Antitumor Effects Research Articles

Multiomics Analysis of Interleukin-21 as a Potential Immunologic and Biomarker in Hepatocellular Carcinoma.

Interleukin-21 (IL-21) is an important antitumor cytokine that contributes to the proliferation and differentiation of CD8+ T cells. It has been proven to enhance the response to immune checkpoint inhibitors (ICIs) in various solid tumors. However, its role in hepatocellular carcinoma (HCC) has not yet been clarified. In this research, we aimed to investigate the antitumor effect of IL-21 in HCC and its effect on ICI treatment. Through transcriptome sequencing analysis and immunohistochemistry validation, we found that patients with high IL-21 expression had a better prognosis. HCCs with high expression of IL-21 had higher infiltration of CD8+ T cells, increased expression of immune checkpoints, and an improved response to ICI treatment. In conclusion, IL-21 can enhance the efficacy of ICI treatment and improve the prognosis of patients by promoting the infiltration of CD8+ T cells and the expression of immune checkpoint-related genes.

β-Lapachone promotes the recruitment and polarization of tumor-associated neutrophils (TANs) toward an antitumor (N1) phenotype in NQO1-positive cancers

ABSTRACT NAD(P)H:quinone oxidoreductase 1 (NQO1) is overexpressed in most solid cancers, emerging as a promising target for tumor-selective killing. β-Lapachone (β-Lap), an NQO1 bioactivatable drug, exhibits significant antitumor effects on NQO1-positive cancer cells by inducing immunogenic cell death (ICD) and enhancing tumor immunogenicity. However, the interaction between β-Lap-mediated antitumor immune responses and neutrophils, novel antigen-presenting cells (APCs), remains unknown. This study demonstrates that β-Lap selectively kills NQO1-positive murine tumor cells by significantly increasing intracellular ROS formation and inducing DNA double strand breaks (DSBs), resulting in DNA damage. Treatment with β-Lap efficiently eradicates immunocompetent murine tumors and significantly increases the infiltration of tumor-associated neutrophils (TANs) into the tumor microenvironment (TME), which plays a crucial role in the drug’s therapeutic efficacy. Further, the presence of β-Lap-induced antigen medium leads bone marrow-derived neutrophils (BMNs) to directly kill murine tumor cells, aiding in dendritic cells (DCs) recruitment and significantly enhancing CD8+ T cell proliferation. β-Lap treatment also drives the polarization of TANs toward an antitumor N1 phenotype, characterized by elevated IFN-β expression and reduced TGF-β cytokine expression, along with increased CD95 and CD54 surface markers. β-Lap treatment also induces N1 TAN-mediated T cell cross-priming. The HMGB1/TLR4/MyD88 signaling cascade influences neutrophil infiltration into β-Lap-treated tumors. Blocking this cascade or depleting neutrophil infiltration abolishes the antigen-specific T cell response induced by β-Lap treatment. Overall, this study provides comprehensive insights into the role of tumor-infiltrating neutrophils in the β-Lap-induced antitumor activity against NQO1-positive murine tumors.

Citral in lemon myrtle, lemongrass, litsea, and melissa essential oils suppress the growth and invasion of breast cancer cells

ObjectiveAlthough cancer therapy suppresses recurrence and prolongs life, it may be accompanied by strong side effects; thus, there is a strong demand for the development effective treatments with fewer side effects. Cancer therapy using plant-derived essential oils is attracting attention as one promising method. This study investigated the antitumor effects of essential oil volatiles on breast cancer cells and identifies four essential oils that display antitumor activity.MethodsBreast cancer cells were cultured in a 96-well plate, then one of twenty essential oils was added dropwise to the central well. The plate was incubated at 37 °C for 48 h and the effect of the volatile components of each essential oil on the surrounding breast cancer cell growth ability was examined using an MTT assay. Gas chromatography was used to investigate the concentration of the transpiration components that may affect cancer cells.ResultsOf the 20 essential oils, Lemongrass, Lemon myrtle, Litsea, and Melissa displayed strong anti-tumor effects. These essential oils inhibited the growth of nearby breast cancer cells, even when diluted more than 500-fold. The transpiration component of lemon Myrtle showed the strongest antitumor effect, but was the least cytotoxic to mononuclear cells in normal peripheral blood (PBMC). Each of these essential oils contained a very large amount of citral. The IC50 against breast cancer cells when citral was volatilized from each essential oil was 1.67 µL/mL for geranial and 1.31 µL/mL for neral. Volatilized citral alone showed strong anti-proliferation and infiltration-inhibiting effects.ConclusionThe transpiration components of Lemongrass, Lemon myrtle, Litsea, and Melissa are thought to inhibit breast cancer cell proliferation due to their high levels of citral.

IL-33 and IL-33-derived DC-based tumor immunotherapy.

Interleukin-33 (IL-33), a member of the IL-1 family, is a cytokine released in response to tissue damage and is recognized as an alarmin. The multifaceted roles of IL-33 in tumor progression have sparked controversy within the scientific community. However, most findings generally indicate that endogenous IL-33 has a protumor effect, while exogenous IL-33 often has an antitumor effect in most cases. This review covers the general characteristics of IL-33 and its effects on tumor growth, with detailed information on the immunological mechanisms associated with dendritic cells (DCs). Notably, DCs possess the capability to uptake, process, and present antigens to CD8+ T cells, positioning them as professional antigen-presenting cells. Recent findings from our research highlight the direct association between the tumor-suppressive effects of exogenous IL-33 and a novel subset of highly immunogenic cDC1s. Exogenous IL-33 induces the development of these highly immunogenic cDC1s through the activation of other ST2+ immune cells both in vivo and in vitro. Recognizing the pivotal role of the immunogenicity of DC vaccines in DC-based tumor immunotherapy, we propose compelling methods to enhance this immunogenicity through the addition of IL-33 and the promotion of highly immunogenic DC generation.

High efficacy of huCD20-targeted AcTaferon in humanized patient derived xenograft models of aggressive B cell lymphoma

Type I interferon (IFN) is a potent antitumoral drug, with an important history in the treatment of hematologic malignancies. However, its pleiotropic nature leads to severe dose-limiting toxicities that blunt its therapeutic potential. To achieve selective targeting of specific immune or tumor cells, AcTakines (Activity-on-Target Cytokines), i.e., immunocytokines utilizing attenuated cytokines, and clinically optimized A-Kines™ were developed. In syngeneic murine models, the CD20-targeted murine IFNα2-based AcTaferons (AFNs) have demonstrated clear antitumoral effects, with excellent tolerability. The current study explores the antitumoral potential of the humanized huCD20-Fc-AFN in 5 different humanized patient derived xenograft (PDX) models of huCD20+ aggressive B non-Hodgkin lymphomas (B-NHLs). The huCD20-Fc-AFN consists of a huCD20-specific single-domain antibody (VHH) linked through a heterodimeric ‘knob-in-hole’ human IgG1 Fc molecule to an attenuated huIFNα2 sequence. An in vitro targeting efficacy of up to 1.000-fold could be obtained, without detectable in vivo toxicities, except for selective (on-target) and reversible B cell depletion. Treatment with huCD20-Fc-AFN significantly increased the median overall survival (mOS) in both non-humanized (mOS 31 to 45 days; HR = 0.26; p = 0.001), and humanized NSG/NOG mice (mOS 34 to 80 days; HR = 0.37; p < 0.0001). In humanized mice, there was a trend for increased survival when compared to equimolar rituximab (mOS 49 to 80 days; HR = 0.73; p = 0.09). The antitumoral effects of huCD20-Fc-AFN were partly due to direct effects of type I IFN on the tumor cells, but additional effects via the human immune system are essential to obtain long-term remissions. To conclude, huCD20-Fc-AFN could provide a novel therapeutic strategy for huCD20-expressing aggressive B-NHLs.Graphical

Genome-wide in vivo CRISPR screen identifies TGFβ3 as actionable biomarker of palbociclib resistance in triple negative breast cancer

Triple negative breast cancer (TNBC) remains exceptionally challenging to treat. While CDK4/6 inhibitors have revolutionized HR + breast cancer therapy, there is limited understanding of their efficacy in TNBC and meaningful predictors of response and resistance to these drugs remain scarce. We conducted an in vivo genome-wide CRISPR screen using palbociclib as a selection pressure in TNBC. Hits were prioritized using microarray data from a large panel of breast cancer cell lines to identify top palbociclib sensitizers. Our study defines TGFβ3 as an actionable determinant of palbociclib sensitivity that potentiates its anti-tumor effects. Mechanistically, we show that chronic palbociclib exposure depletes p21 levels, contributing to acquired resistance, and that TGFβ3 treatment can overcome this. This study defines TGFβ3 as an actionable biomarker that can be used to improve patient stratification for palbociclib treatment and exploits the synergistic interaction between CDK4/6 and TGFβ3 to propose a new combinatorial treatment for TNBC.

Predictive model based on multiple immunofluorescence quantitative analysis for pathological complete response to neoadjuvant immunochemotherapy in lung squamous cell carcinoma.

This study aims to establish a prediction model for neoadjuvant immunochemotherapy (NICT) in lung squamous cell carcinoma to guide clinical treatment. This retrospective study included 50 patients diagnosed with lung squamous cell carcinoma who received NICT. The patients were divided into the pathological complete response (PCR) group and the non-PCR group. HE staining and multiple immunofluorescence (mIF) techniques were utilized to analyze the differences in the immune microenvironment between these groups. LASSO regression and optimal subset regression were employed to identify the most significant variables and construct a prediction model. The PCR group showed higher densities of lymphocyte nuclei and karyorrhexis based on HE staining. Furthermore, based on mIF analysis, the PCR group showed higher cell densities of CD8+, PD-L1+, and CD8+PD-L1+ in the tumor region, while showing lower cell densities of CD3+Foxp3+, Foxp3+, and CD163+. Logistic univariate analysis revealed CD8+PD-L1+, PD-L1+, CD8+, CD4+LAG-3+, lymphocyte nuclei, and karyorrhexis as significant factors influencing PCR. By using diverse screening methods, the three most relevant variables (CD8+, PD-L1+, and CD8+PD-L1+ in the tumor region) were selected to establish the prediction model. The model exhibited excellent performance in both the training set (AUC=0.965) and the validation set (AUC=0.786). In the validation set, In comparison to the conventional TPS scoring criteria, the model attained superior accuracy (0.85), specificity(0.67), and sensitivity (0.92). NICT treatment might induce anti-tumor effects by enriching immune cells and reactivating exhausted T cells. CD8+, PD-L1+, and CD8+PD-L1+ cell abundances within the tumor region have been closely associated with therapeutic efficacy. Incorporating these three variables into a predictive model allows accurate forecasting of treatment outcomes and provides a reliable basis for selecting NICT treatment strategies.

Orchestrated codelivery of peptide antigen and adjuvant to antigen-presenting cells by using an engineered chimeric peptide enhances antitumor T-cell immunity.

The intrinsic pharmacokinetic limitations of traditional peptide-based cancer vaccines hamper effective cross-presentation and codelivery of antigens and adjuvants, which are crucial for inducing robust antitumor CD8+ T-cell responses. Here, we report the development of a versatile strategy that simultaneously addresses the different pharmacokinetic challenges of soluble subunit vaccines composed of antigens and CpG to modulate vaccine efficacy via translating an engineered chimeric peptide, eTAT, as an intramolecular adjuvant. Linking antigens to eTAT enhanced cytosolic delivery of the antigens. This, in turn, led to improved activation and lymph node-trafficking of antigen-presenting cells and antigen cross-presentation, thus promoting antigen-specific T-cell immune responses. Simple mixing of eTAT-linked antigens and CpG significantly enhanced codelivery of antigens and CpG to the antigen-presenting cells, and this substantially augmented the adjuvant effect of CpG, maximized vaccine immunogenicity and elicited robust and durable CD8+ T-cell responses. Vaccination with this formulation altered the tumor microenvironment and exhibited potent antitumor effects, with generally further enhanced therapeutic efficacy when used in combination with anti-PD1. Altogether, the engineered chimeric peptide-based orchestrated codelivery of antigen and adjuvant may serve as a promising but simple strategy to improve the efficacy of peptide-based cancer vaccines.

Imidazolyl Lipids Enhanced LNP Endosomal Escape for Ferroptosis RNAi Treatment of Cancer.

Treatments for cancer that incorporate small interfering RNA (siRNA) to target iron-dependent ferroptosis are thought to be highly promising. However, creating a reliable and clinically feasible siRNA delivery system continues to be a major obstacle in the field of cancer treatment. Here, three imidazole-based ionizable lipid nanoparticles (LNPs) with pH-sensitive effects are rationally designed and synthesized for siRNA delivery. LNPs formulated with the top-performing lipid (O12-D3-I3) encapsulating FVII siRNA (FVII@O-LNP) elicited greater gene silencing than those with the benchmark Onpattro lipid DLin-MC3-DMA (MC3) due to its stronger endosomal escape. Moreover, Fc-siRNA@O-LNPs encapsulated with ferrocene (Fc) and SLC7A11/Nrf2-targeted siRNA is formulated. The outcomes demonstrate optimal safety profiles and a significant anti-tumor effect by inducing long-lasting and efficient ferroptosis through a synergistic action in vivo. In summary, this work shows that imidazolyl lipid-prepared LNPs are efficient delivery vehicles for cancer therapy and ferroptosis-targeting siRNA administration, both of which have extensive clinical application potential.

Intratumoral Fc-optimized agonistic CD40 antibody induces tumor rejection and systemic antitumor immunity in patients with metastatic cancer.

While CD40 agonism is an attractive approach for activating antigen-presenting cells and initiating antitumor responses, previous attempts have encountered limited clinical efficacy coupled with toxicity. We previously demonstrated that interactions between the antibody Fc domain and the inhibitory receptor FcγRIIB are critical for enhanced antitumor activity. Here, we present the results of a phase 1 study on intratumoral administration of an anti-CD40 agonistic antibody (2141-V11) Fc-engineered to enhance FcγRIIB binding. Primary endpoints included safety, maximum tolerated dose (MTD), and recommended phase 2 dose. Secondary objectives included preliminary clinical activity and correlative studies from biospecimens. 2141-V11 was well-tolerated without dose-limiting toxicities and MTD was not reached. In ten evaluable patients with metastatic cancer, the overall response rate was 20%, with complete responses in two patients (melanoma and breast carcinoma) and stable disease in six patients. 2141-V11 induced tumor regression in injected and non-injected lesions, with increased leukocyte infiltration and tertiary lymphoid structures (TLS) formation in post-treatment biopsies. In a humanized mouse model for CD40 and FcγRs, 2141-V11 induced TLS formation in mice bearing orthotopic breast carcinoma, correlating with local and abscopal antitumor effects, systemic immune activation, and immune memory. These findings support the safety and efficacy of 2141-V11, warranting phase 2 studies and suggesting a unique mechanism of action for this Fc-enhanced immunotherapy (NCT04059588).

Exploring the Potential Lipid-Lowering and Weight-Reducing Mechanisms of FH06 Fermented Beverages Based on Non-Targeted Metabolomics and Network Pharmacology

Investigating the intricate pathways through which FH06 fermentation broth exerts lipid-lowering and weight-loss effects is pivotal for advancing our comprehension of metabolic regulation and therapeutic interventions. Ultrahigh-performance liquid chromatography quadrupole electrostatic field orbit trap mass spectrometry (UHPLC-QE-MS) detection and the ChEMBL database were used to determine the effective compounds in the FH06 fermentation broth and predict their targets. The TTD database and DisGeNET database were used to query obesity-related targets. The STRING database was used to construct protein interaction information. The Gene Ontology (GO) database and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used to perform biological function annotation (GO) and KEGG pathway enrichment analyses of the targets. Results: A total of 85 effective compounds were screened from the fermentation broth of FH06; these compounds may act on TP53, PPARG, TNF, and other targets through 10 signaling pathways, such as the chemical carcinogenesis-receptor activation and lipid and atherosclerosis pathways, and exert pharmacological effects, such as hypoglycemic effects and weight loss. They also have anti-inflammatory, antioxidant, antitumor, and immunoregulatory effects. These findings reveal the active ingredients of FH06 fermentation broth and its multi-target and multi-channel characteristics in lipid lowering and weight loss. This study has positive implications for the clinical treatment of obesity using FH06, providing a theoretical and scientific basis for further developing of FH06-assisted lipid-lowering products.

Assessment of silver nanoparticles' antitumor effects: Insights into cell number, viability, and morphology of glioblastoma and prostate cancer cells

Silver nanoparticles (AgNPs) hold promise for cancer therapy. This study aimed to evaluate their impact on tumor and non-tumor cell number, viability, and morphology. Antitumor activity was tested on U-87MG (glioblastoma) and DU-145 (prostate cancer) cell lines. Treatment with AgNPs notably reached a reduction of U-87MG and DU-145 cell growth by 89.30% and 79.74%, respectively, resulting in slower growth rates. AgNPs induced DNA damage, evidenced by reduced nuclear area and DNA content via fluorescent image-based analyses. Conversely, HFF-1 non-tumor cells displayed no significant changes post-AgNPs exposure. Viability assays revealed substantial reductions in U-87MG and DU-145 cells (79% and 63% in MTT assays, 30% and 52.2% in high-content analyses), while HFF-1 cells exhibited lower sensitivity. Tumor cells had notably lower IC50 values than non-tumor cells, indicating selective susceptibility. Transmission electron microscopy (TEM) showed morphological changes post-AgNPs administration, including increased vacuoles, myelin figures, membrane ghosts, cellular extravasation, and membrane projections. The findings suggest the potential of AgNPs against glioblastoma and prostate cancer, necessitating further exploration across other cancer cell lines.

Morusin Inhibits RANKL-induced Osteoclastogenesis and Ovariectomized Osteoporosis.

Postmenopausal osteoporosis (PMOP) is a classic type of osteoporosis that has gradually become a significant health problem worldwide. There is an urgent need for a safe alternative therapeutic agent considering the poor therapeutic strategies currently available for this disease. The roots and bark of the Morus australis tree (Moraceae) are used to make a traditional Chinese medicine known as "Morusin", and accumulating evidence has demonstrated its multiple activities, such as anti-inflammatory and anti-tumor effects. In this study, we aim to explore the effect of Morusin on mouse osteoclasts and its mechanism. In this study, we explored the inhibitory effects of Morusin on murine osteoclasts in vitro and its mechanism, and the protective effect of Morusin on an ovariectomy (OVX)-induced osteoporosis model in vivo. The results showed that Morusin prevented OVX-induced bone loss and dramatically decreased RANKL-induced osteoclastogenesis. Morusin interfered with RANKL-activated NF- κB, MAPK, and PI3K/AKT signaling pathways. The expression of three master factors that control osteoclast differentiation, c-Fos, NFATc1, and c-Jun, was reduced by Morusin treatment. Collectively, in vitro results indicated that Morusin has a protective effect on OVX-induced bone loss in a mouse model. Our data provide encouraging evidence that Morusin may be an effective treatment for PMOP.

Myricetin inhibits 4 T1 breast tumor growth in mice via induction of Nrf-2/GPX4 pathway-mediated Ferroptosis

Ferroptosis is a recently identified form of programmed cell death that is iron-dependent and closely involved in the pathogenesis of breast cancer. Past studies have identified myricetin as being able to inhibit breast cancer growth through its targeting of apoptotic mechanisms, but the precise mechanisms whereby it exerts its antitumoral effects in breast cancer remain to be characterized in detail. Here, the effects of myricetin on the induction of ferroptosis in breast cancer cells were investigated. It was found that myricetin was able to significantly inhibit 4 T1 tumor cell viability and colony forming activity, increasing the level of MDA, Fe2+, and ROS within these cells. From a mechanistic perspective, myricetin was found to induce ferroptotic 4 T1 cell death via downregulating Nrf-2 and GPX4. In vivo experimentation demonstrated that myricetin treatment was sufficient to reduce the growth of subcutaneous breast tumors in female mice as evidenced by decreases in tumor weight and volume, while significantly inhibiting Nrf-2 and GPX4 expression within the tumors of treated mice. Myricetin is capable of readily suppressing breast tumor growth in mice via the induction of ferroptotic activity through the Nrf-2/GPX4 pathway. Myricetin may thus offer utility as a therapeutic agent for the management of breast cancer in clinical settings.

Use of integrated multi-omics analysis to determine antitumor efficacy of donafenib in combination with FADS2 inhibition in hepatocellular carcinoma.

e15083 Background: Pharmacotherapy remains a pivotal treatment strategy for patients with advanced hepatocellular carcinoma (HCC). Donafenib, globally the first targeted therapy proven to offer superior survival benefits and safety over sorafenib in a large-scale phase III clinical trial with head-to-head comparison, has been adopted for first-line treatment of advanced HCC. Given the limited basic research on Donafenib both domestically and internationally, and its constrained clinical efficacy, this study aims to explore combination drug therapy for HCC in an attempt to achieve further breakthroughs and enhance the anticancer activity of Donafenib. Methods: A xenograft tumor model in nude mice was established to observe the in vivo antitumor effects of Donafenib. Transcriptomics and proteomics were employed to characterize the gene expression changes in HCC cell lines after Donafenib intervention and to screen for key molecules. A series of assays, including the Cell Counting Kit-8 (CCK8), flow cytometry, scratch assays, Transwell assays, Western blotting, and in vivo tumorigenicity in nude mice, were conducted to determine the impact of the hub gene on the malignant behavior of HCC and to evaluate the combined antitumor effects of the molecular inhibition and Donafenib treatment in HCC. Results: Treatment with Donafenib significantly slowed tumor growth in a nude mouse xenograft model, resulting in a marked reduction in final tumor volumes compared to control subjects. Proteomic analyses post-Donafenib treatment identified FADS2 as the protein with the highest absolute log2 fold change among all differentially expressed proteins in HCC cells. This finding was corroborated by transcriptomic data, which indicated a concurrent downregulation of FADS2 at the mRNA level. Subsequent in vitro and in vivo assays validated the inhibitory effect of FADS2 blockade on the malignant biological behaviors of HCC cells. Importantly, the concomitant administration of Donafenib and the FADS2 inhibitor SC-26196 exhibited a synergistic antitumor action, significantly enhancing therapeutic efficacy in both HCC cell lines and xenografted tumors in nude mice. Conclusions: Combination therapy, a prevalent approach in clinical oncology, has the potential to augment treatment efficacy while simultaneously diminishing toxicity. This study employed an extensive multi-omics approach complemented by a range of both in vitro and in vivo experiments. It revealed that the synergistic use of Donafenib and SC-26196 markedly enhances Donafenib-induced the inhibition of cancer cell growth and metastasis, demonstrating a significant synergistic antitumor effect. These findings from our preclinical investigations may offer a promising new combinational drug regimen for the therapeutic management of HCC patients.

First-in-human study of ZGGS15, a dual specific antibody targeting LAG-3 and TIGIT, as monotherapy in patients with advanced solid tumors.

e14678 Background: ZGGS15 is a recombinant humanized bispecific antibody composed of a monoclonal antibody against LAG-3 and TIGIT. It can specifically bind LAG-3 and TIGIT. It reverses LAG-3 and TIGIT immune suppression to T and NK cells, relieves suppression of Treg to T and NK cells, promotes the activation and proliferation of T and NK cells, and cytokines release. ZGGS15 restores T and NK cell functions to kill tumor cells. In non-clinical studies, we found that ZGGS15 had significant anti-tumor effects and synergistic anti-tumor effects with Anti-PD-1 antibody. Therefore, we conducted a Phase 1 clinical study to assess the tolerability, safety, and preliminary efficacy of this drug as monotherapy in patients with advanced solid tumors. Methods: During the dose escalation stage, an accelerated titration design (ATD) in the first dose (1 subject if ≤Grade 2 treatment-related adverse event occurred) and the standard "3+3" design in the rest doses were used. Subjects were patients with advanced solid tumors who failed to the available standard treatments. The dose groups were set from 0.3 to 30 (0.3, 1, 3, 10, 20, 30) mg/kg, intravenous infusion, once every 3 weeks. The first 21-day period was defined as the dose-limiting toxicity (DLT) observation period. Subjects could continue to receive ZGGS15 until treatment discontinuation criteria were met. Tumor response was assessed by RECIST1.1 and iRECIST. Pharmacokinetics (PK) and receptor occupancy (RO) profiles were also assessed in this study. Results: A total of 12 patients (4 males and 8 females) completed the DLT observation in the first four dose groups (0.3 to 10 mg), with a median age of 57 years. Eight (66.7%) of them received at least 3 lines of antineoplastic therapies, and five (41.7%) have been exposed to PD-1 or PD-L1 inhibitors. There was no DLT event observed. Thirty-eight treatment-related adverse events (TRAEs) occurred. Up to now, TRAEs were all grade 1 ~ 2. The most frequent TRAEs were proteinuria (33.3%, 4/12), hypokalemia (25%, 3/12), hypoalbuminemia (25%, 3/12), and hypertriglyceridemia (25%, 3/12). The Cmax and AUC increased from 0.3-1 mg/kg approximately in dose proportion. Nearly complete and sustained RO of T and NK cells in PBMCs was observed at 1 mg/kg dose of ZGGS15 for at least 3 weeks. Conclusions: The first four dose levels of ZGGS15 were very well tolerated. A good PK profile, and a long-lasting RO were observed in this escalation study. It is anticipated that this product could provide an additional benefit only when use in combination with an anti-PD-1 antibody. Clinical trial information: NCT05864573 .

Examination of in vitro and in vivo antitumor activity of PARP1 selective inhibitor D0112-005.

e15115 Background: The approved PARP inhibitors have demonstrated efficacy in ovarian, breast, prostate, and pancreatic cancers, particularly in homologous recombination repair (HRR)-deficient tumors, including BRCA mutant tumors. The severe hematological toxicity of first-generation PARPi in combination with chemotherapy is due to the lack of selectivity for PARP2. So, we developed selective PARP1 inhibitor D0112-005 with the aim to improve safety and efficacy. Methods: The selectivity of D0112-005 on PARP1, PARP2 was measured by chemiluminescent assay kit and PARP trap™ assay kit. The anti-proliferative ability of D0112-005 was evaluated by CCK-8 in BRCA mutant cell lines. In vivo anti-tumor effect of D0112-005 was evaluated in MDA-MB-436 BRCA1 mutant xenograft model. The potential for hematologic toxicity was evaluated in rats for 28 days repeat-dose and dogs for 14 days repeat-dose. Results: D0112-005 is a highly potent and selective inhibitor of PARP1 with 2000-fold selectivity for PARP1 over PARP2 and PARP3. D0112-005 trapped PARP1-DNA with an EC50 value of 6.49 nM. In contrast, the EC50 of PARP2-DNA trapping was 430μM. These data demonstrate that D0112-005 is a potent and selective PARP1 trapper. D0112-005 showed high anti-proliferation activity against BRCA mutant MDA-MB-436 and HCC1395 cell lines, with IC50 values of 2.06 nM and 2.446 nM, respectively. In the MDA-MB-436 BRCA1m xenograft model, a dose-dependent anti-tumor effect was observed after 21 days administration, with TGIs of 38.77%, 106.7% and 111.73% at the dose of 0.1, 0.3 or 1 mg/kg once daily by oral, respectively. In the 1 mg/kg group, the anti-tumor efficacy was maintained after 14 days of drug withdrawal. D0112-005 showed well tolerance without bodyweight loss. In rat 28 days repeat dose study, D0112-005 100mg/kg had less changes on hematologic parameters compared with Olaparib. The main toxic reaction of D0112-005 (10mg/kg) was reversible thrombocytopenia in dog 14 days repeated toxicity study. Conclusions: D0112-005 potently and selectively inhibits PARP1 resulting in excellent anti-tumor activity in vivo and in vitro, which also good safety profile in the repeated toxicity study in rats and dogs.

An exhaustive pre-clinical study of a circRNA drug candidate (cmRNA1210) encoding IL-12sc for anti-tumor therapeutics.

e14569 Background: Circular RNA as emerging technology hasn't been approved of acceptance of any IND application. What’s the pharmacokinetics, pharmacodynamics and toxicity study of circular RNA drug candidate would be like is still unpublished. Here we provide an exhaustive pre-clinical study of a novel circular RNA drug candidate encoding IL-12sc, the immune stimulatory effector. Methods: Here, we performed an exhaustive pre-clinical study of a circular RNA drug candidate(cmRNA1210) encoding IL-12sc, including pharmacokinetics, pharmacodynamics, pharmacology and toxicology study. Meanwhile, we explored the bioactivity and cross reactivity of cmRNA1210 in vitro and the pharmacokinetics in vivo. Results: cmRNA1210 and its replace molecule exhibits robust therapeutic effect and good safety in syngeneic mouse tumor model and humanized human tumor mouse model. In situ administration of cmRNA1210 achieves abscopal effect and inhibits lung metastasis progression. Treatment with cmRNA1210 leads to cytokines change in plasma and tumor, cytotoxic immune cells accumulation in tumor and tumor tissue necrosis. Dynamic transcriptome profiling of tumor tissue post cmRNA1210 treatment. Combination with checkpoint inhibitor exhibits synergy effect and significantly decreases tumor relapse rate. Kaplan-Meier survival curve in TCGA dataset shows strong correlation of high IL-12A/B expression and long survival. Conclusions: The bioactivity and cross reactivity in rodents and primates of cmRNA1210 were validated in vitro. The advantage of pharmacokinetics of circular RNA was confirmed in vivo. cmRNA1210 exhibits robust anti-tumor effect in various tumor models, indicating its wild application situation in clinical. In situ treatment achieves abscopal effect and significantly inhibits lung metastasis progression. Systemic anti-tumor effect has been successfully initiated. The pharmacodynamics study reveals there are cytokine changes, immune cells activation or accumulation at tumor site and tumor tissue necrosis after cmRNA1210 treatment. cmRNA1210 as immune stimulatory effector works synergistically with checkpoint inhibitor. Kaplan-Meier survival analysis of IL-12 expression based on TCGA dataset shows there is a strong correlation of high expression and long survival. Overall, this pre-clinical study provides elaborate results of cmRNA1210 as anti-tumor therapeutics and demonstrates the great potential to enter clinical trial in the future.

Use of IL-3RB to mediate antitumor effect of CAR-T cells through the JAK-STAT signaling pathway.

2541 Background: The application of CAR-T in solid tumors has not yet demonstrated significant clinical benefits. The continuous proliferation and decreased terminal differentiation of CAR-T cells in vivo are considered potential strategies that could alter this predicament. Cytokines, as the 3rd signal for T-cell activation, play a crucial role in the lifecycle and fate of T cells. In this study, we innovatively incorporated the IL-3 receptor β chain (IL-3RB) into the CAR structure to enhance signal transduction, potentially activating the anti-tumor function of CAR-T and maintaining the persistent proliferative activity. Methods: Transcriptional profiling analysis performed on HER2 or VEGFR1 CAR-T cells, with a specific focus on the gp140 cytokine family (IL-3/IL-5/CSF-2) and the receptor expression, validated by ELISA and Western blot. By cloning CAR genes containing different structural domains of the IL-3RB into the lentiviral vector pWPXLd, CAR expression, activation, depletion, proliferation, cytotoxicity, pSTAT3 and pSTAT5, and cytokine release of CAR-T cells were assessed using flow cytometry (FCM), Western blot (WB), RTCA, CCK-8, and ELISA. Subcutaneous/abdominal injections of SKOV3 cells or SKOV3-Luc cells into NSG mice, followed treated by CAR-T cells. Tumor burden was regularly monitored, and FCM and Q-PCR were utilized to assess the quantity and phenotype of CAR-T cells in vivo. Results: RNA-sequence, ELISA and WB revealed that upon activation of CAR-T cells, the expression of genes such as IL-3/IL-5/CSF-2 rapidly increased. However, their receptors were hardly expressed. IL-3RB modification did not affect the phenotype of resting CAR-T cells. But, co-cultured with target cells, IL-3RB significantly enhanced the anti-tumor effects of CAR-T cells. This was manifested by more rapid activation, promotion of persistent proliferation, delayed exhaustion phenotype, secretion of higher levels of cytokines (IL-2, IFN-γ, TNF-α) and efficient cytotoxicity. IL-3RB-expressing CAR-T cells exhibited activation of the JAK kinase and STAT3, STAT5 transcription factor signaling pathways in vitro. We established solid tumor models of subcutaneous (SKOV3) and intraperitoneal metastasis, where HER2/VEGFR1 CAR-T cells partially inhibited tumor cell growth in vivo. In contrast, IL-3RB-enhanced CAR-T cells completely eradicated tumor cells, and even achieving a state of complete remission. FCM and Q-PCR also confirmed that IL-3RB significantly improved the proliferation and survival of CAR-T cells in vivo. Conclusions: IL-3RB is scarcely expressed in T cells and not regulated by T cell activation. IL-3RB-modified CAR-T cells exhibit superior persistence and significant anti-tumor advantages in a solid tumor through the JAK-STAT signaling pathway. Our innovative CAR structure design has the potential to demonstrate promising anti-tumor effects in clinical translational applications.

Effect of IL-15 on anti-tumor immune response to radio-immunotherapy through systemic immune activation.

e14641 Background: In this study, we propose to investigate the key role of IL-15 in radio-immunotherapy and its mechanism in regulating systemic immune activation by radio-immunotherapy. And further explore the potential of IL-15 combined with radio-immunotherapy. Methods: LLC5 and MC38 cells were used in this study. Mouse model and co-culture model were constructed to analyze the effect of IL-15 after radio-immunotherapy. Anti-IL-15 antibody was used to clarify the role of IL-15 on radio-immunotherapy. Flow cytometry and multiplex immunofluorescence were used to clarify IL-15Rα lever in macrophage after radio-immunotherapy. The effect of IL-15 in tumor and secondary lymphoid organ(SLO) was observed after macrophage removal. Macrophage IL-15 level were tested by ELISA. Tumor growth, TME, and memory effect after IL-15 combined with radio-immunotherapy were observed. In co-culture model, CD8+ T cell function were tested. Clearance macrophage of mice to determine the role of macrophage in combination therapy. After macrophages co-cultured with tumor cells, cytokine microarray was used to analyze the cytokine secretion of macrophages in combination treatment. Results: IL-15 secretion was significantly upregulated in TME after radio-immunotherapy. IL-15 and IL-15Rα were most enriched on macrophages and IL-15Rα was significantly increased after radio-immunotherapy. IL-15 inhibition significantly attenuated tumor killing and systemic immune activation and resulted in decreased IL-15Rα expression on macrophages. Removal of macrophages resulted in a significant reduction of IL-15 in tumor and spleen. Radio-immunotherapy in the co-culture model induced a significant increase of IL-15 secreted by macrophages.IL-15 combined with radio-immunotherapy can significantly controlled tumor growth, stimulated systemic immune activation, and CD8+ T cells and memory T cells were significantly increased in the circulation of long-term survival mice. In co-culture experiments, combination treatment stimulated proliferation and secretion of IFN-γ+CD8+ T cells. And it stimulated macrophages secrete more chemokines, especially CCL5, recruit more CD8+ T cells aggregate around macrophages. Macrophage clearance and co-culture experiments confirm that the synergistic effect of this combination therapy is mainly dependent on macrophages. Conclusions: IL-15 plays a critical role in systemic immune activation induced by radio-immunotherapy. Macrophages are the main cellular site of IL-15 effect in the microenvironment of tumor and SLO after radio-immunotherapy. IL-15 combined with radio-immunotherapy had a synergistic antitumor effect, and this combination treatment induced a further enhancement of memory T-cell-mediated systemic immune activation. Macrophage-secreted chemokines such as CCL5 played an important role in the antitumor effect of this combination therapy strategy.