tumor cluster. F, DEG analysis of the top genes in the EGFRHIGH/METHIGH tumor subcluster showing genes related to immunomodulation, tumor metastasis, drug resistance and cancer stemness in volcano plot. G, Multiplex IHC of the tumor tissue showing reduced expression of EGFR in tumor treated with combination treatment. EGFR+ site, colored green, represents tumor regions that were stained positive for EGFR and does not directly translate to the level of expression. Tumor with damaged or indistinct regions of tumor nest was treated as an outlier and was removed from each group. H, Fluorescence intensity of EGFR in randomized region of interest sites converted into average H-scores. H-scores ranged from 0 (H-0, blue) to 3 (H-3, red), with H-3 representing the highest intensity as shown in the top. Average H-score of EGFR expression in pembrolizumab treated mice was significantly higher than the other groups (P < 0.01).

Multiplex IHC showing T cell subpopulations in the tumor microenvironment of HNSCC PDX (YHIM-3003) tumor after combination treatment of amivantamab and pembrolizumab. A, Whole slide images of tumor scan showing segmentation of tissues and cells. Helper CD4 and cytotoxic CD8 T cells were marked with FoxP3 (regulatory T cells) and GZMB (active cytotoxic T cells). Cancer cells were stained with Pan-CK. B, Stained whole slide images were quantified and presented in bar plots showing GZMB+ CD8 T cells and regulatory T cells in the tumor microenvironment, tumor nest and stroma in different treatment groups. Proportion of GZMB+ CD8 T cells in the total TME and the tumor nest was significantly increased in the combination group (P < 0.05). Each bar in the bar plot represents five mice that were internally sacrificed.

IntroductionTo understand the immune system within the tumor microenvironment (TME) of non-small cell lung cancer (NSCLC), it is crucial to elucidate the characteristics of molecules associated with T cell activation.MethodsWe conducted an in-depth analysis using single-cell RNA sequencing data obtained from tissue samples of 19 NSCLC patients. T cells were classified based on the Tumor Proportion Score (TPS) within the tumor region, and molecular markers associated with activation and exhaustion were analyzed in T cells from high TPS areas.ResultsNotably, tetraspanins CD81 and CD82, belonging to the tetraspanin protein family, were found to be expressed in activated T cells, particularly in cytotoxic T cells. These tetraspanins showed strong correlations with activation and exhaustion markers. In vitro experiments confirmed increased expression of CD81 and CD82 in IL-2-stimulated T cells. T cells were categorized into CD81highCD82high and CD81lowCD82low groups based on their expression levels, with CD81highCD82high T cells exhibiting elevated activation markers such as CD25 and CD69 compared to CD81lowCD82low T cells. This trend was consistent across CD3+, CD8+, and CD4+ T cell subsets. Moreover, CD81highCD82high T cells, when stimulated with anti-CD3, demonstrated enhanced secretion of cytokines such as IFN-γ, TNF-α, and IL-2, along with an increase in the proportion of memory T cells. Bulk RNA sequencing results after sorting CD81highCD82high and CD81lowCD82low T cells consistently supported the roles of CD81 and CD82. Experiments with overexpressed CD81 and CD82 showed increased cytotoxicity against target cells.DiscussionThese findings highlight the multifaceted roles of CD81 and CD82 in T cell activation, cytokine production, memory subset accumulation, and target cell cytolysis. Therefore, these findings suggest the potential of CD81 and CD82 as promising candidates for co-stimulatory molecules in immune therapeutic strategies for cancer treatment within the intricate TME.

Abstract Background: The role of tetraspanins, particularly CD81 and CD82, in T cell activation, shedding light on their contribution to anti-tumor effects in the tumor microenvironment. The findings provide insights for potential applications of these tetraspanins in immunotherapy. Method: The study employed 10x Genomics for single-cell RNA sequencing and processed the data using Seurat. It focused on the tumor microenvironment, categorizing cell types, especially TIL subsets. T cells were sorted based on CD81 and CD82 expression and activated, and their activation markers and cytokine production were analyzed. Bulk RNA sequencing was performed on both sorted T cells and tumor samples, and the TCR repertoire was examined. Cytotoxicity of CD81 and CD82 transduced cells was measured using the xCELLigence RTCA HT system. Result: Tetraspanins have been linked to T cell co-stimulation and activation, with CD81 and CD82 playing a significant role in TCR signaling and activation. Single-cell and bulk RNA sequencing data from lung cancer patients reveal high expression of CD81 and CD82 in the tumor microenvironment, especially in T cells, correlating with T cell activation and cytokine production. In vitro experiments confirm the activation and effector cytokines, especially IFN-γ, TNF-α, and IL-2, producing potential of T cells expressing CD81 and CD82, suggesting their importance in anti-cancer immunity. Overexpression of CD81 and CD82 enhances T cell cytotoxicity against cancer cells, further supporting their role in T cell function. Despite some limitations, these findings shed light on the significant impact of tetraspanins in modulating immune responses, particularly in the context of cancer immunotherapy. Conclusion: We report that CD81 and CD82 serve as specific markers for activated T cells within the TME. CD81 and CD82 are correlated with the activation and regulation of T cells, and they may also mediate the regulation of relevant cytokines. Overall, our findings provide better insights into the molecular mechanisms regulating TILs present in the TME of non-small cell carcinoma and propose CD81 and CD82 as associated markers. These discoveries could serve as a foundation for the development of new immunotherapies targeting the TME. Citation Format: Kwangmin Na, Seul Lee, Dong Kwon Kim, Young Seob Kim, Joon Yeon Hwang, Seong-san Kang, Sujeong Baek, Chai Young Lee, Seung Min Yang, Yu Jin Han, Mi hyun Kim, Heekyung Han, Youngtaek Kim, Jae Hwan Kim, Seunghyun Jeon, Youngseon Byeon, Jii Bum Lee, Sun Min Lim, Min Hee Hong, Kyoung-Ho Pyo, Byoung Chul Cho. CD81 and CD82 in tumor infiltrating lymphocytes within the NSCLC tumor microenvironment play a crucial role in T cell activation and cytokine production [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6805.

LDHA and SLC16A3 are important regulators of glycolytic pathways and were upregulated in EGFRHIGH/METHIGH tumor subcluster of HNSCC PDX (YHIM-3003). A, Correlation between expression of EGFR and biomarkers based on TCGA database. LDHA and SLC16A3 expression was positively correlated with expression of EGFR in both HNSCC and LUSC. B, Expression of LDHA and SLC16A3 was significantly increased in EGFRHIGH/METHIGH tumor subcluster (EMHIGH) of HNSCC PDX (left). Additionally, expression of LDHA and SLC16A3 increased in pembrolizumab treated group. C, Regulators of glycolysis (HK2, GPI, ALDOA, PGK1, PGAM1, ENO1, ENO2) comparatively increased in the EMHIGH tumor subcluster (left) and pembrolizumab treated group (right). D, Regulators of hypoxia (HIF1A, HDAC1, KDM1A, KDM2A) and downstream signaling markers (CA9, VEGFA, TWIST1) increased in the EMHIGH tumor subcluster (left) and pembrolizumab treated group (right). E, Total protein and surface expression of EGFR and MET demonstrated strong correlation in HNSCC and LUSC cell lines (left). H1703, LUSC human cancer cell, was treated with IFNγ for 24 hours to mimic the physiological response of pembrolizumab in the TME (right). Induction of IFNγ, though not significant, upregulated the expression of pEGFR, while pMET level significantly increased (P < 0.01). F, Protein expression of EGFR/p-EGFR, MET/p-MET, MCT4 (SLC16A3), and LDHA after 72 hours of amivantamab at 10 mg/mL in H1703. IFNγ was treated at 100 ng/mL for 24 hours (left). Amivantamab reduced the expression of these genes under IFNγ-induced conditions (right).

Abstract Background Live biotherapeutic products (LBPs) are known to enhance immune responses through the GUT-TME axis. Here, we investigate the GUT-TME-related immune cell profiling and signals associated with the anti-cancer effects of CJRB-101. Methods Tumors from NSCLC patients (anti-PD-1 refractory) were transplanted in Hu-CD34-NSG to establish humanized patient-derived xenograft (PDX) models. CJRB-101 was administered at 1x109 CFU (p.o., BID) or combination with anti-PD-1 (10 mpk, i.p., BIW). TME was analyzed using multiplex IHC, flow cytometry and scRNA sequencing. Samples were collected from C3PQ syngeneic mice at multiple timepoints for GUT-TME immune cell profiling. For depletion assay, immune cells were individually depleted during the combination treatment. TLR4-mediated mechanism was evaluated using ex vivo and in vivo assay treated with CJRB-101 or cell membrane of CJRB-101. Results CJRB-101 combined with pembrolizumab effectively suppressed tumor growth in anti-PD-1 resistant PDX models. Further analysis revealed a correlation between the activity of NK cells and angiogenesis inhibition. Abundance of NK/NKT was higher in the CJRB-101 treated group compared to the vehicle group in multiple PDX models. The expression of GZMB and IFNG in NK/NKT cells was significantly higher in the CJRB-101 treated group compared to the vehicle group in YHIM2014 (p<0.001). CJRB-101 treated group showed significantly higher expression of antiangiogenic IL1B (p<0.001) while the expression of pro-angiogenic VEGFA (p=0.002) was lower compared to the vehicle group in YHIM2014 cancer cells. Macrophages in the intestine were increased at Day 3 in the CJRB-101 group compared to anti-PD1, while NK cells, granulocytes, CD3+, CD4+, CD8+ T cells increased at Day 10 in the syngeneic model. Depletion assay confirmed that macrophages, CD8+ T cells and neutrophils were pivotal in the anti-cancer effects of CJRB-101. We observed that TAK242 and MD2 reduced the IL-6 secretion of Raw264.7 in a dose-dependent manner. The cell membrane played a key role in increasing BMDM M1 polarization and repolarization of M2 to M1, and that inhibition of TLR4 resulted in a decrease in BMDM repolarization. TLR inhibition also demonstrated that TGI decreased from 34% to 20% when treated with cell membrane + TAK242 compared to cell membrane monotherapy. Conclusions This study showed that macrophages were the dominant immune population in the early stages then T cells (CD3, CD4, CD8), NK cells and granulocytes became more active in the latter stages of the GUT-TME-axis immune response. In vivo results indicated that anti-cancer efficacy of CJRB-101 is immune-cell driven by TLR4-dependent stimulation of key immune cell populations (macrophages, CD8+ T cells, neutrophils) modulated by the cell membrane of CJRB-101. CJRB-101 is currently undergoing clinical investigation for treatment of patients with advanced NSCLC. Citation Format: Arim Min, Bo-eun Kwon, Seong-san Kang, Sujeong Baek, Junwon Yang, Hyunkyung Park, Jieun Im, Hyunjeong Kim, Jaemin Kim, Jieun Kwon, Dong Kwon Kim, Jii Bum Lee, Hyeonseok Oh, Seung Min Yang, Yu Jin Han, Mi hyun Kim, Heekyung Han, Kwangmin Na, Young Taek Kim, Mi Ran Yun, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho. CJRB-101 induces immune responses through the GUT-TME axis in immune cell-driven mechanism in lung cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4993.

Abstract Immune checkpoint inhibitors are effective first-line therapy for solid cancers. However, low response rate and acquired resistance over time has led to the need for additional therapeutic options. Here, we evaluated synergistic antitumor efficacy of EGFR × MET targeting bispecific antibody, amivantamab with PD-L1 immunotherapy, pembrolizumab in head and neck squamous cell carcinoma (HNSCC) and lung squamous cell carcinoma tumor–bearing humanized patient-derived xenograft (PDX) models. We demonstrated that pembrolizumab or amivantamab alone was ineffective and that combination treatment induced a significant reduction of tumor growth in both models (P < 0.0001 and P < 0.01, respectively). It appeared that combination of amivantamab and pembrolizumab significantly enhanced infiltration of granzyme B–producing CD8 T cells was in the TME of HNSCC PDX (P < 0.01) and enhanced neoantigen-associated central memory CD8 T cells in circulating immune cells. Analysis of single-cell RNA transcriptomics suggested that the tumor cells dramatically upregulated EGFR and MET in response to PD-L1 immunotherapy, potentially creating a metabolic state fit for tumor persistence in the tumor microenvironment (TME) and rendered pembrolizumab ineffective. We demonstrated that EGFRHIGHMETHIGH subcluster displayed an increased expression of genes implicated in production of lactate [SLC16A3 and lactate dehydrogenase A (LDHA)] compared to the EGFRLOWMETLOW cluster. Accumulation of lactate in the TME has been associated with immunosuppression by hindering the infiltration of tumor killing CD8 T and NK cells. This study proved that amivantamab reduced glycolytic markers in the EGFRHIGHMETHIGH subcluster including SLC16A3 and LDHA and highlighted remodeling of the TME by combination treatment, providing rationale for additional therapy of amivantamab with PD-1 immunotherapy. Significance: Amivantamab in synergy with pembrolizumab effectively eradicated EGFRHIGHMETHIGH tumor subcluster in the tumor microenvironment of head and neck squamous cell carcinoma and overcame resistance against anti-PD-1 immunotherapy.

Abstract Backgrounds: Live biotherapeutic products (LBPs) emerged as potential therapeutics to overcome the limitation of ICIs. This research shows that CJRB-101, a novel bacterial strain, can improve anti-tumor effects in synergy with pembrolizumab in non-small cell lung cancer (NSCLC). Objectives and Methods: Tumors from NSCLC patients (anti-PD-1 refractory and resistant) were transplanted into Hu-CD34-NSG to establish humanized patient-derived xenograft (PDX) mice models. Five models (YHIM-2003, 2004, 2009, 2010 and 2014) were treated with CJRB-101 at low (5 × 107 CFU) or high (109 CFU) doses, or with pembrolizumab (10 mg/kg, i.p., Q5D) or in combination. Tumor growth inhibition (TGI) rate was measured. Tumor microenvironment (TME) was analyzed using multiplex IHC, flow cytometry and single cell RNA sequencing. Ex-vivo assays were performed to validate in silico findings. Results: Tumor in PDX models was unresponsive to pembrolizumab alone, however, in combination with CJRB-101 effectively suppressed tumor growth. The synergy was highlighted in YHIM-2009 where TGI was 10-fold higher (56%) than pembrolizumab group (5%). Immune profiling revealed that macrophages may be responsible for the anti-tumor effects of CJRB-101. IHC showed significantly increased antigen presenting specialized DCs (CD16+CD68−CD11c+) and granzyme B+ CD8+ T cells in the tumor by CJRB-101 compared to pembrolizumab (p<0.01). This suggested that CJRB-101 induced infiltration of cytotoxic CD8 T cells into the tumor nest by enhancing antigen presenting machinery. Trajectory analysis showed that CJRB-101 induced repolarization of M2 to M1 macrophages, characterized by high expression of CXCL9/10. CXCL9+/10+ M1 macrophages were comparatively more abundant in the combination group (23.11%) than the pembrolizumab group (0.91%). CXCL9/CXCL10 expression in macrophages was higher in the CJRB-101 group compared to the pembrolizumab group (p<0.0001). The combination group (10.84%) had a higher relative abundance of CD8+ T cells compared to the pembrolizumab group (1.58%) and higher IFNγ expression in CD8+ T cells compared to the pembrolizumab group (p=0.0152), suggesting that CJRB-101 repolarized macrophages and recruited active CD8+ T cells. Co-culture assays using bone marrow-derived macrophages validated that CJRB-101 drove differentiation towards F4/80+ or MHC II+ expressing M1 macrophage (p<0.0001) and repolarized existing M2 (CD206+) to M1 (p=0.0002). Conclusion: Combination treatment of CJRB-101 with anti-PD-1 showed synergistic anti-tumor effects via repolarization of M2 to M1 macrophages, leading to activation of CD8+ T cells in TME. Especially, CXCL9+/10+ M1 macrophage playing a key role in TGI induced by CJRB-101 in NSCLC models. Findings from this study provided rationale for clinical investigation of CJRB-101. Citation Format: Arim Min, Chun-bong Synn, Seong-san Kang, Bo-eun Kwon, Junwon Yang, Hyunkyung Park, Jieun Im, Hyunjeong Kim, Sujeong Beak, Dong Kwon Kim, Jii Bum Lee, Hyeonseok Oh, Seung Min Yang, Yu Jin Han, Mi hyun Kim, Heekyung Han, Kwangmin Na, Young Taek Kim, Sungwoo Lee, Mi Ran Yun, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Ji Yun Lee, Chang Gon Kim, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho. A novel bacterial strain, CJRB-101, induces anti-cancer effects by repolarization of M2 to CXCL9 and CXCL10 dual expressing M1 macrophages in humanized non-small cell lung cancer mice models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6433.

Background: The Aryl hydrocarbon receptor (AhR) is one of the most predominant regulators of cancer metabolism. The AhR exerts important immunosuppressive functions by activating Treg cells and myeloid-derived suppressor cells and repressing CD8+ effector T cells. Here, we propose that a best-in-class AhR inhibitor, DA-4505, improves anti-tumor efficacy via modulation of tumor immune surveillance compared to BAY2416964, an AHR antagonist drug candidate being studied in the clinical phase. Methods: To evaluate anti-tumor effects of DA-4505 and BAY2416964, the two AhR inhibitors were dosed at 10 mg/kg once daily alone or in combination with aPD-1 (10 mg/kg) in surgical and chemotherapy models, and a PDX model (YHIM2004). Tumor volume, relapse, and survival were evaluated, and immune profiles were analyzed with IHC, flow cytometry, and scRNAseq. Results: A significant tumor reduction appeared in the CT26 and 4T1 tumor models after the DA-4505 treatment compared to vehicle group (P<0.05). In contrast, DA-4505 treatment did not induce significant tumor regression compared to vehicle group in tumor-bearing NOG mice, suggesting that anti-tumor effects of DA-4505 were driven by immunologic mechanisms. To evaluate the role of DA-4505 in conjunction with surgery, DA-4505 alone or in combination with anti-PD-1 was given prior to and following resection of the tumors in 4T1 tumor-bearing mice. Survival of mice treated with DA-4505 alone or DA-4505 combined with anti-PD-1 was significantly prolonged after resection compared to aPD-1 treatment group (P<0.05). In addition, there were four mice that did not have a relapse by treating DA-4505 with or without aPD-1 after surgery (4/5). A tumor regression also appeared in the YHIM2004-engrafted humanized mouse study. A tumor reduction was shown by treating DA-4505 alone or in combination with pembrolizumab compared to vehicle group (P<0.05). Next, we co-administered an AhR inhibitor and aPD-1 as a partner to improve the antitumor effects of chemotherapy. The DA-4505 add-on group showed tumor regression when compared with the combination therapy group treated with aPD-1 and chemotherapy (P<0.0001). In addition, a significant increase in survival rate was shown in the group treated with a DA-4505 add-on compared to vehicle group (P<0.001). Analysis of scRNAseq showed that M1 macrophage expressing CCL7 and CCL8 were increased in DA-4505 treated group compared to the vehicle and aPD-1 groups. This suggests that immune modulatory effect of DA-4505 may be due to enhanced recruitment of immune cells into the tumor site by macrophages with high chemotactic activity. Conclusion: The AhR inhibitor DA-4505 demonstrated an improvement in anti-tumor efficacy. In addition, it has shown a synergistic effect when combined with aPD-1. Discoveries from this study provide a preclinical rationale for future clinical implications in solid tumor. Citation Format: DongKwon Kim, Sujeong Baek, Seung Min Yang, Yu Jin Han, Seong-san Kang, Chun-Bong Synn, Mi Hyun Kim, Heekyung Han, Kwangmin Na, Young Taek Kim, Sungwoo Lee, Taedong Han, Hyounmie Doh, Jongho Cho, Dajeong Kim, Daewon Cha, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Mi Ran Yun, Ji Yun Lee, Jii Bum Lee, Chang Gon Kim, Min Hee Hong, Sun Min Lim, Byoung Chul Cho, Kyoung-Ho Pyo. A novel AhR inhibitor ‘DA-4505’ improved the anti-cancer efficacy of surgical and chemotherapy via synergistic anti-tumor effects of aPD-1. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5107.

Prostaglandin E2 (PGE2) is widely recognized as one of the major bioactive lipids that, with the striking regenerative potential, promote drug-resistance in cancer cells as well as immune evasion in the tumor microenvironment (TME). Primarily driven by apoptotic cell death, PGE2 is thought to elicit wound-healing responses to help provide an immunosuppressive and proliferative niche that supports cancer stem cell repopulation and thereby therapy-resistance. While COX1/2 inhibitors that attenuate PGE2 production have shown promising anti-cancer effects in various (pre-)clinical settings, the gastrointestinal- and cardiotoxicities precluded their development as anti-cancer agents. It is anticipated that specific targeting of PGE2 signaling via its cognate receptors constitutes a safer and potentially more effective approach. Of the receptor subtypes EP1-4, Gα,s-coupled EP2 and EP4 are believed to be directly involved in immunosuppressive effects of PGE2.OCT-598 is a novel, highly potent and selective EP2/EP4 dual antagonist with Ki values of 23 nM and 0.2 nM vs EP2 and EP4, respectively. PGE2 inhibited normal differentiation of human monocytes into CD1a+CD16- dendritic cells under the presence of GM-CSF and IL-4 and promoted differentiation towards CD1a-CD16+ macrophages in vitro. However, EP2/EP4 dual inhibition by OCT-598 reversed this phenomenon to a greater extent than either EP2- or EP4-specific inhibitor alone. In vivo, OCT-598 effected tumor growth inhibition in multiple syngeneic mouse models as a single agent as well as in combination with an immune checkpoint blocker (ICB). Furthermore, the addition of OCT-598 to the lung cancer standard-of-care regimen (anti-PD-1 plus chemotherapy) in TC-1 mouse lung adenocarcinoma model gave rise to complete tumor regression. In conclusion, dual blockade of EP2 and EP4 by OCT-598 is shown to be a compelling strategy to reinforce antitumor effects by thwarting PGE2-mediated therapy resistance and immune evasion.Findings from this study provide a rationale for clinical development of OCT-598 as a therapeutic option for human malignant cancers. Citation Format: Youngrae Lee, Sujeong Baek, Dong Kwon Kim, Yeri Lee, Donggeon Kim, Seongin Jo, Sang Kyun Lim, Young Sook Shin, Soonsang Kwon, Seung Min Yang, Young Taek Kim, Seong-San Kang, Chun-Bong Synn, Kwangmin Na, Mi Hyun Kim, Heekyung Han, Yu Jin Han, Sungwoo Lee, Jae Hwan Kim, Mi Ran Yun, Youngseon Byeon, Young Seob Kim, Ji Yun Lee, Jii Bum Lee, Chang Gon Kim, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho, Taeyoung Yoon. OCT-598, a novel EP2/EP4 dual antagonist, promotes anti-tumor immune responses in syngeneic mouse tumor models in combination with standard-of-care chemo- and immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3234.