Jeuk (South Korea)

• Two-layered prestressing system to avoid stress concentration at ends of girder • Bottom layer of tendons with conventional anchorages over whole length of girder • Top layer of shorter tendons anchored with anchorage dead-end (onion) • Use of anchorage dead and live-end makes prestressing closer to moment distribution • Significant reduction of prestress-induced stresses like bearing and bursting stresses This study introduces a two-layered tendon system to prestress more efficiently the prestressed steel-concrete composite girder by separating the anchorages traditionally concentrated at the girder ends. The bottom layer uses live ends and runs over the length of the girder while the top layer is shorter with dead ends disposed nearer to the central part of the girder. Such disposition makes it possible to apply the prestressing force where needed, reduces prestressing stresses, and mitigates the congestion of reinforcement and anchorages at the girder ends. Specifically, the anchorage dead-ends of the shorter tendons in the upper layer focus on the largest tensile stresses at mid-span caused by the positive moment. The proposed system is investigated at the element-level on a 4.6 m-long half-girder girder to verify the behavior of the dead end-anchored tendons. Then, the system is examined at the structure-level on a 72 m-long rigid-frame bridge. For the 72 m-long girder, a preliminary parametric study revealed that the appropriate length of the dead-end anchored tendon in the upper layer was 75% of the girder's length. The results show that the proposed system works as desired and relieves significantly the girder from undesirable stresses like bearing and bursting stresses.

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.

Introduction:Despite the availability of several epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), most patients with non-small-cell lung cancer (NSCLC) eventually develop resistance to these agents. Notably, EGFR_C797S mutations confer resistance to the third-generation EGFR-TKI osimertinib and no approved post-osimertinib targeted pharmacology options are currently available. BLU-945 is a novel, reversible, and orally available next-generation EGFR-TKI that selectively targets EGFR-activating (EGFRm) and resistance mutations (including EGFR_C797S) with nanomolar potency while sparing wild-type EGFR in vitro.Methods:In vitro activity of BLU-945 as a single agent and in combination with osimertinib was tested in engineered EGFR-mutant cell lines as well as patient-derived cells and patient-derived organoids. In vivo activity was evaluated in osimertinib-resistant patient-derived xenograft mouse models. Three patient cases from the global, first-in-human, phase I/II SYMPHONY trial (NCT04862780) demonstrating the clinical efficacy of BLU-945 were reported.Results:In vitro BLU-945 demonstrated inhibited cell viability and growth of EGFR-mutant/osimertinib-resistant cell lines. BLU-945 demonstrated in vivo tumor shrinkage in osimertinib-resistant models of NSCLC (osimertinib second line: EGFR_L858R/C797S and third line: EGFR_ex19del/T790M/C797S and L858R/T790M/C797S) both as monotherapy and in combination with osimertinib. BLU-945 also demonstrated tumor shrinkage in patients from the SYMPHONY trial.Conclusion:Our findings demonstrate the preclinical and early clinical activity of BLU-945 in EGFRm NSCLC progressing on previous EGFR-TKIs.

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.

Abstract Introduction: EGFR mutations holds the major targets for drug in lung adenocarcinoma (LUAD). Despite the tremendous study of EGFR mutant (MT) LUAD, the comprehensive interpretation of the heterogeneous character of LUAD harboring EGFR MT remains a key challenge. Here, we investigated the heterogeneity of EGFR MT LUAD and explored the tumor microenvironment (TME) in EGFR MT LUAD. Method: We performed single-cell RNA sequencing (scRNA-seq) from 135 LUAD patients which consist of normal(n=24), EGFR wild (WT)(n=18), and MT(n=93). Also, we used whole genome sequencing and bulk-RNA sequencing to validate with scRNA-seq results. From 898,648 cells, main cell types were classified. To explore the various characteristics of MT LUAD tumor cells, we used two ways: i) We re-clustered epithelial cells populating the normal, WT, and MT. ii) We re-clustered only MT epithelial cells. In each analysis, we identified the tumor character in the clusters using differential expressed genes analysis, lineage tracing, clinical information, mutation, and trajectory analysis. Also, we extracted each main cell type except epithelial cells, and identified subtypes of main cell types. Finally, we revealed the interaction of cellular components in TME. Results: In the analysis of epithelial cells, we identified characteristics of specific EGFR MT by comparing of EGFR WT and MT tumors in clusters with similar biological features. The cluster represented by alveolar type 2 (AT2) known as initiation of LUAD was populating normal, WT, and MT. In this cluster, MT- and WT-associated pathway shared but differently significant between MT and WT in the pathway analysis. The cluster represented by proliferative is mostly comprised tumor cells and we found significantly increased the expression of MDK, CD24 in the MT of the cluster. In the analysis of only MT epithelial cells, 2 of clusters were stage-specific cluster: i) The cluster annotated as early stage cluster, ii) The cluster annotated as advanced stage cluster. Trajectory showed that there is a pseudotemporal continuum, following the stage from early stage cluster to advanced stage cluster. Also, based on the lineage tracing, 2 of clusters revealed lineage-specific clusters: i) The cluster annotated as AT2 was enriched from early stage cells, ii) The cluster annotated as basal cell known as origin of lung squamous cell carcinoma(LUSC) was enriched from advanced stage cells. Psedotemporal ordering of these cluster revealed AT2 cluster transdifferentiate into basal cell cluster which implied the possibility of LUAD to LUSC transition by drug resistance. In the interaction of MT and WT TME, the number of signaling received epithelial cells from myeloid cells, endothelial cells, and fibroblasts as sender increased compared with the interaction of normal. Conclusion: We shed light on the ecosystem of TME according to clinical and biological feature of tumor in EGFR mutant LUAD. Citation Format: You Won Lee, Eun Ji Lee, Seung Yeon Oh, Kyoung-Ho Pyo, Seong Gu Heo, YoungJoon Park, Su-Jin Choi, Kyumin Lim, Ju-hyeon Lee, Jae Hwan Kim, Jii Bum Lee, Ji Yoon Lee, Sun Min Lim, Chang Gon Kim, Min Hee Hong, Mi Ran Yun, Byoung Chul Cho. Phenotype profiling of tumor microenvironment in EGFR mutant lung adenocarcinoma with multi-omics data. [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 5935.

A recently developed treatment strategy for lung cancer that combines immune checkpoint inhibitors with chemotherapy has been applied as a standard treatment for lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), and it has improved the outcomes of chemotherapy. Maintenance treatment with anti-PD-1 antibody (aPD-1) enhances the effect of immunochemical combination therapy and improves therapeutic efficacy, which contributes toward a significant improvement in patient survival rates. The AXL receptor tyrosine kinase (AXL), which is expressed in tumor cells, plays an essential role in the resistance of cancers to chemotherapy and immunotherapy, and stimulates signaling associated with epithelial-mesenchymal transition (EMT) in metastatic cancer. AXL is thus an attractive target for controlling resistance to anti-tumor therapies. In this study, we examined the effect of AXL inhibitors on immune activation and tumor growth in TC1 and C3PQ mouse tumor models, in the context of clinical immunotherapy/chemotherapy and maintenance treatment, using an aPD-1 with/without pemetrexed. To determine the optimal timing for administration of SKI-G-801, an AXL inhibitor, we investigated its anti-tumor effects based on inclusion at the immunochemotherapy and maintenance therapy stages. We also performed flow cytometry-based immune profiling of myeloid cells and lymphoid cells at different points in the treatment schedule, to investigate the immune activation and anti-tumor effects of the AXL inhibitor. The addition of SKI-G-801 to the immune checkpoint inhibitor and chemotherapy stage, as well as the maintenance therapy stage, produced the best anti-tumor results, and significant tumor growth inhibition was observed in both the TC1 and C3PQ models. Both models also exhibited increased proportion of effector memory helper T cells and increased expression of CD86+ macrophages. Especially, regulatory T cells were significantly reduced in the TC1 tumor model and there was an increase in central memory cytotoxic T cell infiltration and an increased proportion of macrophages with high CD80 expression in the C3PQ tumor model. These results suggest increased infiltration of T cells, consistent with previous studies using AXL inhibitors. It is expected that the results from this study will serve as a stepping stone for clinical research to improve the existing standard of care.

Abstract Introduction: Neoadjuvant therapy suggests the systemic treatment of cancer prior to surgical therapy. The main purpose of neoadjuvant therapy is to improve surgical outcomes in patients for whom a primary surgical approach is difficult to practice. Recently, Anti-PD-1 has been applied to neoadjuvant and adjuvant therapy, but the anti-cancer responses are not sufficient to prevent tumor recurrence. According to our previous study, a novel AXL inhibitor, SKI-G-801 strongly inhibits metastasis of syngeneic tumor with immune responses. The primary goal of this study is to overcome limitation of anti-PD-1 based neoadjuvant and adjuvant therapy with SKI-G-801. Methods: 4T1 cancer bearing mouse models were established for neoadjuvant or adjuvant treatment. Mice were treated with single anti-PD-1 (BIW), SKI-G-801 (QD) or a combination for 6 days, followed by the surgical dissection of tumor tissue in a subcutaneous xenograft model. In five mice per group, their survival rates were monitored for 50 days. We used flow cytometry and immunohistochemistry for comprehensive immune profiling and tumor-infiltrating lymphocytes (TILs) from three mice per group surgically removed tumor tissues. Three tumors per group were stained, and CD3-positive cells per unit area (mm2) were analyzed using Vectra Polaris. Results: According to results, the median survival of control group (only surgical operation without treatment) was 16 days (N=5), whereas, the longest survival group was neoadjuvant-combination (median survival = 37 days). Two out of five mice were survived until end of experiment (D-50). Most of the groups had similar outcomes excluding neoadjuvant-combination group. The median survival of adjuvant-anti-PD-1 group was 30 days, and adjuvant-SKI-G-801 and neoadjuvant-anti-PD-1 group were 25 days. The Neoadjuvant-SKI-G-801 was recorded 23.5 days. Statistically, survival rate of neoadjuvant-combination therapy was significantly higher than of neoadjuvant-anti-PD-1 and neoadjuvant-SKI-G-801 (log-rank test, p <0.01).The reason for neoadjuvant therapy is to improve immune responses before surgical operation. We investigated the effects of SKI-G-801 or anti-PD-1 alone and combination therapy on neoadjuvant treatment in the tumor microenvironment. IHC and flow cytometry were performed to elucidate tumor infiltrating immune cell populations with the tumor obtained by surgery. In IHC result, CD3+ cells population was significantly increased in SKI-G-801 alone and combination of neoadjuvant therapy compared to control group (p<0.05, p < 0.001, respectively), however, anti-PD-1 group was not significant. IFN-γ+ Tc cells, CD3+CD44+ memory Tc, and PD-1+Tc cells were significantly infiltrated into the tumor in the combination of neoadjuvant therapy (p < 0.01). Conclusion: This study confirmed that a potential combination with aPD-1 and SKI-G-801 applies to neoadjuvant therapy, as evidenced by increased T cell infiltration and function. Our findings provide a rationale for further clinical investigations. Keywords: Axl inhibitor, Neoadjuvant therapy, Adjuvant therapy, Immunotherapy, Anti-PD-1 Citation Format: Ha Ni Jo, Wongeun Lee, Chun-Bong Synn, Hee Kyu Lee, Jae-Hwan Kim, Yeongseon Byeon, Sung Eun Kim, Ji Min Lee, Do Hee Kim, Jung-Ho Kim, Beung-Chul Ahn, Min Hee Hong, Hye Ryun Kim, Kyoung-Ho Pyo, Byoung Chul Cho. Neoadjuvant and adjuvant anti-PD-1 based combination immunotherapy with a novel AXL inhibitor, SKI-G-801 in syngeneic tumor model: A combined analysis of immune profiling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5189.

Purpose: Patients with advanced lung adenocarcinoma often lack large clinical specimens required for molecular testing which limits next therapeutic options. Patient-derived cells (PDC) from malignant effusions can predict patient drug responses and provide a valuable tool for studying drug resistance mechanisms. In this study, we created an Advanced Lung Adenocarcinoma Cell Bank (ALACB) consisting of 28 unique PDCs established from patients who progressed on various tyrosine kinase inhibitor TKIs including 3rd generation EGFR TKI osimertinib.Experimental design: Patient malignant effusion samples were tested for malignancy, processed, observed by light microscopy, and cultured with appropriate media and supplements. The criteria for established PDCs include the following: sharing the same driver mutation as the patient; free of stromal fibroblasts confirmed by FACS staining; recapitulating patient’s drug response; and can be cryopreserved and re-grown. Established PDCs were further authenticated by STR profiling and microplasma testing to maintain cell quality. We were able to perform various in vitro assays and next generation sequencing for detailed analysis of individual PDC.Results: We were able to successfully establish 28 PDCs incorporating unique patient characteristics. Among the total 146 samples, we observed a success rate of 30% only accounting 95 malignancy positive samples. Established PDCs consists of 20 EGFR mutation positive cell lines, 3 with ALK rearrangements, 6 with ROS1 rearrangements, 1 with BRAF K601E mutation and 1 with KRAS G12D mutation. Seven PDCs were established from patients who progressed on osimertinib, two from olmutinib (HM61713), and others from 1st and 2nd generation EGFR TKI such as gefitininb and erlotinib. We were able to newly generate 3 PDCs harboring a T790M mutation with an activating EGFR mutation. Moreover, we are one of the few labs to successfully establish an EGFR del19/T790M/C797S triple mutant PDC showing resistance to osimertinib treatment in vitro. Other established PDCs harbored rare EGFR mutations including exon 20 insertion, L861Q and G719X/S768I. PDC with EGFR G719X/S768I compound mutation showed high sensitivity to afatinib but was resistant to gefitininb and osimertinib treatment. Three ALK-positive PDCs were derived from crizotinib-resistant, alectinib-resistant and ceritinib-resistant tumors. Furthermore, of 6 ROS1 fusion positive PDCs, 3 were derived from TKI-naïve tumors, and 3 from crizotinib-resistant tumors.Conclusions: Successfully established PDCs reflecting patient genomic profiles and drug response is a valuable resource for biological assays, generating in vitro drug-resistant models, and evaluating novel drugs and therapeutic targets. Further advances in drug development combined with a library of cell bank will facilitate lung cancer translational research.Citation Format: Hyeong-Seok Joo, Dong Hwi Kim, Seok-Young Kim, Ji-Yeon Lee, Mi-Ran Yun, Han-Na Kang, Byoung Chul Cho, Hye Ryun Kim. Advanced lung adenocarcinoma cell bank (ALACB) : A comprehensive preclinical platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3999.

Introduction: MERTK is thought to play a role in cancer immune evasion mechanism based on M2 type macrophage-driven immunosuppression in tumor microenvironment (TME). Thus, MERTK is a highly promising target for anti-cancer therapy under high tumor associated macrophages (TAMs) condition. We evaluated the anti-tumor effects and immune reaction of combination or single MERTK inhibitor in two lung squamous patients derived xenograft (PDX) tumor engrafted Hu-CD34-NSG models.Methods: The aPD-1 sensitive (YHIM2004) and aPD-1 non-sensitive (YHIM2009) squamous cell carcinoma PDX models were selected from pre-test with pembrolizumab. The selected two models were composed four groups; control, pembrolizumab, MERTK inhibitor, and combination. We applied pembrolizumab as aPD-1 blockade (10 mpk, q2w, i.p. inj.). The dose of MERTK inhibitor was 50 mpk, q1d by orally. The treatment was started when tumor size reached 100 mm3. Whole exome sequencing (WES), microarray (mRNA), flow cytometry, and multispectral image analysis were performed on the tumors. To elucidate the involvement of MERTK inhibitor in tumor, the tumor mutation burden, tumor microenvironment and immune cell phenotype were deconvoluted.Results: The TMB of YHIM2004 and YHIM2009 was 3.23 and 2.64 (mutations/Mb) respectively. The PD-L1 and TIL level of YHIM2004 were relatively higher than YHIM2009. Both two models were observed high macrophage in tumor. The two PDX-models, YHIM2004 (aPD-1 sensitive), YHIM2009 (aPD-1 non-sensitive) were observed anti-tumor effects in MERTK inhibitor single treatment groups (TGI = 44.23%, 67.40% respectively, p < 0.05, t-test). Especially, YHIM2004 tumor was reduced after combination therapy more than single treatment. Of note, the NOG-PDX model demonstrated no anti-tumor effect in MERTK inhibitor monotherapy, suggesting that this drug involves anti-cancer immune responses. Flow cytometry data showed increased PD-1+ and granzyme B+ T cells (p <0.05, t-test) in the combined group (YHIM2004) and MERTK inhibitor monotherapy group (YHIM2009) as much as the tumor suppressive effect.Conclusion: We concluded that MERTK signaling involves regulation of anti-cancer immune responses. Our results suggest an anti-cancer therapeutics of MERTK selective inhibitor on aPD-1 sensitive or non-sensitive tumors under high TAM tumor microenvironment.Citation Format: Kyoung-Ho Pyo, Ha-Ni Cho, Chun-Feng Xin, Jae Seok Cho, Han Na Kang, Jiyeon Yun, Hye Ryun Kim, Byoung Chul Cho. Efficacy of MERTK inhibitor in combination with pembrolizumab in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4081.