Molecular Cancer Therapeutics最新文献

Gene-directed enzyme prodrug therapy represents a promising antitumor strategy owing to its low systemic toxicity. However, clinical translation has been hindered by challenges in suicide gene delivery. Artificially engineered circular RNA (circRNA) demonstrates exceptional potential for gene delivery when combined with lipid nanoparticle technology, exhibiting high stability, prolonged protein-coding capacity, and cost-effective production. We developed an in vitro synthesized circRNA encoding the cytosine deaminase-uracil phosphoribosyltransferase (circCDUPRT). Upon intratumoral administration, circCDUPRT achieved sustained intratumoral expression with minimal systemic toxicity. The combination of circCDUPRT and prodrug 5-fluorocytosine showed significant antitumor efficacy in both in vitro and in vivo tumor models. In advanced melanoma models, combining circCDUPRT/5-fluorocytosine with IL-15-expressing circRNA potently enhanced the expansion and activation of CD8+ T and NK cells. Collectively, these findings establish the synthetic circRNA platform as a cost-effective, high-efficiency delivery system for gene-directed enzyme prodrug therapy and immunotherapy applications.

Drug repurposing is the process of reusing existing pharmaceuticals for novel clinical purposes, which offers advantages such as streamlined clinical trial access and reduced drug development costs. Clarithromycin (CAM), a member of the macrolide antibiotics family, is a promising candidate for repurposing in cancer therapy due to its known preclinical and clinical immunomodulatory and anticancer properties. In the current study, we investigated whether CAM could be repurposed as a preventive treatment for KRAS-mutant lung cancer, a subtype of lung adenocarcinoma that is strongly associated with heavy smoking. CCSPCre; LSL-KrasG12D mice at an early stage of tumor development were treated with different doses of CAM for 10 weeks. While exhibiting an excellent safety profile, CAM was able to prevent the development of premalignant and malignant lung lesions in a dose-dependent manner. In addition, CAM significantly reduced the infiltration of neutrophils/polymorphonuclear myeloid-derived suppressor cells and inhibited the mRNA expression of protumor inflammatory cytokines IL-6, TNFα, and IL-1β, as well as M2 macrophage markers Fizz1 and Arginase1 in the lung tumor microenvironment. Moreover, we investigated the effect of CAM in reshaping the intestinal and lung microbiome. Long-term CAM usage decreased intestinal microbiome diversity but, more notably, significantly increased the abundance of the probiotic genus Muribaculaceae while decreasing the abundance of Desulfovibrio, a genus associated with the promotion of various malignancies. Taken together, we conclude that CAM could provide promising cancer prevention efficacy in KRAS-mutant lung cancer due to its immunomodulatory properties on the tumor microenvironment and its regulatory effects on the microbiome.

Bispecific antibodies (BsAb) are a rapidly advancing class of biopharmaceuticals with substantial potential for cancer immunotherapy. Although BsAbs have shown notable success in treating certain hematologic malignancies, their application for solid tumors remains limited. The extra domain B (EDB) of fibronectin represents a promising pan-tumoral stromal target, offering an attractive alternative to conventional cellular tumor antigens, which often face limitations with respect to specificity in solid tumors. In this study, we describe the generation and characterization of a T cell-engaging BsAb that targets murine CD3 using the 2C11 clone and EDB with the L19 clone. Specifically, the BsAb consists of a Fab fragment (targeting CD3) fused with two single-chain Fv fragments (targeting EDB) at the C-terminus. The BsAb was produced in Chinese hamster ovary cells and purified to homogeneity. To compare stromal and cellular targeting, two murine tumor cell lines naturally secreting EDB in the stroma were transduced to express the target on the cell surface. In both cell lines, biodistribution analysis revealed increased tumor uptake in the cellular model compared with the stromal one. Similarly, treating immunocompetent cellular EDB tumor-bearing mice with the BsAb improved anticancer activity. By contrast, no significant therapeutic benefit was observed in the stromal model. These findings underscore the importance of direct tumor cell targeting compared with stromal targeting for effective BsAb therapy.

Cancer-associated fibroblasts (CAF) are key components of the tumor microenvironment (TME) that promote tumor progression either directly through tumor-CAF interactions or indirectly by influencing tumor-infiltrating immune cells, thereby creating an immunosuppressive TME. High stromal signatures have been associated with reduced therapeutic efficacy and resistance to immune checkpoint blockades. Adipocyte enhancer-binding protein 1 (AEBP1) is predominantly expressed in myofibroblasts, and its expression is further increased in CAFs that produce the extracellular matrix. It has two isoforms: The extracellular isoform binds to collagen and promotes collagen remodeling, whereas the intracellular isoform modulates transcription and signaling. We observed the expression of both isoforms in primary human CAFs. Our data showed that the combined knockout (KO) of both AEBP1 isoforms via gene editing decreased CAF proliferation, collagen gel contractility, and CAF-mediated tumor cell proliferation in vitro. AEBP1 KO mouse fibroblasts demonstrated reduced activity in both in vitro assays and in vivo within a coimplantation mouse model. RNA sequencing revealed that AEBP1 KO downregulated the collagen biosynthesis and extracellular matrix organization-related pathways in mouse fibroblasts and human CAFs. Importantly, AEBP1 loss in fibroblasts led to significant alterations in tumor cell phenotypes, including a marked reduction of tumor cells exhibiting an epithelial-mesenchymal transition signature in vivo. Furthermore, AEBP1 KO in CAFs enhanced the anti-PD-1-induced effector T-cell function and the anti-PD-1 efficacy. Our findings indicate that AEBP1 plays a crucial role in regulating the function of CAFs within the TME. Targeting AEBP1 could be a promising strategy to inhibit the tumor-promoting activities of CAFs and to overcome resistance to anti-PD-1 immunotherapy.

TUB-010 is a next-generation antibody-drug conjugate (ADC) targeting CD30 expressed on various hematopoietic malignancies such as Hodgkin lymphoma. Among the therapeutic options for patients with relapsed and refractory CD30-positive cancers is brentuximab vedotin (Adcetris), a monomethyl auristatin E (MMAE)-delivering anti-CD30 ADC with a mean drug-to-antibody ratio of 4. Adcetris exhibits a high response rate at the cost of significant toxicities, likely driven by the payload MMAE and the instability of the maleimide conjugation chemistry. TUB-010 uses the same antibody and payload as Adcetris but is based on the Tub-tag conjugation strategy, which stably attaches MMAE to the hydrophilic Tub-tag peptides on the light chains via chemoenzymatic conjugation. This new technology enables the generation of a homogeneous and site-specific drug-to-antibody ratio 2 ADC with unique biophysical properties. TUB-010 demonstrates similar binding and lysosomal release characteristics as Adcetris, which translates into comparable in vitro cytotoxicity on CD30-positive cell lines when normalized to the MMAE concentration. Importantly, TUB-010 exhibits higher stability with negligible premature deconjugation in circulation and reduced aggregation, as well as lower nonspecific cytotoxicity on target-negative cells compared with Adcetris. As a consequence, TUB-010 induces superior tumor control compared with Adcetris when dosed at equal MMAE concentrations in vivo and also lower toxicity and higher tolerability in rodents and nonhuman primates. Taken together, TUB-010 is a novel, potential best-in-class anti-CD30 ADC with improved biophysical properties designed to deliver MMAE with higher precision and a wider therapeutic window than Adcetris using Tub-tag technology. Therefore, TUB-010 may increase the clinical benefit of anti-CD30 ADC therapies.

Histone deacetylase (HDAC) inhibitors have been considered as anti-leukemic agents but have shown poor efficacy in clinical trials. In this study, we investigated the immediate transcriptional response to the HDAC inhibitor SAHA (vorinostat) in healthy CD34+ blood stem/progenitor cells and myeloblasts from patients with primary acute myeloid leukemia (AML) carrying TET2 and NPM1 mutations. We found that although healthy CD34+ and AML cells differed substantially at the transcriptional level, they responded very similarly to 10-minute SAHA treatment. HDAC inhibition led to a global increase in histone acetylation; however, only 150 to 250 genes were upregulated. These were involved in oxidative stress, metabolism, chromatin regulation, cell cycle control, and cell death, and the vast majority was upregulated in both healthy and AML cells. Upregulated genes were more highly acetylated compared with average expressed genes and had higher levels of promoter-proximal paused RNA polymerase II (Pol II) before treatment. Upon HDAC inhibition, upregulated genes increased BRD4 occupancy the most and released paused Pol II into transcription elongation. Our results suggest that the immediate effect of HDAC inhibition is to trigger release of paused Pol II into elongation. We speculate that the similar transcriptional response in healthy and leukemic cells may contribute to the poor efficacy of HDAC inhibitors in patients with hematologic malignancies.

Diffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain tumor with limited therapeutic progress due to its infiltrative brainstem location, blood-brain barrier (BBB), and resistance to systemic agents. We present a novel strategy for targeted central nervous system (CNS) delivery of ATSP-7041, a stapled peptide dual HDM2/HDMX inhibitor, using the Minimally Invasive Nasal Depot (MIND) technique. In p53-wild-type, PPM1D-mutant DIPG neurospheres (BT869), ATSP-7041 exhibited ~125-fold greater anti-tumor activity than the HDM2-selective antagonist RG7388, consistent with elevated HDMX expression. MIND delivery in mice achieved sustained ATSP-7041 distribution across brain regions, including the pons, with peak levels at 72 hours and persistence for up to 14 days. In a patient-derived orthotopic xenograft (PDX) model of DIPG, a single MIND-administered ATSP-7041 depot reduced tumor burden and prolonged survival compared to controls. This feasibility study provides proof-of-concept for on-target p53 reactivation in DIPG using a BBB-penetrant dual HDM2/HDMX inhibitor delivered by the MIND platform. The findings support a translational path for ALRN-6924, the clinical analog of ATSP-7041, in DIPG and potentially other brain tumors that retain wild-type p53 but remain incurable due to drug resistance and restricted CNS access.

EGFR Exon20 insertions (Exon20Ins) constitute the third most common EGFR activating mutation in non-small cell lung cancer. We developed a semi-mechanistic pharmacodynamic (PD) model for irreversible inhibitors of EGFR Exon20Ins mutations by integrating kinetic data of proprietary compounds with a mechanistic description of EGFR turnover and phosphorylation to investigate the preclinical relationship between phosphorylated EGFR (phosEGFR) reduction and efficacy, and its translation to the clinical setting. In engineered NCI-H2073 cells hosting the Exon20 SVDIns mutation, EGFR turnover was studied via SILAC MS and phosEGFR time-course analysed via ELISA. Kinetic parameters were determined from a biochemical binding assay. These data were integrated into the model to describe phosEGFR inhibition in vitro and in vivo. Tumour volume data from xenograft studies were then used to quantify the relationship between phosEGFR inhibition and anti-tumour activity. We found that sustained >84% phosEGFR inhibition is required for tumour regression. Clinical phosEGFR simulations were generated for 2 proprietary inhibitors, providing an early estimation of their active human doses. We also explored clinical phosEGFR reduction induced by the 3rd generation TKI osimertinib, suggesting that limited target engagement may explain modest response achieved in EGFR Exon20Ins at the clinically investigated doses. The developed model is a valuable tool to understand the impact of kinetic characteristics on phosEGFR reduction and related efficacy, select a target engagement-based criterion for therapeutic dose predictions, and provide interpretation and insights on observed clinical efficacy of irreversible inhibitors in EGFR Exon20Ins.

Mitogen-activated protein kinase kinase (MEK) is a component of an important signaling pathway involved in the development and progression of pancreatic ductal adenocarcinoma (PDAC). However, MEK-targeted therapeutics are not effective, and therefore not indicated, for patients with PDAC. We have found that Annexin A8 (ANXA8) is involved in resistance to MEK inhibitor therapy in PDAC. Expression of ANXA8 was induced at both the mRNA and protein levels early by MEK inhibitor treatment in PDAC cells and the level of ANXA8 mRNA expression was inversely correlated with sensitivity to the MEK inhibitor. Furthermore, downregulation of ANXA8 enhanced the inhibitory effect of MEK inhibitor on PDAC cell proliferation, suggesting that ANXA8 could be a potential therapeutic target for PDAC. To achieve a therapeutic strategy targeting ANXA8, we have identified all-trans retinoic acid (ATRA) as a compound exerting ANXA8-inhibitory effects in PDAC cells. Combination of the MEK inhibitor and ATRA demonstrated additive anti-tumor effects in PDAC cells in vitro and in vivo. Immunohistochemical analysis revealed that ANXA8 was frequently upregulated in PDAC showing poor differentiation relative to PDAC with high or moderate differentiation. Furthermore, patients with ANXA8-positive PDAC were found to have a significantly poorer prognosis than those with ANXA8-negative PDAC. In summary, our findings suggest that ANXA8 plays a role in MEK inhibitor resistance in PDAC, and that a combination of MEK inhibition with ANXA8-targeted therapy could be a novel effective strategy for PDAC.

The ganglioside GD2 is an attractive cancer target due to its high expression in neuroblastoma and other solid tumors, with limited normal tissues distribution. Despite regulatory approvals of three anti-GD2 antibodies, clinical efficacy remains limited by neurotoxicity, suboptimal affinity for ADCC, and immunogenicity. Developing a highly effective, less toxic anti-GD2 agent remains an unmet need. In this study, a novel anti-GD2 murine antibody, CA450, was identified by immunizing mice with GD2 conjugated to KLH or Qβ virus-like particles (VLP), followed by phage display screening. The humanized version, hCA450-21, displayed higher cell-binding activity than ch14.18 and Hu3F8, along with excellent specificity and internalization capacity. To overcome the limitations of traditional anti-GD2 antibody therapy, hCA450-21 was engineered and conjugated to Exatecan to create an antibody-drug conjugate (ADC), which may reduce or avoid neurotoxicity by employing a mechanism distinct from antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The hCA450-21.1-LA-68B ADC demonstrated potent in vitro cytotoxicity against glioblastoma, melanoma, and breast cancer cell lines, and in vivo tumor growth inhibition in LN229 and SK-MEL-5 xenograft models. Toxicity studies in mice showed a favorable safety profile, with reduced neurotoxicity compared to naked antibody therapy by ch14.18-IgG1 and hCA450-21.1-IgG1. The crystal structure of the hCA450-21.1 Fab-GD2 complex was resolved at 1.69 Å, revealing unique hydrogen bonds and hydrophobic interactions that contribute to its high specificity and affinity. Overall, the novel hCA450-21.1-LA-68B ADC shows preclinical efficacy and reduced toxicity, particularly neurotoxicity, indicating potential as a safer and more effective therapy for GD2-positive pediatric and adult tumors.