Prolonged intratumoral treatment with TLR7/8 agonist R848 regulates the tumor immune microenvironment resulting in enhanced antitumor activity

Background: Hepatocellular carcinoma (HCC) is a primary malignancy of the liver. It represents the third leading cause of cancer deaths worldwide, with over 500,000 people affected each year. The incidence of HCC is particularly high in China, accounting for more than 50% of the global incidences. The development of targeted anticancer agents effective against HCC for patients who do not tolerate, or have failed or relapsed from sorafenib treatment would meet a major unmet medical need. ENMD-2076 is an orally bioavailable small molecule inhibitor of Aurora A and angiogenic kinases including VEGFR 2 and FGFR 1 with pro-apoptotic, antiproliferative and angiogenic activities against a variety of human cancers. ENMD-2076 has been tested in multiple Phase I and II clinical trials, with partial responses (PR) and prolonged progression free survival (PFS) observed in multiple cancers including liver cancer. Objective: This in vivo study was designed to compare the efficacy of ENMD-2076 with that of standard of care agents including sorafenib, doxorubicin, and 5-FU in 3 different cell-line derived human HCC xenograft models. The tolerability and efficacy of combinations of ENMD-2076 with doxorubicin or 5-FU were also determined. Experimental Design: Three different human HCC cell lines, SMMC-7721, QGY-7703 and HepG 2 were used for establishing subcutaneous tumor xenograft models in nude mice. After tumors grew to more than 100 mm3, mice were randomly assigned into one of the groups receiving vehicle, sorafenib, ENMD-2076 alone or in combination with chemotherapy agents, including doxorubicin or 5-FU, respectively, for 20 days. Results: In all HCC models tested, ENMD-2076 induced tumor growth inhibition significantly greater than that of sorafenib. When treated with ENMD-2076 at 100 mg/kg, the tumor growth inhibition (TGI) rates were 73.67%, 76.59% or 80.12% in the SMMC-7721, QGY-7703 or HepG 2 models, respectively, which were significantly higher than those observed in sorafenib treated groups (48.63%, 51.43%, or 57.60%, respectively;P<0.01 in all three models). Tumor growth inhibition rates were similarly higher in the ENMD-2076 treated groups than in groups treated with doxorubicin at 1 mg/kg and 5-FU at 30 mg/kg. The combined treatment of ENMD-2076 with either doxorubicin or 5-FU was well tolerated. Higher TGI rates were observed in combination groups compared to single agents of chemotherapies but were not statistically different. Conclusion: ENMD-2076 showed robust antitumor activity against three cell line-derived xenograft models of HCC superior to that of sorafenib, doxorubicin, and 5FU, supporting clinical investigation of this agent in HCC patients who do not tolerate, or have failed or relapsed from other systemic treatment such as sorafenib, doxorubicin or 5 FU. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C265. Citation Format: Peipei Yin, Hongqi Lu, Mark R. Bray, Bingsheng Li, Ken Ren. Significant in vivo activity of ENMD-2076, a novel multi-targeted kinase inhibitor, towards xenograft models of human hepatocellular carcinomas . [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C265.

Background: IGFBP2, known to enhance the invasion capacity of ovarian cancer cells, has been suggested that its inhibition could be potentially a treatment strategy of ovarian cancer. AST-201 (pUMVC3-hIGFBP2) is a therapeutic cancer vaccine using a plasmid DNA encoding IGFBP2 N-terminus. In a phase 1 study (NCT01322802) completed, 100 μg AST-201 (intradermal immunization, id) showed not only a significant efficacy by inducing the Th1-cell immunity against IGFBP2 but also safety and tolerability profiles in ovarian cancer patients. The primary objective of this study is to evaluate whether the administration of AST-201 alone and the combination with pembrolizumab could show anti-tumor efficacy and/or synergic effect in the ID8-Luc2 ovarian cancer mouse model. Also, immunological responses were observed as explorative endpoint. Methods: AST-201 (100 μg/animal, id, mixed with mGM-CSF as an immune adjuvant) was immunized to mice (C57BL/6) once a week for a total of 4 times on different days, either alongside pembrolizumab (10 mg/kg, intraperitoneal injection) twice a week for a total of 3 times on different days. Also, AST-201 was immunized was a mono treatment. The efficacy was evaluated by a tumor growth inhibition (TGI) rate at the last day, and immune cell profiling via FACS analysis was conducted with splenocytes and tumor tissues collected at 8 weeks after the first injection. Results: As a primary endpoint, a TGI rate at Day 55 of AST-201 mono treatment was 67%, comparing to a control group (p<0.05). The anti-tumor effect of AST-201 combining with pembrolizumab was better than standard dose pembrolizumab, based on a TGI rate at Day 55 (78% vs 66%, not significant). Immune profiling showed that AST-201 and pembrolizumab combination regimen could inhibit a tumor growth by transforming TME into inflamed-type (‘hot’) form the low immunoreactivity, which was supported by increased CD4+TEM, and CD8+TEM and T helper cells in splenocyte and TIL analysis. Conclusion: A study demonstrated that AST-201 (IGFBP2 cancer vaccine) showed an anti-tumor effect as mono treatment and would be potentially leading to a synergistic effect with a combination regimen of pembrolizumab. Phase 2 randomized-controlled study of AST-201 in ovarian cancer will be initiated under the MRCT strategy (CornerStone-004 study, NCT05794659). Citation Format: Jinback Lim, Jinho Kang, Hyo-Hyun Park, Jin Kyeong Choi, Jee Hyun Choi, Seong-Yong Jang, Min-Ah Kim, Myeong-Kyu Park, Mary L Disis, Eunkyo Joung, Ashley Yongmin Kim, Hun Jung. AST-201 (pUMVC3-hIGFBP2 N-terminus) demonstrates anti-tumor effect in an ovarian cancer mouse model [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 4114.

BackgroundIGFBP2, known to enhance the invasion capacity of ovarian cancer cells,1 has been suggested that its inhibition could be potentially a treatment strategy of ovarian cancer. AST-201 (pUMVC3-hIGFBP2) is a...

To study the effects of QHF-cisplatin on H22 hepatocellular carcinoma (HCC) and their mechanisms of action. Sixty BALB/c mice were randomly divided into a model group (n = 48) and a normal control group (n = 12). An HCC xenograft tumor was created by injecting H22 cells directly into the liver parenchyma of the mice. The 48 BALB/c mice in the model group were randomly divided into four groups: QHF, DDP (cisplatin), QHF plus DDP, and model control. The inhibitory effects of these drugs on tumor growth were evaluated by calculating the rate of tumor growth inhibition. The mice were examined by observing their general condition, body weight and survival time. Changes in tumor tissue were observed under an optical microscope. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and α-fetoprotein (AFP) levels in serum were measured. Hepatocyte growth factor (HGF), c-mesenchymal-epithelial transition (c-Met) factor, phosphorylated (p)-c-Met, p38, p-p38, extracellular signal-regulated kinase (ERK), p-ERK and vascular endothelial growth factor (VEGF) levels were evaluated in tumor and liver tissues using western blotting. Compared with the DDP group, a lower incidence of toxic reactions and a higher survival time were observed in the QHF plus DDP group. Tumor weight was significantly lower in the QHF, DDP and QHF plus DDP groups than in the model control group (0.24 ± 0.07, 0.18 ± 0.03 and 0.14 ± 0.01 g vs 0.38 ± 0.05 g, respectively), and the differences were statistically significant (P < 0.01). The rate of tumor growth inhibition in the QHF, DDP and QHF plus DDP groups was 38.7%, 52.6% and 63.5%, respectively. AST, ALT and AFP levels in serum were significantly lower in the QHF, DDP and QHF plus DDP groups compared to the model control group (P < 0.05). Similarly, HGF, p-c-Met, p-p38, p-ERK and VEGF levels in tumor tissue were significantly lower in the QHF, DDP and QHF plus DDP groups (P < 0.05). QHF and DDP have an antiangiogenic effect on H22 HCC in mice. QHF inhibits tumor growth via blocking the HGF/c-Met signaling pathway, inhibiting p38, ERK and VEGF signaling.

To study the in vitro and in vivo antitumor effect of lidamycin (LDM) on hepatoma and the active moiety of its molecule. MTT assay was used to determine the growth inhibition of human hepatoma BEL-7402 cells, SMMC-7721 cells and mouse hepatoma H22 cells. The in vivo therapeutic effects of lidamycin and mitomycin C were determined by transplantable hepatoma 22 (H22) in mice and human hepatoma BEL-7402 xenografts in athymic mice. In terms of IC(50) values, the cytotoxicity of LDM was 10 000-fold more potent than that of mitomycin C (MMC) and adriamycin (ADM) in human hepatoma BEL-7402 cells and SMMC-7721 cells. LDM molecule consists of two moieties, an aproprotein (LDP) and an enediyne chromophore (LDC). In terms of IC(50) values, the potency of LDC was similar to LDM. However, LDP was 10(5)-fold less potent than LDM and LDC to hepatoma cells. For mouse hepatoma H22 cells, the IC(50) value of LDM was 0.025 nmol/L. Given by single intravenous injection at doses of 0.1, 0.05 and 0.025 mg/kg, LDM markedly suppressed the growth of hepatoma 22 in mice by 84.7%, 71.6% and 61.8%, respectively. The therapeutic indexes (TI) of LDM and MMC were 15 and 2.5, respectively. By 2 iv. injections in two experiments, the growth inhibition rates by LDM at doses of 0.1, 0.05, 0.025, 0.00625 and 0.0125 mg/kg were 88.8-89.5%, 81.1-82.5%, 71.2-74.9%, 52.3-59.575%, and 33.3-48.3%, respectively. In comparison, MMC at doses of 5, 2.5, and 1.25 mg/kg inhibited tumor growth by 69.7-73.6%, 54.0-56.5%, and 31.5-52.2%, respectively. Moreover, in human hepatoma BEL-7402 xenografts, the growth inhibition rates by LDM at doses of 0.05 mg/kg X2 and 0.025 mg/kg X2 were 68.7% and 27.2%, respectively. However, MMC at the dose of 1.25 mg/kg X2 showed an inhibition rate of 34.5%. The inhibition rate of tumor growth by LDM was higher than that by MMC at the tolerated dose. Both LDM and its chromophore LDC display extremely potent cytotoxicity to hepatoma cells. LDM shows a remarkable therapeutic efficacy against murine and human hepatomas in vivo.

Vascular endothelial growth inhibitor (VEGI) has been identified as an anti‑angiogenic cytokine. However, the effects of VEGI174 protein, and its functional domain peptidesV7andV8, on renal cell carcinoma (RCC) remain unknown. In the present study, the protein and peptides were biosynthesised as experimental agents. The A498 and 786‑O RCC cell lines, and an established mouse xenograft model, were separately treated with VEGI174, V7orV8. Cellular functions, including proliferation, migration and invasion, were subsequently detected. Cell migration and invasion were monitored using the xCELLigence system. Furthermore, tumour growth and mouse behaviours, including mobility, appetite and body weight, were assessed. The results demonstrated that VEGI174, V7 and V8 inhibited the proliferation, migration and invasion of A498 and 786‑O cell lines when administered at concentrations of 1and100pM, 10nM and 1µM. The inhibitory effects exhibited dose‑ and time‑dependent antitumour activity. Furthermore, VEGI174, V7andV8 inhibited tumour growth in A498 and 786‑O xenograft mice. In the A498 xenografts, the tumour growth inhibition (TGI) rates in the VEGI174‑, V7‑andV8‑treated groups were71, 20and31%, respectively. In the 786‑O xenografts, the TGI rates in the VEGI174‑, V7‑andV8‑treated groups were34, 26and31%, respectively. There was no significant loss in body weight and no cases of mortality were observed for all treated mice. In conclusion, VEGI174, V7 and V8 exhibited potential antitumour effects and were well tolerated invivo. V7andV8, as functional domain peptides of the VEGI174 protein, may be studied for the future treatment of RCC.

BackgroundIn gastric cancer, the HER2 overexpression (6% to 29.5% of gastric cancers) is considered to increase proliferation activity and suppress apoptosis of the cancer cells.1 However, HER2-targeting therapy in gastric...

Recombinant adenoviruses, carrying herpes simplex virus thymidine kinase (HSVtk) genes, were developed to evaluate the possibility of tissue-specific gene therapy for thyroid carcinomas. The HSVtk gene was driven by a minimal thyroglobulin (TG) promoter (AdTGtk) and a tandemly repeated minimal TG promoter (Ad2 x TGtk) to obtain thyroid-specific cell killing ability. The transduction of HSVtk genes by infection with Ad2 x TGtk followed by ganciclovir (GCV) treatment showed more powerful cytotoxicity for TG-producing FRTL5 cells, a rat normal thyroid cell line, and FTC-133 cells, a human follicular thyroid carcinoma cell line, than when infected with AdTGtk in vitro. The cell killing ability of Ad2 x TGtk was 10- to 30-fold higher than that of AdTGtk and similar to that of AdCMVtk, which carries HSVtk under the control of CMV promoter. Whereas after treatment with adenovirus/GCV to non-TG-producing cell lines (undifferentiated thyroid carcinoma cell lines and carcinoma cell lines from other tissues), Ad2 x TGtk and AdTGtk needed more than 100-fold concentrated GCV to reach IC(50) compared to AdCMVtk. We confirmed the enhanced efficacy of Ad2 x TGtk for tissue-specific cytotoxicity in vivo. After adenovirus/GCV treatment for FTC-133 tumor-bearing nude mice, Ad2 x TGtk enhanced tumor growth inhibition and survival rates compared to AdTGtk. Tumor growth inhibition and survival rates by Ad2 x TGtk were similar to that by AdCMVtk. Moreover, any toxic effect for rat normal tissues was not revealed after intravenous injections with Ad2 x TGtk and intraperitoneal administrations with GCV in vivo, whereas severe liver damages were observed after treatment with AdCMVtk/GCV. These data indicate a beneficial effect of Ad2 x TGtk for tissue-specific gene therapy for TG-producing thyroid carcinomas without toxicity for normal tissues.

We have examined, in a prospective randomized controlled trial, the effect of 8- and 16-week initial steroid treatment on the course of idiopathic nephrotic syndrome (INS). Patients with a first episode of INS were randomized to receive standard 8-week prednisolone (2 mg/kg daily for 4 weeks, then 1.5 mg/kg on alternate days for 4 weeks) or prolonged 16-week prednisolone treatment (2 and 1.5 mg/kg daily each for 4 weeks, then 1.5 and 1 mg/kg on alternate days each for 4 weeks). Relapses were treated with prednisolone, 2 mg/kg daily for 2 weeks, then 1.5 mg/kg on alternate days for 4 weeks. Of 45 patients, 23 received standard therapy and 22 prolonged therapy. The mean duration of follow-up was 29.2 and 27.3 months in the standard and prolonged treatment groups, respectively. The time to first relapse was longer in the prolonged treatment (mean 222.2 days, median 120.0 days) than the standard group (mean 134.3 days, median 96.5 days). The percentage of patients with no relapse at 6 and 12 months after prednisolone withdrawal was 40.9% and 27.3% in the prolonged treatment and 21.7% and 8.7% in the standard groups, respectively. The inability to show statistically significant differences between the two groups was probably related to the small number of patients studied. Prolonged therapy did not affect the subsequent relapse rates and proportion of patients with frequent relapses and steroid dependence. The mean dose of prednisolone received, for the initial episode and relapses during the next year, was higher and associated with significant steroid toxicity in the prolonged treatment group. Our findings suggest that 16-week prednisolone treatment for the initial episode of INS may delay occurrence of the first relapse, but results in significant side effects. Prolongation of initial therapy may be useful in developing countries where frequent infections often induce early relapses.

To validate the usefulness of a human tumor-nude mice xenograft system as a potential model in the secondary screening of anticancer agents, the antitumor activity of 17 anticancer drugs has been studied in the treatment of a human breast cancer tumor (MX-1) transplanted to nude mice. For evaluation of the antitumor activity of the drugs we employed the LD10 predetermined in BDF1 mice as a standard therapeutic dose. Drugs were administered IV, IP, or PO, and antitumor activity was assessed by drug-induced growth inhibition measured by caliper. Among the 17 anticancer drugs, the most active compounds (maximum inhibition rate of tumor growth: greater than or equal to 90%) are mitomycin C, chromomycin A3, vincristine, vinblastine, vindesine, and hexamethylmelamine. Another group of compounds showed moderate activity (maximum inhibition rate of tumor growth: 89%-50%), these being adriamycin, daunomycin, mitoxantrone, bleomycin, 5-FU, 6-TG, and ftorafur. The remaining four drugs (peplomycin, cytosine arabinoside, 6-MP, and methotrexate) were inactive against the MX-1 tumor. These results suggested that in the nude mouse-human tumor xenograft system of the MX-1 tumor there was a good correlation between the antitumor activity of various anticancer drugs and their clinical efficacy; this system is therefore expected to be a useful model for the secondary screening system.

This study investigated the effect of TNP-470 in combination with carmustine (BCNU) on the growth of subcutaneously implanted human glioblastoma xenografts in nude mice. Human glioblastoma U-251 cells (1×10(7)) were injected into 24 nude mice subcutaneously. The tumor-bearing mice were randomly divided into 4 groups on the seventh day following tumor implantation: TNP-470 group, in which TNP-470 was given 30 mg/kg subcutaneously every other day 7 times; BCNU group, in which 20 mg/kg BCNU were injected into peritoneal cavity per 4 days 3 times; TNP-470 plus BCNU group, in which TNP-470 and BCNU were coadministered in the same manner as in the TNP-470 group and the BCNU group; control group, in which the mice were given 0.2 mL of the mixture including 3% ethanol, 5% acacia and 0.9% saline subcutaneously every other day 7 times. The tumor size and weights were measured. The tumor microvessel density (MVD) was determined by immunostaining by using goat-anti-mouse polyclonal antibody CD105. The results showed that on the 21th day following treatment, the volume of xenografts in the TNP-470 plus BCNU group was (108.93±17.63)mm(3), markedly lower than that in the TNP-470 group [(576.10±114.29)mm(3)] and the BCNU group [(473.01±48.04)mm(3)] (both P<0.01). And the xenograft volume in these 3 treatment groups was even much lower than that in the control group [(1512.61±470.25) mm(3)] (all P<0.01). There was no significant difference in the volume of xenografts between the TNP-470 group and the BCNU group (P>0.05). The inhibition rate of the tumor growth in the TNP-470 plus BCNU group was (92.80±11.37)%, notably higher than that in the TNP-470 group [(61.91±6.29)%] and the BCNU group [(68.73±9.65)%] (both P<0.01) on the 21th day following treatment. There was no significant difference in the inhibition rate of tumor growth between the TNP-470 group and the BCNU group (P>0.05). The MVD of xenografts in the TNP-470 plus BCNU group was decreased significantly as compared with that in the TNP-470 group or the BCNU group (both P<0.05). The MVD of xenografts in the 3 treatment groups was markedly reduced as compared with that in the control group (all P<0.05). No significant changes in weights were observed before and after the treatment in each group (all P>0.05). It was concluded that the combination of TNP-470 and BCNU can significantly inhibit the growth of human glioblastoma xenografts in nude mice without evident side effects.

&lt;p&gt;PDF file - 376K, Inhibition rates (IR) of tumor growth (A) and response rates at IR30 (chemosensitivity) (B). The IRs and IR30 of a single bevacizumab or cetuximab regimen were significantly less than those of the other four combination regimens (P � 0.001). IR, inhibition rate; FRB, FOLFIRI + bevacizumab; FXB, FOLFOX + bevacizumab; FRC, FOLFIRI + cetuximab; FXC, FOLFOX + cetuximab.&lt;/p&gt;

&lt;p&gt;PDF file - 376K, Inhibition rates (IR) of tumor growth (A) and response rates at IR30 (chemosensitivity) (B). The IRs and IR30 of a single bevacizumab or cetuximab regimen were significantly less than those of the other four combination regimens (P � 0.001). IR, inhibition rate; FRB, FOLFIRI + bevacizumab; FXB, FOLFOX + bevacizumab; FRC, FOLFIRI + cetuximab; FXC, FOLFOX + cetuximab.&lt;/p&gt;

To investigate the effects of autophagy gene Beclin 1 on growth of cervical cancer HeLa cells in vitro and vivo. The eukaryotic expression vector of Beclin1 was constructed and transfected via lipofectamine into HeLa cells. The experimental cells were classified into 3 groups: pcDNA3.1(+)-Beclin1 group,pcDNA3.1(+) group and HeLa group. Real time-ploymerase chain reaction and Western blot were used for detecting expression of Beclin1 mRNA and protein in the transfected cells. Flow cytometry (FCM) was employed to observe the effect of transfection on the apoptosis of HeLa cells, and proliferation was analyzed by MTT assay. The formation of autophagic vacuoles was measured by MDC staining. HeLa cells transfected with plasmid pcDNA3.1(+)-Beclin1 and pcDNA3.1(+) were inoculated subcutaneously in nude mice. The carcinogenic and growth activities of cancer cells in vivo were observed. Eukaryotic expression vector pcDNA3.1(+)-Beclin1 was constructed successfully. It significantly improved the expression of Beclin1 mRNA and protein in HeLa cells. The proliferation of HeLa cells was inhibited, and the inhibition rate was 58.7%. FCM investigation showed that the apoptotic rate was (28.22 ± 2.34)% of pcDNA3.1(+)-Beclin1 group, significantly higher than the (14.6 ± 4.6)% in the pcDNA3.1(+) group and (11.2 ± 3.0)% in the HeLa group (P < 0.05). The monodansylcadaverin (MDC) staining showed significantly more autophagic vacuoles in the pcDNA3.1(+)-Beclin1 group (10.9%) than that in the pcDNA3.1(+) group (3.1%) and HeLa group (2.5%) (P < 0.05). After transfected with vector pcDNA3.1(+)-Beclin1, the carcinogenic activity of HeLa cells was decreased in nude mice, and the inhibition rate of tumor growth was 52.2%. Autophagy gene Beclin 1 overexpression can inhibit the proliferation and growth of HeLa cells in vitro and vivo,while promote autophagy and apoptosis of HeLa cells. So it might be one of new gene therapy strategies for cervical carcinoma.

To investigate the anti-tumor effect of acridone against breast cancer in vivo and provide a therapeutic agent for treatment of breast cancer. The nude mice xenografted tumor model was established by MCF-7 cells. The mice were randomly divided into four groups. The mice in each group (n=6) were intraperitoneally injected with 0.1 mg/kg saline (low-dose), 0.5 mg/kg (middle-dose) and 1.0 mg/kg (high-dose) of acridone for 21 days, respectively. At the end of the animal experiment, the weight of tumors was recorded to calculate the tumor inhibition rate. The serum hormone levels in peripheral blood were determined using ELISA. Hematoxylin and eosin (HE) staining was used to analyze the histopathological changes. The expression of ABCG2 protein and mRNA were determined by Western blot and RT-PCR, respectively. The inhibition rates of tumor growth in the high-dose, middle-dose, and low-dose groups were 29.18%, 17.21%, and 4.27%, respectively. Compared with control and low-dose group, the tumors growth rate in high-dose and middle-dose groups were decreased significantly. Histologically, the tumors were inhibited in the growth rate, the tissue structure was broken. Estrogen in all groups with acridone treatment decreased, the progesterone in high-dose and middle-dose groups increased remarkably. The expression of ABCG2 protein and ABCG2 mRNA decreased after treatment with acridone. We showed that acridone could induce cell apoptosis, inhibited ABCG2 (ATP-binding cassette sub-family G member 2) protein and adjusted hormone level. The results suggested that acridone could serve as a chemotherapeutic agent for treatment of breast cancer in vivo.