e15547 Background: Kirsten rat sarcoma (KRAS) mutations lead to aberrant activation of the RAS/RAF/MEK/ERK signaling pathway, promoting cell proliferation and metastasis, and are among the most common oncogenic mutations in colorectal cancer (CRC) patients. Frequent point mutations include G12D, G12V, and G13D. Patients with these mutations often have poor prognosis and exhibit drug resistance. Immunotherapy may become a potential treatment to improve the current status.Oncolytic virus vaccines are genetically engineered viruses targeting tumor-specific antigens, which enhance tumor cytotoxicity and immune responses. This study aims to initially construct an oncolytic virus vaccine targeting KRAS mutations and validate its preclinical efficacy, addressing the lack of treatment options for advanced colorectal cancer and improving the poor response of CRC to immunotherapy. Methods: Based on the wild-type herpes simplex virus HSV-1 (F strain), we constructed KRAS-mutant oncolytic virus vaccine(KR10) by incorporating the KRAS mutation gene(G12D, G12C,etc). KR11, with GFP inserted, and KR12, with Flu-MP inserted, were used as control groups. Western blot was applied to verify the success of the construction. CCK-8 assay were performed to assess the selective cytotoxic effects of KR10 on normal cells and tumor cells.A subcutaneous xenograft tumor model (CT26 colorectal cancer) in mice was developed to evaluate the anti-tumor efficacy of KR10 at different viral doses. Pharmacokinetic and pharmacological studies were then conducted in large animal models(crab-eating macaques). Results: The KR10 was engineered successfully. Cytotoxicity assays showed the selective killing of tumor cells and the safety of KR10 on normal cells. In the CT26 (KRAS -G12D) model, KR10 significantly reduced tumor size and volume compared to the control groups, with effects increasing with dose within a certain range.Furthermore, re-administration of KR10 in mice demonstrated sustained anti-tumor effects. For crab-eating macaques, intravenous and subcutaneous KR10 administration showed no signs of severe toxicity, death, or distress, and there were no significant abnormalities in any safety indicators. Pharmacokinetic studies revealed dose-dependent viral clearance with minimal drug accumulation, and limited viral shedding via the respiratory/digestive tracts. Immunological assays indicated mild immune activation, evidenced by elevated IgG and IgM levels and complement consumption. Conclusions: This study is a preclinical investigation of KRAS-mutant oncolytic virus vaccine for the treatment of metastatic colorectal cancer. It is the first to construct an oncolytic virus (KR10) targeting the G12D mutation on KRAS, with in vitro efficacy validation and animal experiments. The funding for this study was supported by the Shanghai Shenkang Project (No.SHDC2022CRT009).

Identifying strategies to improve the efficacy of the immune checkpoint blockade (ICB) remains a major clinical need. Based on the high tissue penetration capability of hydrogen molecules (H2) and their immunomodulatory effects, this work proposes a local gas delivery strategy targeting the tumor microenvironment for high-efficacy hydrogen immunotherapy. We synthesized hepta-fluorinated zeolitic imidazolate frameworks nanosheet (F7-ZIF) with high H2 payload, sustained acid-responsive gas release property, and biodegradation. H2-loaded F7-ZIF (F7-ZIF/H2) effectively released H2 and Zn2+ to induce significant mitochondrial damage and cell apoptosis. More importantly, F7-ZIF/H2 considerably upregulated the expression of CD47 ("do not eat me" signal) on tumor cells, which increases target accessibility for anti-CD47 antibody, thereby enhancing their binding efficiency. RNA-seq suggests that the CD47 antibody not only blocks the "do not eat me" signal (mediated by CD47-SIRPα interactions) but also engages Fc receptors on macrophages through its Fc region to trigger antibody-dependent cellular phagocytosis. In melanoma tumor models with small (∼50 mm3) and large sized established tumors (∼200 mm3), the combination of F7-ZIF/H2 and anti-CD47 reaches 90% and 83% of tumor inhibition rate, respectively, compared to free anti-CD47. When further combined with anti-PD-L1, the therapeutic system triggers systemic T cell immunity that rejects the progression of both primary and distal tumors. This work provides insights into gas-assisted cancer immunotherapy.

Tumor-Infiltrating Lymphocytes (TILs) immunotherapy is a highly promising treatment for Non-small Cell Lung Cancer (NSCLC), which is responsible for 18% of all cancer-related deaths. The heterogeneity of TILs remains poorly understood. Here, we utilized combined single-cell RNA (scRNA)/T cell receptor sequencing (scTCR-seq) data from lung adenocarcinoma (LUAD) patients. Naïve CD4+ and effector memory CD8+ T cells were increased in tumor tissue compared with circulating blood samples. Activated signaling pathways were detected, and GZMA was identified as a potential novel diagnostic biomarker. During the transitional phase, macrophages (FTL) and dendritic (AIF1) cells transported the most CD3 TCR clones to T cells, while cytotoxicity CD8+ T (NKG7) cells transported to terminal exhausted CD8+ T cells. In both transition and expansion phases, T helper cells (CXCL13) are transported to regulatory T cells (Tregs). Additionally, we investigated the expression profiles of key cytokines, checkpoint receptors, and their ligands. Cytotoxicity CD8+ T cells (CCL5 and IFNG), T helper cells (FTL, TNFRSF4, and TIGIT), and regulatory T cells (CTLA4, TIGIT and FTL) exhibited functional roles in both primary and metastatic tumor stages. Taken together, our study provides a single-cell resolution of the TIL immune landscape and suggests potential treatment strategies to overcome drug resistance.

3122 Background: Advanced hepatocellular carcinoma (HCC) is a lethal malignancy with limited treatment options. DX1002 is a novel, specific, small-molecule, tubulin inhibitors, that targets the beta subunit of tubulin in tumor vascular endothelial cells. It has been designed to destroy tumor blood vessels which has been demonstrated in preclinical efficacy models. The objectives of this trial were to assess safety, tolerability and preliminary efficacy of DX1002 in patients (pts) with pre-treated advanced HCC or gastric cancer. Here, we present the results from the HCC subpart. Methods: This is a prospective, single-arm open-label multi-center phase Ib/IIa trial (CTR2400080296) to investigate DX1002 in patients with pre-treated advanced HCC or gastric cancer. Patients received DX1002 600 mg orally once daily, continuously in 28-day cycles until progressive disease or intolerable toxicity. Primary endpoints were safety and objective response rate (ORR); other endpoints included progression-free survival (PFS), overall survival (OS), disease control rate (DCR), pharmacokinetics and safety. Results: From Jun-2021 to Jan-2024, 40 patients with advanced HCC were enrolled. The median age was 54 (36-71) years. As of 31-Jan-2024, of the 40 pts, 4 pts (10.0%) showed PR and 15 (37.5%) had SD. ORR was 21.1% and DCR was 47.5%. The median PFS was 3.55 (95% CI 2.89-8.31) months and median OS was 13.4 (95% CI 11.0-NA) months. Any-grade TRAEs occurred in 75% pts (30/40), with 22.5% Grade 3 (9/40). The most common TRAEs were Aspartate aminotransferase increased (22.5%), total bilirubin increased (20%), albuminuria (20%), Hypoalbuminemia (15%) and gamma-glutamyl transferase increased (15%). Four patients (10%) discontinued DX1002 due to TRAEs. No death-related cases occurred. Conclusions: Initial data shows DX1002 demonstrated manageable safety profile and promising anti-tumor potential in pre-treated advanced hepatocellular carcinoma. Further exploration of DX1002 in HCC is warranted. Clinical trial information: ChiCTR2400080296.

The ubiquitin protein ligase E3C (UBE3C) has been reported to play an oncogenic role in breast cancer (BRCA). This work further investigates the effect of UBE3C on the radioresistance of BRCA cells. Molecules linking to radioresistance in BRCA were identified by analyzing two GEO datasets, GSE31863 and GSE101920. UBE3C overexpression or knockdown was induced in parental or radioresistant BRCA cells, followed by irradiation treatment. The malignant properties of cells in vitro, and the growth and metastatic activity of cells in nude mice, were analyzed. Downstream target proteins, as well as upstream transcriptional regulators of UBE3C, were predicted by bioinformatics tools. Molecular interactions were confirmed by immunoprecipitation and immunofluorescence assays. Furthermore, artificial alterations of TP73 and FOSB were induced in the BRCA cells for functional rescue assays. According to bioinformatics analyses, UBE3C expression was linked to radioresistance in BRCA. UBE3C knockdown in radioresistant BRCA cells reduced while its overexpression in parental BRCA cells increased the radioresistance of cells in vitro and in vivo. UBE3C, which induced ubiquitination-dependent protein degradation of TP73, was transcriptionally activated by FOSB. The radioresistance of cancer cells was blocked by TP73 overexpression or FOSB knockdown. Additionally, LINC00963 was found to be responsible for the recruitment of FOSB to the UBE3C promoter for transcription activation. This work demonstrates that LINC00963 induces nuclear translocation of FOSB and the consequent transcription activation of UBE3C, which enhances radioresistance of BRCA cells by inducing ubiquitination-dependent protein degradation of TP73.

Tumor-infiltrating lymphocytes (TILs) have shown remarkable clinical responses in some patients with advanced solid tumors. As a rare subset of TILs, CD4−/CD8− double-negative T cells (DNTs) were poorly known. This study aims to investigate the characteristics and function of CD3+CD4−CD8− TILs (double-negative TIL, DN-TILs) derived from solid tumor. DN-TILs were derived and expanded ex vivo from resected gastric carcinoma tissue and phenotyped by flow cytometry. The cytotoxicity of DN-TILs was determined against established tumor cell lines in vitro or through in vivo adoptive transfer into xenograft models. K562 cells were transferred with the HLA gene to verify whether the cytotoxicity of DN-TILs was MHC-independent. Flow cytometric analysis revealed a high-purity population of DN-TILs (> 97

Objective:Low coverage whole genome sequencing was performed on patients with metastatic gastric adenocarcinoma to explore the occurrence, development and genomic changes of chemotherapy resistance in gastric cancer.Methods:Nine patients with gastric adenocarcinoma were recruited since July to December 2019 in Tenth People's Hospital Affiliated to Tongji University. Peripheral blood samples were collected before and after chemotherapy. Plasma cfDNA (circulating free DNA) was isolated and send to low-coverage genome-wide sequencing, followed by chromosomal instability (CIN) analyses by a customized workflow ultrasensitive chromosomal aneuploidy detection (UCAD).Results:According to the efficacy of chemotherapy, the patients were divided into two groups, the chemotherapy effective group and the chemotherapy ineffective group. The level of CIN after treatment was lower than that before in the chemotherapy effective group [(1.186±1.642) % : (5.639±7.666) %, P=0.017]. The level of CIN after treatment was seemingly higher than that before in the chemotherapy ineffective group (0.11% : 0.08%). Frequent chromosomal copy number variations included 7p, 8q, 10q, 11q, 12p, 17q gain, 8p loss, 17p loss, et al. Two (22.2%) patients were found with additional copy number gains after chemotherapy, including 11q gain, and 5p gain. Further analyses showed those patients were treatment resistance. Conclusions:Dynamic detection of chromosomal instability based on plasma cfDNA might be used as a predictive biomarker to evaluate the efficacy of metastatic gastric cancer and the emergence of chemotherapy resistance.

e14610 Background: The findings on the clinical effect of the therapy with anti-PD-1 Abs plus angiogenic receptor inhibitors in the treatment of MSS/pMMR mCRC have been inconsistent. The aim of this study was to evaluate the prognostic and predictive role of NLR and IFN-γ in mCRC treated with anti-PD-1 and angiogenic receptor inhibitors. Methods: A total number of 35 patients with MSS/pMMR colorectal cancer treated with anti-PD-1 and angiogenic receptor inhibitors at Shanghai Tenth People’s Hospital (Shanghai, China) between December 2019 and January 2023 were retrospectively evaluated. We reviewed the tumor response and progression-free survival (PFS), and evaluated the associations among neutrophil-to-lymphocyte ratio (NLR), cytokines, and outcomes in the treatment of MSS/pMMR mCRC. A cut-off value of 3 was adopted to discriminate patients with low (NLR < 3) versus high (NLR > 3) NLR. A cut-off value of 2.7 was employed to discriminate patients with low (IFN-γ < 2.7) versus high (IFN-γ > 2.7) IFN-γ. Tissue samples from the mCRC patients were stained with CD4, CD8 to establish the immune status. Results: Of the remaining 25 mCRC patients, 14 received sintilimab and fruquintinib, 3 received sintilimab and regorafenib, 6 received camrelizumab and fruquintinib, and 2 received raltizumab and fruquintinib. Two patients were treated with PEG-rhG-CSF, and one patient had myelosuppression after previous chemotherapy. Thus, the NLR ratio could not be calculated in these three patients. Meanwhile, among the 25 patients, 4 patients no cytokine test had been performed before the treatment with anti-PD-1 and VEGF inhibitors. Thus, cytokine analyses were conducted only in the remaining 21 patients. Partial response and stable disease were found in 5 (20%) and 8 (32%) patients, respectively. The disease control rate was 52%. The pretreatment NLR ratio and IFN-γ level of the responder and stable patients were higher than those in progressive patients ( P = 0.0010, n = 22; P = 0.0267, n = 21, respectively). Higher baseline levels of NLR ratio and IFN-γ was associated with longer progression-free survival in mCRC patients receiving anti-PD-1 and angiogenic receptor inhibitors. NLR and IFN-γ were also positively correlated with PFS (R 2 = 0.3334, P = 0.0049; R 2 = 0.4063, P = 0.0019) respectively. Patients getting clinical benefits (PR+SD) were found CD4+ T cells infiltrated (P = 0.0074). Conclusions: Higher NLR ratio and IFN-γ level are prognostic factors associated with longer progression-free survival and better response of MSS/pMMR mCRC patients to anti-PD-1 and angiogenic receptor inhibitors. NLR ratio and IFN-γ level pretreatment could be predictive factors of the response to anti-PD-1 and angiogenic receptor inhibitors in MSS/pMMR mCRC patients. Clinical trial information: ChiCTR2300067767 .

免疫检查点抑制剂在肺癌肝转移患者中的临床疗效远远不能令人满意[1]。既往研究显示溶瘤病毒可增强抗PD-1/PD-L1单抗体内外的疗效[2, 3]。本研究旨在评估溶瘤病毒重组人5型腺病毒注射液(H101)联合特瑞普利单抗对表皮生长因子受体-酪氨酸激酶抑制剂(epidermal growth factor receptor-tyrosine kinase inhibitor, EGFR-TKI)、化疗或免疫检查点抑制剂治疗后进展的晚期肺癌肝转移患者的安全性和有效性。本研究已在2022年12月7—9日瑞士日内瓦举行的欧洲肿瘤内科学会免疫肿瘤大会上进行壁报交流。上海市科学技术委员会"产学研医项目"(18DZ1910102)。肺癌是目前发病率和死亡最高的恶性肿瘤之一, 晚期患者治疗存在挑战, 尤其是肺癌肝转移患者, 预后差, 治疗手段有限, 病死率高[1]。PD-1抑制剂现已成为包括晚期肺癌在内的多种恶性肿瘤的标准治疗方法, 但是仍有相当部分患者对于PD-1抑制剂治疗反应不佳[2], 寻求新的治疗途径以提高疗效成为必然。而溶瘤病毒治疗是众多有极具治疗前景的有希望的免疫调节疗法之一。就溶瘤病毒而言, 其是一类趋向于感染肿瘤细胞, 通过自身在肿瘤细胞中的复制从而杀死并裂解肿瘤细胞的病毒, 同时进一步诱导机体抗肿瘤免疫反应。溶瘤病毒瘤内注射可以重塑肿瘤局部免疫微环境, 使"冷肿瘤"转变为"热肿瘤", 提高免疫治疗疗效[3-4], 其与抗PD-1抗体联合应用已经在国内外的一些临床研究中取得了很好的临床研究结果[5-7]。

BACKGROUND:Transmembrane emp24 domain containing (TMED) proteins are known to play pivotal roles in normal development, but have been reported to be implicated in pancreatic disease, immune system disorders, and cancers. As far as TMED3 is concerned, its roles in cancers are controversial. However, evidence describing TMED3 in the context of malignant melanoma (MM) is scarce.RESULTS:In this study, we characterized the functional significance of TMED3 in MM and identified TMED3 as a tumor-promoting factor in MM development. Depletion of TMED3 arrested the development of MM in vitro and in vivo. Mechanistically, we found that TMED3 could interact with Cell division cycle associated 8 (CDCA8). Knocking down CDCA8 suppressed cell events associated with MM development. On the contrary, elevating CDCA8 augmented cell viability and motility and even reversed the inhibitory effects of TMED3 knockdown on MM development. On the other hand, we found that the levels of P-Akt and P-PI3K were decreased in response to TMED3 downregulation, which was partially abolished following SC79 treatment. Thus, our suspicion was that TMED3 exacerbates MM progression via PI3K/Akt pathway. More notably, previously decreased P-Akt and P-PI3K in TMED3-depleted cells were rescued after overexpressing CDCA8. Also, previously impaired cell events due to CDCA8 depletion were ameliorated after SC79 addition, implying that TMED3 regulates PI3K-AKT pathway via CDCA8, thereby promoting MM development.CONCLUSIONS:Collectively, this study established the link between TMED3 and MM, and provides a potential therapeutic intervention for patients with MM harboring abundant TMED3.

Distant metastasis and primary tumor relapse are the two main hurdles to the success of surgical treatment for cancer patients. Circulating tumor cells (CTCs) and incomplete surgical resection are the primary cause of distant metastasis and recurrence of tumors respectively. Chimeric antigen receptor (CAR)-modified T cells targets residual carcinomas and CTCs hold the potential to inhibit primary recurrence and reduce tumor metastasis, but the experimental evidence is lacking. Here, we developed a surgery-induced tumor metastasis model in immunocompetent mice to investigate the efficacy of CAR-T cells therapy in preventing metastasis and local recurrence. We observed that subcutaneous tumor resection has induced a large number of CTCs intravasated into circulation. EpCAM-specific CAR-T was effective in clearing CTCs following surgical removal of the tumor. This resulted in less pulmonary metastasis and longer survival in mice when compared to mice treated with surgery followed by Mock-T cells. In addition, the local relapse was obviously inhibited at the surgical site followed by EpCAM-CAR-T cell treatment. This study demonstrated that CAR-T cell therapy can be an adjuvant treatment following surgery to prevent tumor metastasis and inhibit primary tumor relapse for cancer patients.

The efficacy of CAR-T cells for solid tumors is unsatisfactory. EpCAM is a biomarker of epithelial tumors, but the clinical feasibility of CAR-T therapy targeting EpCAM is lacking. Here, we report pre- and clinical investigations of EpCAM-CAR-T cells for solid tumors. We demonstrated that EpCAM-CAR-T cells costimulated by Dectin-1 exhibited robust antitumor activity without adverse effects in xenograft mouse models and EpCAM-humanized mice. Notably, in clinical trials for epithelial tumors (NCT02915445), 6 (50%) of the 12 enrolled patients experienced self-remitted grade 1/2 toxicities, 1 patient (8.3%) experienced reversible grade 3 leukopenia, and no higher-grade toxicity reported. Efficacy analysis determined two patients as partial response. Three patients showed >23 months of progression-free survival, among whom one patient experienced 2-year progress-free survival with detectable CAR-T cells 200 days after infusion. These data demonstrate the feasibility and tolerability of EpCAM-CAR-T therapy.

Distant metastasis and primary tumor relapse are the two main hurdles to the success of surgical treatment for cancer patients. Circulating tumor cells (CTCs) and incomplete surgical resection are the primary cause of distant metastasis and local recurrence of tumors, respectively. Chimeric antigen receptor (CAR)-modified T cells target residual carcinomas and CTCs hold the potential to inhibit primary recurrence and reduce tumor metastasis, but the experimental evidence is lacking. Here, we developed a surgery-induced tumor metastasis model in immunocompetent mice to investigate the efficacy of CAR-T cells therapy in preventing metastasis and local recurrence. We observed that subcutaneous tumor resection has induced a large number of CTCs intravasated into circulation. EpCAM-specific CAR-T was effective in clearing CTCs following surgical removal of the tumor. This resulted in less pulmonary metastasis and longer survival in mice when compared to mice treated with surgery followed by Mock-T cells infusion. In addition, the local relapse was obviously inhibited at the surgical site followed by EpCAM-CAR-T cell treatment. This study demonstrated that CAR-T cell therapy can be an adjuvant treatment following surgery to prevent tumor metastasis and inhibit primary tumor relapse for cancer patients.

Anti-CD19 chimeric antigen receptor (CAR) T-cells are an effective treatment for refractory B-cell lymphoma, but CD19 deletion is prone to relapse. We conducted this study to find more effective dual CAR19/20 T-cells to target B-cell lymphoma and prevent antigen loss leading to recurrence. In this study, we transduced CD19 and CD20 CARs into human T cells in parallel and compared parallel dual CAR19/20, single CAR, and tandem CAR19/20 in vitro and in vivo. After transduction with the corresponding vectors, CD19 and CD20 CARs were dually expressed in human T cells. It was observed that parallel CAR19/20 T-cells contained a substantial proportion of naive subpopulations and were able to proliferate in vitro. Treatment with parallel CAR19/20, single CAR, or tandem CAR19/20 T-cells sustainably induced complete lysis of leukemia cells in a 5 : 1 ratio. Compared with single or tandem CAR T-cell-transplanted mice, parallel CAR19/20 T-cell-transplanted mice exhibited smaller tumor volume, more stable body weight, and longer survival. This suggests that parallel CAR19/20 has superior antilymphoma activity in vivo. In addition, parallel CAR19/20 T-cells were also able to kill patients' lymphoma cells in vitro. Therefore, it can be considered that parallel CAR19/20 is equally effective against single CAR and tandem CAR19/20 in vitro but more effective against lymphoma cells in vivo. This is a promising treatment to prevent the recurrence of antigen loss following CD19-targeted therapy in B lymphoma.

Abstract Radiation resistance and the etiology of gastric cancer (GC) is significantly influenced by the DNA damage response, particularly DNA double-strand breaks (DSBs). A repair method for dealing with DNA damage is homologous recombination repair (HRR). The stability of the genome may be directly harmed by the homologous recombination deficiency (HRD) or blockage of the HRR pathway, inducing a slight increase in chromosome aberration and eventually leading to malignant tumors, including gastric adenocarcinoma. Therefore, to clarify the mechanism of DNA damage repair and create GC therapeutic targets, it is crucial to investigate the feedback mechanism of HRR to DSB in GC. Through bioinformatics analysis, the differentially expressed genes related to GC were found, the core genes were screened by the WGCNA method, the ceRNA network F10-AS1/hsa-miR-146b-5p / RAD54L was predicted and constructed by mirDIP and LncBase database, and the inhibitory effect of knockdown F10-AS1 on RAD54L was verified by in vivo and in vitro experiments. Results exhibited that knockdown of F10-AS1 raised the inhibitory effect of F10-AS1 on hsa-miR-146b-5p, promoted the combination of miR-146b-5p and RAD54L, downregulated the expression of RAD54L, and induced GAC cell death in vitro and slowed tumor growth in vivo. In the HR route of DNA repair, the F10-AS1/hsa-miR-146b-5p/RAD54 axis is crucial. Knockdown of F10-AS1 means that it inhibits homologous recombination in cancer cells and the maintenance efficiency of the DNA repair mechanism, induces the accumulation of spontaneous DSB, blocks DNA repair in cancer cells, achieves the best effect of anticancer drugs, and inhibits tumor development.

Copyright © 2022 Yuan, Zhu, Mao, Wang, Qian, Wu, Guo and Xu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

This article reported the mechanism of Anlotinib in gastric cancer treatment. Gastric cancer cells were treated with Anlotinib (8 μM) and transfected by STING shRNA and STING vectors. Cell counting kit-8 assay, wounding healing assay, and Transwell experiment were applied for proliferation, migration, and invasion detection. PD-L1 fluorescence intensity in gastric cancer cells was explored by flow cytometry. IFN-β level was researched by enzyme-linked immunosorbent reaction. Xenograft tumor experiment was performed by administering mice with Anlotinib and anti-PD-L1 antibody. Immunohistochemistry and western blot were used for proteins expression detection. Quantitative real-time reverse transcription-polymerase chain reaction was applied for mRNA expression detection. Hematoxylin and eosin staining was conducted on lung, liver, kidney, and cerebral cortex of mice. Gastric cancer cells treated with Anlotinib exhibited reduced proliferation, migration, and invasion (p<0.01). Anlotinib treatment reduced PCNA, CDK1, and MMP2 protein expressions and increased E-cadherin protein expression in gastric cancer cells (p<0.01). Anlotinib treatment suppressed PD-L1 expression and activated the cGAS-STING/IFN-β pathway in gastric cancer cells (p<0.01). STING knockdown partially reversed the inhibition of Anlotinib on gastric cancer cells proliferation, migration, invasion, and immune escape (p<0.05 or p<0.01). However, STING overexpression exhibited the opposite effect. Anlotinib synergistically improved anti-tumor efficacy of anti-PD-L1 in vivo. Anlotinib synergistic anti-PD-L1 increased CD3+, CD8+ T cells, and activated the cGAS-STING/IFN-β pathway in xenograft tumor. Anlotinib was non-toxic to lung, liver, cortex, and kidney. Anlotinib suppressed gastric cancer cells proliferation, migration, and immune escape by activating the cGAS-STING/IFN-β pathway.

e21503 Background: Treatment options are limited for patients with advanced melanoma and refractory melenoma, especially for acral and mucosal melanoma. Previous data showed that the objective response rate (ORR) of second-line PD1 monotherapy for acral and mucosal melanoma were 14-15.8% and 0-13.3%, respectively. We evaluate the safety and efficacy of recombinant human adenovirus 5 injection (oncolytic virus) plus toripalimab (PD-1 antibody) for patients who have progressed on checkpoint inhibitors, chemotherapy and anti-angiogenesis. Preliminary analysis of efficacy- related factors. Methods: Patients received the recombinant human adenovirus 5 injection (2ml) local lesions plus toripalimab 240mg systemic therapy every 2 weeks until progression or intolerable toxicity. The primary objective was safety and ORR. Secondary objective included progression-free survival (PFS) and disease control rate (DCR). Efficacy assessments were performed every 2 months according to RECIST v1.1 criteria. Blood samples were collected from patients prospectively. Serum interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10, Tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were measured at baseline and after two cycles of treatment via flow cytometry. The peripheral blood neutrophils and lymphocytes were collected simultaneously. Results: Ten cases of patients were enrolled from November 2020 to January 2022. Baseline character:primary site (1 cutaneous melanoma; 5 acral melanoma; 4 mucosal melanoma), prior therapy (anti-PD1 100%; anti-angiogenesis 60%; chemotherapy 60%; second line and above 40%), 50% liver/bone lesions. All patients evaluated received at least 2 cycles of the combination regimen. The treatment-related adverse event was fever, accounting for 80%, and there were no other adverse events. The ORR was 20% (2PR) and the DCR was 70% (2PR, 5SD, 3PD). The 2-month PFS was 70%, and the median PFS has not yet been reached. Disease progression in 3 patients including liver and bone lesion. Higher baseline lymphocyte levels and lower neutrophil-to-lymphocyte ratio (NLR) values were found in the respongders compared with non-responders. The serum IL-6, IL-8, IL-10, and TNF-α levels were significantly increased in the responders after 2 cycles of treatment. Conclusions: The preliminary data showed that the combination of recombinant human adenovirus 5 injection plus toripalimab demonstrated acceptable toxicity and promising antitumor efficacy in patients with advanced and refractory melanoma, and promoted favorable changes in serum IL-6, IL-8, IL-10 and TNF-α levels in clinical responders. High baseline lymphocyte and low NLR values may be predictors of treatment response. Long-term survival data remain to continue with follow-up. It is worth to further validate the efficacy in a randomized prospective trial. Clinical trial information: 2000033959.

INTRODUCTION:Anlotinib (AL3818) is a novel multi-target tyrosine kinase inhibitor (TKI) targeting vascular endothelial growth factor receptor (VEGFR) and suppressing tumor growth. Modulation of tumor suppressive immune microenvironment via the inhibition of vascular endothelial growth factor may augment the activity of immune checkpoint inhibitors. Here we described the results of safety, and clinical efficacy of anlotinib combined with immunotherapy in patients with advanced solid tumors, the serum cytokine levels, and peripheral blood T lymphocyte populations were detected simultaneously.METHODS:Twenty six cases with advanced late-stage cancers including lung, gallbladder, endometrial, gastric, pancreatic, penile cancers and melanoma were treated since January 2019. Patients received a combination of anlotinib (12mg) once daily on day 1 to day 14 (21 days as a course) plus anti-PD-1 antibodies every 3 weeks until progression or intolerable toxicity. Imaging was performed every 6 weeks for the first year of therapy. Blood samples were collected from patients prospectively. Serum interleukin (IL)-2, IL-4, IL-6, IL-10, Tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and circulating immune cell subsets were measured at baseline and after two cycles of treatment via flow cytometry.RESULTS:There were ten tumor types enrolled with lung, gallbladder, cholangiocarcinoma and soft tissue sarcoma being the most common. Most patients had received front line treatments for metastatic disease (80.8%). The objective response rate (ORR) was 23.1%, including one complete response (CR) (3.8%) and five partial responses (PR) (19.2%) and a disease control rate (DCR=CR+PR+SD) of 80.8% (21 of 26). The median PFS was 8.37 months (95% CI: 6.5-10.0 months). Three patients (11.5%) had grade 3 treatment-related adverse events. There were no grade 4 or 5 treatment-related adverse events. Grades 3 toxicities included hand-foot syndrome (n=2) and hypertension (n=1). Higher serum IL-2, IL-4, IL-10, TNF-α, IFN-γ levels and lower ratios of CD4/CD8 T cells were found in the responders compared with non-responders.CONCLUSIONS:The preliminary data showed that the combination of anlotinib and anti-PD-1 antibodies demonstrated promising durable antitumor efficacy with acceptable toxicity in patients with various advance tumors, and promoted favorable changes in serum IL-2, IL-4, IL-10, TNF-α, IFN-γ levels and circulating immune cell subsets in clinical responders. It is worth to further validate the efficacy in a randomized prospective trial.