Target Tumor Tissue Research Articles

Genetically Engineered Filamentous Bacteriophages Displaying TGF-β1 Promote Angiogenesis in 3D Microenvironments

Combined 3D cell culture in vitro assays with microenvironment-mimicking systems are effective for cell-based screening tests of drug and chemical toxicity. Filamentous bacteriophages have diverse applications in material science, drug delivery, tissue engineering, energy, and biosensor development. Specifically, genetically modified bacteriophages have the potential to deliver therapeutic molecules or genes to targeted tumor tissues. The engineered bacteriophages in this study significantly enhanced endothelial cell migration and tube formation within the extracellular matrix (ECM). Compared to TGF-β1 alone and non-modified phages, the presence of TGF-β1 on the bacteriophages demonstrated superior performance as a continuous stimulant in the microenvironment, effectively promoting these angiogenic processes. Assays, including RT-qPCR, ELISA, and fluorescence microscopy, confirmed the expression of angiogenic markers such as CD31, validating the formation of 3D angiogenic structures. Our findings indicate that the TGF-β1 displayed by bacteriophages likely acted as a chemotactic factor, promoting the migration, proliferation, and tube formation of endothelial cells (ECs) within the ECM. Although direct contact between ECs and bacteriophages was not explicitly confirmed, the observed effects strongly suggest that TGF-β1-RGD bacteriophages contributed to the stimulation of angiogenic processes. The formation of angiogenic structures by ECs in the ECM was confirmed as three-dimensional and regulated by the surface treatment of microfluidic channels. These results suggest that biocompatible TGF-β1-displaying bacteriophages could continuously stimulate the microenvironment in vitro for angiogenesis models. Furthermore, we demonstrated that these functionalized bacteriophages have the potential to be utilized as versatile biomaterials in the field of biomedical engineering. Similar strategies could be applied to develop angiogenic matrices for tissue engineering in in vitro assays.

Intelligent Modular DNA Lysosome-Targeting Chimera Nanodevice for Precision Tumor Therapy.

Lysosome targeting chimeras (LYTACs) have emerged as a powerful modality that can eliminate traditionally undruggable extracellular tumor-related pathogenic proteins, but their low bioavailability and nonspecific distribution significantly restrict their efficacy in precision tumor therapy. Developing a LYTAC system that can selectively target tumor tissues and enable a modular design is crucial but challenging. We here report a programmable nanoplatform for tumor-specific degradation of multipathogenic proteins using an intelligent modular DNA LYTAC (IMTAC) nanodevice. We employ circular DNA origami to integrate predesigned modular multitarget protein binding sites and pH-responsive protein degradation promoters that specifically recognize cell-surface lysosome-shuttling receptors in tumor tissues. By precisely manipulating the stoichiometry and modularity of promoters and ligands targeting diverse proteins, the IMTAC nanodevice enables accurate localization and delivery into tumor tissues, where the acidic tumor microenvironment triggers degradation switch activation, multivalent binding, and efficient degradation of various prespecified proteins. The tissue-specificity and multiple ligands in IMTACs significantly improve the drug utilization rate while reducing off-target effects. Importantly, this system demonstrates the capability of collabo-rative degradation of EGFR and PDL1 in tumor tissue for combined targeting and immunity therapy of hepatocellular carcinoma (HCC), resulting in obvious tumor necrosis and inhibition of tumor growth in vivo even at low concentrations. This study presents a unique strategy for building a general, intelligent, modular, and simple encoded nanoplatform for designing precision medicine degraders and developing proprietary antitumor drugs.

Multi-functional nanosonosensitizer-engineered bacteria to overcome tumor hypoxia for enhanced sonodynamic therapy

BackgroundUltrasound-triggered sonodynamic therapy (SDT), with high safety and acceptance, has become a promising tumor treatment. However, the dense stroma, hypoxic microenvironment of tumor, and the unpredictable treatment timing limit the effectiveness of sonosensitizers and the antitumor therapeutic effect. Thus, it is crucial to develop an imaging-guided sensitization strategy for hypoxic tumor sonosensitization to improve the efficacy of SDT. MethodsIn this study, we developed a biohybrid system CB@HPP, which genetically engineered bacteria to express catalase (CB) and modified nanosonosensitizers (HPP) to the surface of these bacteria. Tumor hypoxia relief, tumor targeting, biocompatibility, and antitumor efficacy were evaluated through in vitro and in vivo experiments. In addition, the photoacoustic (PA), ultrasound (US), and fluorescence (FL) imaging effects of CB@HPP were evaluated in vivo and in vitro. ResultsAfter intravenous injection, CB@HPP was able to target tumor tissue. CB@HPP possessed efficient catalase activity and successfully degraded hydrogen peroxide to produce oxygen. Increased oxygen levels relief intratumoral hypoxia, thereby enhancing CB@HPP-mediated. In addition, CB@HPP showed FL/PA/US multimodal imaging capabilities, which reflects the aggregation effect of CB@HPP in the tumor and suggest the timing of treatment. ConclusionThe biohybrid system CB@HPP significantly alleviates tumor hypoxia, and multimodal imaging-mediated oxygen-producing SDT effectively suppresses tumors. This integrated imaging and therapeutic biohybrid system provides a more efficient and attractive cancer treatment strategy for SDT. Statement of significanceThis study developed a sensitizing SDT strategy for imaging-guided drug-targeted delivery and in situ oxygen production. We designed a biohybrid system CB@HPP, which was hybridized by the engineered bacteria with catalytic oxygen production and nanosonosensitizer with multimodal imaging capability. CB@HPP significantly alleviates tumor hypoxia, and multimodal imaging-mediated oxygen-producing SDT effectively suppresses tumors. This integrated imaging and therapeutic biohybrid system provides a more efficient and attractive cancer treatment strategy for SDT.

Comparative Analysis of Cystamine and Cysteamine as Radioprotectors and Antioxidants: Insights from Monte Carlo Chemical Modeling under High Linear Energy Transfer Radiation and High Dose Rates.

This study conducts a comparative analysis of cystamine (RSSR), a disulfide, and cysteamine (RSH), its thiol monomer, to evaluate their efficacy as radioprotectors and antioxidants under high linear energy transfer (LET) and high-dose-rate irradiation conditions. It examines their interactions with reactive primary species produced during the radiolysis of the aqueous ferrous sulfate (Fricke) dosimeter, offering insights into the mechanisms of radioprotection and highlighting their potential to enhance the therapeutic index of radiation therapy, particularly in advanced techniques like FLASH radiotherapy. Using Monte Carlo multi-track chemical modeling to simulate the radiolytic oxidation of ferrous to ferric ions in Fricke-cystamine and Fricke-cysteamine solutions, this study assesses the radioprotective and antioxidant properties of these compounds across a variety of irradiation conditions. Concentrations were varied in both aerated (oxygen-rich) and deaerated (hypoxic) environments, simulating conditions akin to healthy tissue and tumors. Both cystamine and cysteamine demonstrate radioprotective and strong antioxidant properties. However, their effectiveness varies significantly depending on the concentration employed, the conditions of irradiation, and whether or not environmental oxygen is present. Specifically, excluding potential in vivo toxicity, cysteamine substantially reduces the adverse effects of ionizing radiation under aerated, low-LET conditions at concentrations above ~1 mM. However, its efficacy is minimal in hypoxic environments, irrespective of the concentration used. Conversely, cystamine consistently offers robust protective effects in both oxygen-rich and oxygen-poor conditions. The distinct protective capacities of cysteamine and cystamine underscore cysteamine's enhanced potential in radiotherapeutic settings aimed at safeguarding healthy tissues from radiation-induced damage while effectively targeting tumor tissues. This differential effectiveness emphasizes the need for personalized radioprotective strategies, tailored to the specific environmental conditions of the tissue involved. Implementing such approaches is crucial for optimizing therapeutic outcomes and minimizing collateral damage in cancer treatment.

The Chemo-Immunotherapeutic Roles of Tumor-Derived Extracellular Vesicle-Based Paclitaxel Delivery System in Hepatocarcinoma.

As a first-line chemotherapeutic agent, albumin-bound paclitaxel (PA) has a considerable effect on the treatment of various cancers. However, in chemotherapy for hepatocarcinoma, the sensitivity to PA is low owing to the innate resistance of hepatocarcinoma cells; the toxicity and side effects are severe, and the clinical treatment impact is poor. In this study, we present a unique nanodrug delivery system. The ultraviolet (UV)-induced tumor-cell-derived extracellular vesicles (EVs) were isolated and purified by differential centrifugation. Then, PA was loaded by coextrusion to create a vesicle drug delivery system (EVPA). By employing the EV-dependent enhanced retention effect and specific homing effect, EVPA would passively and actively target tumor tissues, activate the immune response to release PA, and achieve the combination therapeutic effect of chemo-immunotherapy on hepatocarcinoma. We demonstrated that the tumor-killing effect of EVPA is superior to that of PA, both in vivo and in vitro and that EVPA can be effectively taken up by hepatocarcinoma and dendritic cells, activate the body's specific immune response, promote the infiltration of CD4+ and CD8+ T cells in tumor tissues, and exert a precise killing effect on hepatocarcinoma cells via chemo-immunotherapy.

Light-Triggered PROTAC Nanoassemblies for Photodynamic IDO Proteolysis in Cancer Immunotherapy.

While proteolysis-targeting chimeras (PROTACs) hold great potential for persistently reprogramming the immunosuppressive tumor microenvironment via targeted protein degradation, precisely activating them in tumor tissues and preventing uncontrolled proteolysis at off-target sites remain challenging. Herein, a light-triggered PROTAC nanoassembly (LPN) for photodynamic indoleamine 2,3-dioxygenase (IDO) proteolysis is reported. The LPN is derived from the self-assembly of prodrug conjugates, which comprise a PROTAC, cathepsin B-specific cleavable peptide linker, and photosensitizer, without any additional carrier materials. In colon tumor models, intravenously injected LPNs initially silence the activity of PROTACs and accumulate significantly in targeted tumor tissues due to an enhanced permeability and retention effect. Subsequently, the cancer biomarker cathepsin B begins to trigger the release of active PROTACs from the LPNs through enzymatic cleavage of the linkers. Upon light irradiation, tumor cells undergo immunogenic cell death induced by photodynamic therapy to promote the activation of effector T cells, while the continuous IDO degradation of PROTAC simultaneously blocks tryptophan metabolite-regulated regulatory-T-cell-mediated immunosuppression. Such LPN-mediated combinatorial photodynamic IDO proteolysis effectively inhibits tumor growth, metastasis, and recurrence. Collectively, this study presents a promising nanomedicine, designed to synergize PROTACs with other immunotherapeutic modalities, for more effective and safer cancer immunotherapy.

The efficacy and safety of pH-responsive and photothermal-sensitive multifunctional nanoparticles loaded with cryptotanshinone for the treatment of gastric cancer.

A multifunctional polydopamine/mesoporous silica nanoparticles loaded cryptotanshinone (PDA/MSN@CTS) was synthesized and subjected to investigating its physicochemical properties and anti-gastric cancer (GC) effects. Utilizing network pharmacology and molecular docking techniques, CTS was identified as our final research target. The structural morphology and physicochemical properties of PDA/MSN@CTS were examined. Near-infrared (NIR) laser was employed to evaluate the photothermal properties of the PDA/MSN@CTS, along with pH-responsive and NIR-triggered release assessments. In vitro experiments evaluated the impact of PDA/MSN@CTS on the malignant behavior of AGS gastric cells. A subcutaneous tumor model was further established to evaluate the in vivo safety of PDA/MSN@CTS. Furthermore, the in vivo photothermal efficacy of PDA/MSN@CTS, in addition to its combined effect with photothermal therapy (PTT), was investigated. Uniform and stable PDA/MSN@CTS had been successfully synthesized and demonstrated efficient release under tumor environment and NIR irradiation. Upon increasing NIR laser conditions, in vivo cytotoxicity, apoptosis rate, reactive oxygen species scavenging ability, and suppression of migration and invasion of AGS cells by PDA/MSN@CTS were significantly enhanced. In vivo assessments revealed excellent blood compatibility and biosafety of PDA/MSN@CTS, alongside robust tumor tissue targeting. Combining nanoparticles with PTT facilitated the anti-GC effects of PDA/MSN@CTS. Compared to free drugs, PDA/MSN@CTS exhibits higher selectivity towards cancer cells, demonstrating effective anticancer activity and biocompatibility both in vitro and in vivo. Furthermore, our nanomaterial possesses excellent photothermal properties, and under NIR conditions, PDA/MSN@CTS exhibits synergistic therapeutic effects.

Current Non-Metal Nanoparticle-Based Therapeutic Approaches for Glioblastoma Treatment.

The increase in the variety of nano-based tools offers new possibilities to approach the therapy of poorly treatable tumors, which includes glioblastoma multiforme (GBM; a primary brain tumor). The available nanocomplexes exhibit great potential as vehicles for the targeted delivery of anti-GBM compounds, including chemotherapeutics, nucleic acids, and inhibitors. The main advantages of nanoparticles (NPs) include improved drug stability, increased penetration of the blood-brain barrier, and better precision of tumor targeting. Importantly, alongside their drug-delivery ability, NPs may also present theranostic properties, including applications for targeted imaging or photothermal therapy of malignant brain cells. The available NPs can be classified into two categories according to their core, which can be metal or non-metal based. Among non-metal NPs, the most studied in regard to GBM treatment are exosomes, liposomes, cubosomes, polymeric NPs, micelles, dendrimers, nanogels, carbon nanotubes, and silica- and selenium-based NPs. They are characterized by satisfactory stability and biocompatibility, limited toxicity, and high accumulation in the targeted tumor tissue. Moreover, they can be easily functionalized for the improved delivery of their cargo to GBM cells. Therefore, the non-metal NPs discussed here, offer a promising approach to improving the treatment outcomes of aggressive GBM tumors.

GSH-responsive bithiophene Aza-BODIPY@HMON nanoplatform for achieving triple-synergistic photoimmunotherapy

Photoimmunotherapy represents an innovative approach to enhancing the efficiency of immunotherapy in cancer treatment. This approach involves the fusion of immunotherapy and phototherapy (encompassing techniques like photodynamic therapy (PDT) and photothermal therapy (PTT)). Boron-dipyrromethene (BODIPY) has the potential to trigger immunotherapy owing to its excellent PD and PT efficiency. However, the improvements in water solubility, bioavailability, PD/PT combined efficiency, and tumor tissue targeting of BODIPY require introduction of suitable carriers for potential practical application. Herein, a disulfide bond-based hollow mesoporous organosilica (HMON) with excellent biocompatibility and GSH-responsive degradation properties was used as a carrier to load a bithiophene Aza-BODIPY dye (B5), constructing a sample chemotherapy reagent-free B5@HMON nanoplatform achieving triple-synergistic photoimmunotherapy. HMON, involving disulfide bond, is utilized to improve water solubility, tumor tissue targeting, and PD efficiency by depleting GSH and enhancing host-guest interaction between B5 and HMO. The study reveals that HMON’s large specific surface area and porous properties significantly enhance the light collection and oxygen adsorption capacity. The HMON’s rich mesoporous structure and internal cavity achieved a loading rate of B5 at 11 %. It was found that the triple-synergistic nanoplatform triggered a stronger anti-tumor immune response, including tumor invasion, cytokine production, calreticulin translocation, and dendritic cell maturation, eliciting specific tumor-specific immunological responses in vivo and in vitro. The BALB/c mouse model with 4T1 tumors was used to assess tumor suppression efficiency in vivo, showing that almost all tumors in the B5@HMON group disappeared after 14 days. Such a simple chemotherapy reagent-free B5@HMON nanoplatform achieved triple-synergistic photoimmunotherapy.

A Diketopyrrolopyrrole-Based All-in-One Nanoplatform for Self-Reinforcing Mild Photothermal Therapy Cascade Immunotherapy for Tumors.

Mild photothermal therapy (PTT) has attracted attention for effectively avoiding the severe side effects associated with high-temperature tumor ablation. However, its progress is hindered by the limited availability of high-performance photothermal agents (PTAs) and the thermoresistance of cancer cells induced by heat shock reactions. Herein, this work proposes a new strategy to expand the library of high-performance organic small-molecule PTAs and utilize it to construct a multifunctional nano-theranostic platform. By incorporating additional acceptors and appropriate π-bridges, a diketopyrrolopyrrole-based dye BDB is developed, which exhibits strong absorption and bright fluorescence emission in the near-infrared (NIR)region. Subsequently, BDB is co-coated with the heat shock protein (HSP) inhibitor tanespimycin (17-AAG) using the functional amphiphilic polymers DSPE-Hyd-PEG2000-cRGD to form an all-in-one nanoplatform BAG NPs. As a result, BAG NPs can precisely target tumor tissue, guide the treatment process in real-time through NIR-II fluorescence/photoacoustic/photothermal imaging, and release 17-AAG on demand to enhance mild PTT. Additionally, the mild PTThas been demonstrated to induce immunogenic cell death (ICD) and activate a systemic anti-tumor immune response, thereby suppressing both primary and distant tumors. Overall, this study presents a multifunctional nanoplatform designed for precise mild PTT combined with immunotherapy for effective tumor treatment.

Exploring the Theranostic Applications and Prospects of Nanobubbles.

Anticancer medications as well as additional therapeutic compounds, have poor clinical effectiveness due to their diverse distribution, non-selectivity for malignant cells, and undesirable off-target side effects. As a result, ultrasound-based targeted delivery of therapeutic compounds carried in sophisticated nanocarriers has grown in favor of cancer therapy and control. Nanobubbles are nanoscale bubbles that exhibit unique physiochemical properties in both their inner core and outer shell. Manufacturing nanobubbles primarily aims to enhance therapeutic agents' bioavailability, stability, and targeted delivery. The small size of nanobubbles allows for their extravasation from blood vessels into surrounding tissues and site-specific release through ultrasound targeting. Ultrasound technology is widely utilized for therapy due to its speed, safety, and cost-effectiveness, and micro/nanobubbles, as ultrasound contrast agents, have numerous potential applications in disease treatment. Thus, combining ultrasound applications with NBs has recently demonstrated increased localization of anticancer molecules in tumor tissues with triggered release behavior. Consequently, an effective therapeutic concentration of drugs/genes is achieved in target tumor tissues with ultimately increased therapeutic efficacy and minimal side effects on other non-cancerous tissues. This paper provides a brief overview of the production processes for nanobubbles, along with their key characteristics and potential therapeutic uses.

Progression in low-intensity ultrasound-induced tumor radiosensitization.

Radiotherapy (RT) is a widely utilized tumor treatment approach, while a significant obstacle in this treatment modality is the radioresistance exhibited by tumor cells. To enhance the effectiveness of RT, scientists have explored radiosensitization approaches, including the use of radiosensitizers and physical stimuli. Nevertheless, several approaches have exhibited disappointing results including adverse effects and limited efficacy. A safer and more effective method of radiosensitization involves low-intensity ultrasound (LIUS), which selectively targets tumor tissue and enhances the efficacy of radiation therapy. This review summarized the tumor radioresistance reasons and explored LIUS potential radiosensitization mechanisms. Moreover, it covered diverse LIUS application strategies in radiosensitization, including the use of LIUS alone, ultrasound-targeted intravascular microbubble destruction, ultrasound-mediated targeted radiosensitizers delivery, and sonodynamic therapy. Lastly, the review presented the limitations and prospects of employing LIUS-RT combined therapy in clinical settings, emphasizing the need to connect research findings with practical applications. LIUS employs cost-effective equipment to foster tumor radiosensitization, curtail radiation exposure, and elevate the quality of life for patients. This efficacy is attributed to LIUS's ability to utilize thermal, cavitation, and mechanical effects to overcome tumor cell resistance to RT. Multiple experimental analyses have underscored the effectiveness of LIUS in inducing tumor radiosensitization using diverse strategies. While initial studies have shown promising results, conducting more comprehensive clinical trials is crucial to confirm its safety and effectiveness in real-world situations.

Lysosome-Targeting Bacterial Outer Membrane Vesicles for Tumor Specific Degradation of PD-L1.

Increased expression of immune check point genes, such as PD-L1, is one of the main reasons for immunosuppression, especially for colon cancer. Development of novel therapeutic strategies is of great importance to improve the prognosis. In this study, outer membrane vesicles (OMV) derived from Gram-negative bacteria are engineered to immune checkpoint blockade nanosystem for efficient elicitation of anti-tumor immunity. Briefly, the OMVs are engineered with Lyp1-Traptavidin (S52G, R53D mutant of streptavidin) fusion protein displayed on the surface. The Lyp-1 endows the OMV with the capacity to target tumor tissues, while the Traptavidin ensures easy decoration of biotinylated anti-PD-L1 and biotinylated M6P (mannose 6-phosphate). The simultaneously anchored anti-PD-L1 and M6P (ligand for cation-independent mannose 6-phosphate receptor) on the engineered OMVs coordinately direct the membrane PD-L1 to lysosome for degradation, and thus unleash the anti-tumor immunity. With syngeneic tumor model, the engineered OMVs are confirmed to boost immunity, inhibit cancer growth, and thus prolong survival. Together, A proposed OMV-based modular nanosystem that enables assembly of biotinylated anti-PD-L1 and M6P on the surface for tumor-targeted immune checkpoint blockade.

TROCEPT: An oncolytic virus platform engineered for αvß6 integrin targeted tumor-localised expression of an immune checkpoint inhibitor following intravenous delivery.

e14579 Background: TROCEPT is a novel tumor-selective replicating, oncolytic adenovirus type 5 (Ad5) rationally engineered to remove all natural tissue tropisms. This engineering addresses the main limitation of other viral therapies which infect normal tissues and are rapidly removed by the liver and are therefore limited to local delivery. TROCEPT has been further engineered to specifically bind to αvβ6 integrin which is expressed at high frequency in the majority of epithelial cancers, so that following intravenous delivery the virus targets tumor tissue where it replicates and amplifies. In addition, the TROCEPT platform encodes transgenes which enables in-tumor production of powerful therapeutic drugs. The lead programme, TROCEPT-01, encodes a fully human, full length immune checkpoint inhibitor (ICI) antibody. TROCEPT-01 is currently undergoing IND-enabling studies and is expected to enter First in Human studies in 2024 in multiple solid tumor indications. Methods: Proof of concept studies, both in vitro and in vivo, to demonstrate TROCEPT-01’s tumor selectivity and toxicity profile have been performed. Results: In vitro experiments demonstrate that viral replication, transgene expression and oncolytic cell death following infection with TROCEPT-01 is highly selective for αvβ6 integrin positive tumor cells compared to a panel of normal human primary cells. Intravenous delivery in in vivo SKOV3 ovarian human tumor xenograft mouse models demonstrate virus delivery, replication, and transgene expression in the tumor. In comparison to the parental Ad5 virus, the engineering of TROCEPT-01 leads to more viral delivery to the tumour, less delivery to normal tissue including liver, reduced markers of liver toxicity and reduced inflammatory cytokine release, all of which validate the tumour selectivity and safety profile of the virus. Conclusions: We have demonstrated that TROCEPT engineering enables highly selective tumor targeting and significant tumor exposure following intravenous delivery. TROCEPT-01’s in-tumor selective expression of ICIs enables high local drug concentration in the tumor, potentially reducing systemic toxicity and increasing efficacy. TROCEPT-01 is currently undergoing IND-enabling studies and is expected to enter First in Human studies in 2024 in multiple solid tumor indications.

Tumor versus Tumor Cell Targeting in Metal-Based Nanoparticles for Cancer Theranostics.

The application of metal-based nanoparticles (mNPs) in cancer therapy and diagnostics (theranostics) has been a hot research topic since the early days of nanotechnology, becoming even more relevant in recent years. However, the clinical translation of this technology has been notably poor, with one of the main reasons being a lack of understanding of the disease and conceptual errors in the design of mNPs. Strikingly, throughout the reported studies to date on in vivo experiments, the concepts of "tumor targeting" and "tumor cell targeting" are often intertwined, particularly in the context of active targeting. These misconceptions may lead to design flaws, resulting in failed theranostic strategies. In the context of mNPs, tumor targeting can be described as the process by which mNPs reach the tumor mass (as a tissue), while tumor cell targeting refers to the specific interaction of mNPs with tumor cells once they have reached the tumor tissue. In this review, we conduct a critical analysis of key challenges that must be addressed for the successful targeting of either tumor tissue or cancer cells within the tumor tissue. Additionally, we explore essential features necessary for the smart design of theranostic mNPs, where 'smart design' refers to the process involving advanced consideration of the physicochemical features of the mNPs, targeting motifs, and physiological barriers that must be overcome for successful tumor targeting and/or tumor cell targeting.

Staphylococcus Aureus Membrane Vesicles Kill Tumor Cells Through a Caspase-1-Dependent Pyroptosis Pathway.

Nanosized outer membrane vesicles (OMVs) from Gram-negative bacteria have attracted increasing interest because of their antitumor activity. However, the antitumor effects of MVs isolated from Gram-positive bacteria have rarely been investigated. MVs of Staphylococcus aureus USA300 were prepared and their antitumor efficacy was evaluated using tumor-bearing mouse models. A gene knock-in assay was performed to generate luciferase Antares2-MVs for bioluminescent detection. Cell counting kit-8 and lactic dehydrogenase release assays were used to detect the toxicity of the MVs against tumor cells in vitro. Active caspase-1 and gasdermin D (GSDMD) levels were determined using Western blot, and the tumor inhibition ability of MVs was determined in B16F10 cells treated with a caspase-1 inhibitor. The vesicular particles of S. aureus USA300 MVs were 55.23 ± 8.17 nm in diameter, and 5 μg of MVs remarkably inhibited the growth of B16F10 melanoma in C57BL/6 mice and CT26 colon adenocarcinoma in BALB/c mice. The bioluminescent signals correlated well with the concentrations of the engineered Antares2-MVs (R2 = 0.999), and the sensitivity for bioluminescence imaging was 4 × 10-3 μg. Antares2-MVs can directly target tumor tissues in vivo, and 20 μg/mL Antares2-MVs considerably reduced the growth of B16F10 and CT26 tumor cells, but not non-carcinomatous bEnd.3 cells. MV treatment substantially increased the level of active caspase-1, which processes GSDMD to trigger pyroptosis in tumor cells. Blocking caspase-1 activation with VX-765 significantly protected tumor cells from MV killing in vitro and in vivo. S. aureus MVs can kill tumor cells by activating the pyroptosis pathway, and the induction of pyroptosis in tumor cells is a promising strategy for cancer treatment.

Engineering of an EPHA2-Targeted Monobody for the Detection of Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer worldwide, and is second only to lung cancer with respect to cancer-related deaths. Noninvasive molecular imaging using established markers is a new emerging method to diagnose CRC. The human ephrin receptor family type-A 2 (hEPHA2) oncoprotein is overexpressed at the early, but not late, stages of CRC. Previously, we reported development of an E1 monobody that is specific for hEPHA2-expressing cancer cells both in vitro and in vivo. Herein, we investigated the ability of the E1 monobody to detect hEPHA2 expressing colorectal tumors in a mouse model, as well as in CRC tissue. The expression of hEPHA2 on the surface of CRC cells was analyzed by western blotting and flow cytometry. The targeting efficacy of the E1 monobody for CRC cells was examined by flow cytometry, and immunofluorescence staining. E1 conjugated to the Renilla luciferase variant 8 (Rluc8) reporter protein was used for in vivo imaging in mice. Additionally, an enhanced green fluorescence protein (EGFP) conjugated E1 monobody was used to check the ability of the E1 monobody to target CRC tissue. The E1 monobody bound efficiently to hEPHA2-expressing CRC cell lines, and E1 conjugated to the Rluc8 reporter protein targeted tumor tissues in mice transplanted with HCT116 CRC tumor cells. Finally, E1-EGFP stained tumor tissues from human CRC patients, showing a pattern similar to that of an anti-hEPHA2 antibody. The E1 monobody has utility as an EPHA2 targeting agent for the detection of CRC.

Abstract 2718: A novel TROP2-targeted immune stimulating antibody conjugate (ISAC) with potent anti-tumoral activtiy and accpetable safety

Abstract Immune stimulating antibody conjugates (ISACs) are a unique class of ADC in which antibodies recognizing tumor antigens are conjugated with immune agonists. ISACs target tumor tissue and specifically activate intra-tumoral myeloid cells, unleashing downstream immune response. Like the other immune agonists, balance between efficacy and safety remains a challenge for ISACs. Here we describe a potential first-in-class TROP2 ISAC with rationally selected antibody and TLR7/8 agonist linker-payload. The anti-TROP2 antibody elicited robust ADCP of TROP2+ tumor cells by macrophages. In-vitro, TROP2 ISAC mildly activated myeloid cells only in the presence of TROP2+ tumor cells. In-vivo, the molecule potently suppressed tumor growth in different TROP2+ xenograft tumors, and effectively enhanced killing of an ADC. In terms of safety, the ISAC was tolerated in both WT and hTROP2KI mice, and, importantly, in monkeys. Taken together, our data demonstrate a novel TROP2 ISAC with outstanding efficacy and manageable safety profile, which may benefit patients with TROP2+ tumors. Citation Format: Ye Chen, Fei You, Chenchen Zhu, Yao Xiong, Li Li, Nana Luo, Shuaixiang Zhou, Fushuai Wang, Chenyang Xu, Fan Fei, Chang Li, Jianglu Wang, Jinling Xu, Huizhong Xiong. A novel TROP2-targeted immune stimulating antibody conjugate (ISAC) with potent anti-tumoral activtiy and accpetable safety [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 2718.

Abstract 5307: Preclinical evaluation of a potential FIC 4-1BB/CD24 bispecific antibody IBD0333

Abstract Background: This study demonstrates the preclinical evaluation of a novel 4-1BB/CD24 bispecific antibody IBD0333. 4-1BB’s activation triggers a signaling cascade that activates both innate and adaptive immune systems. Due to the hepatotoxicity of current anti-4-1BB monoclonal antibodies, tumor-associated antigens (TAA) targeting to specifically activate immune cells in the tumor microenvironment (TME) is expected to enhance its safety profile. CD24 is highly expressed in many cancers, such as ovarian and breast cancer and its high expression is often related to poor prognosis. The signaling pathway of CD24/Siglec-10 triggers a “don’t eat me” signal that facilitates immune escape of tumor cells. Therefore, IBD0333 was designed to target CD24 overexpressed tumor cells and activate CD8+ T cells to induce T cell mediated antitumor immunity in TME of the targeted tumor tissue. To further reduce the toxicity risk, the anti-4-1BB moiety is designed to activate the signaling pathway only when IBD0333 binds to the tumor cells that overexpress CD24. Methods: For CD24 terminus, we applied virus like particle (VLP) as antigen for immunization to obtain functional CD24 antibodies. For 4-1BB terminus, competitive ELISA/FACS analysis as well as amino acid point mutation binding ability analysis based on the receptor crystal structure were performed to characterize the binding epitope. The in vivo mice efficacy and toxicity study, as well as cynomolgus monkey toxicity study were also conducted to describe the anti-tumor activity and safety profile. For IBD0333, we verified binding affinity by FACS to cancer cells and the 4-1BB activity in reporter-gene assay. IL2 and INF-γ secretion assay were performed to evaluate the PBMC activation. C57BL/6J-h4-1BB humanized mice bearing MC38-hCD24 colon cancer cells were used to validate the anti-tumor efficacy of IBD0333. Preclinical PK and toxicity study was also performed in cynomolgus monkeys to describe the safety profile of IBD0333. Results: IBD0333 showed excellent binding and PBMC stimulation activities. In toxicology studies of IBD0333, no obvious abnormality in mice or cynomolgus monkeys administered with IBD0333 was observed. The MTD was estimated to be greater than 200 mg/kg with no severe side effects observed in the study, showing that IBD0333 has great potential to achieve significantly improved safety profile. As to efficacy, IBD0333 showed excellent tumor inhibition activities in mice model with 99% tumor growth inhibition at 1 mg/kg and 100% at 3 mg/kg. Conclusion: IBD0333 is a clinical stage, potential first-in-class, 4-1BB/CD24 bsAb that simultaneously stimulates both innate and adaptive immunity to achieve strong synergistic effects with reduced hepatotoxicity. The IND approvals from the FDA and the NMPA have been obtained and the Phase I clinical trial of IBD0333 in locally advanced/metastatic solid tumors or non-Hodgkin’s lymphoma is currently on-going. Citation Format: Xiaoling Jiang, Chongbing Wu, Zi Chen, Liusong Yin. Preclinical evaluation of a potential FIC 4-1BB/CD24 bispecific antibody IBD0333 [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 5307.

Cancer cell-specific and pro-apoptotic SMAC peptide-doxorubicin conjugated prodrug encapsulated aposomes for synergistic cancer immunotherapy

BackgroundImmunogenic cell death (ICD) is a crucial approach to turn immunosuppressive tumor microenvironment (ITM) into immune-responsive milieu and improve the response rate of immune checkpoint blockade (ICB) therapy. However, cancer cells show resistance to ICD-inducing chemotherapeutic drugs, and non-specific toxicity of those drugs against immune cells reduce the immunotherapy efficiency.MethodsHerein, we propose cancer cell-specific and pro-apoptotic liposomes (Aposomes) encapsulating second mitochondria-derived activator of caspases mimetic peptide (SMAC-P)-doxorubicin (DOX) conjugated prodrug to potentiate combinational ICB therapy with ICD. The SMAC-P (AVPIAQ) with cathepsin B-cleavable peptide (FRRG) was directly conjugated to DOX, and the resulting SMAC-P-FRRG-DOX prodrug was encapsulated into PEGylated liposomes.ResultsThe SMAC-P-FRRG-DOX encapsulated PEGylated liposomes (Aposomes) form a stable nanostructure with an average diameter of 109.1 ± 5.14 nm and promote the apoptotic cell death mainly in cathepsin B-overexpressed cancer cells. Therefore, Aposomes induce a potent ICD in targeted cancer cells in synergy of SMAC-P with DOX in cultured cells. In colon tumor models, Aposomes efficiently accumulate in targeted tumor tissues via enhanced permeability and retention (EPR) effect and release the encapsulated prodrug of SMAC-P-FRRG-DOX, which is subsequently cleaved to SMAC-P and DOX in cancer cells. Importantly, the synergistic activity of inhibitors of apoptosis proteins (IAPs)-inhibitory SMAC-P sensitizing the effects of DOX induces a potent ICD in the cancer cells to promote dendritic cell (DC) maturation and stimulate T cell proliferation and activation, turning ITM into immune-responsive milieu.ConclusionsEventually, the combination of Aposomes with anti-PD-L1 antibody results in a high rate of complete tumor regression (CR: 80%) and also prevent the tumor recurrence by immunological memory established during treatments.Graphical