Efficient Cell Killing Research Articles

Evaluation of Potency and Specificity of Cryptophycin-Loaded Antibody-Drug Conjugates.

An enhanced variant of the antimitotic toxin cryptophycin was conjugated to the anti-Her2 monoclonal antibody (mAb) Trastuzumab upon Michael addition. Either antibodies with freed hinge-region cysteines or THIOMAB formats with engineered cysteines in the mAbs light chain were added to a maleimide derivative of cryptophycin. These Antibody-Drug Conjugates (ADCs) showed retained binding to Her2 positive tumor cells and highly efficient cell killing in double-digit pM range on high Her2-expressing SK-BR-3 cells. Two ADCs (DAR6, DAR3) showed superior cell killing of the cell lines JIMT-1 and RT112 with medium receptor expression level in comparison with a DAR6 MMAE ADC serving as reference. The observed cell cytotoxicity is target-dependent since no impact on cell viability was observed for low Her2-expressing MDA-MB468 cells. Particularly the DAR3 ADC in THIOMAB format exhibiting desirable biophysical properties and high potency emerged as a promising candidate for further in vivo investigations.

Multifunctional hyaluronic acid ligand-assisted construction of CD44- and mitochondria-targeted self-assembled upconversion nanoparticles for enhanced photodynamic therapy.

Upconversion nanoparticles (UCNPs) have been used as a potential nanocarrier for photosensitizers (PSs), which have demonstrated a great deal of promise in achieving an effective photodynamic therapy (PDT) for deep-seated tumors. However, overcoming biological barriers to achieve mitochondria-targeted PDT remains a major challenge. Herein, CD44- and mitochondria-targeted photodynamic nanosystems were fabricated through the self-assembly of hyaluronic acid-conjugated-methoxy poly(ethylene glycol)-diethylenetriamine-grafted-(chlorin e6-dihydrolipoic acid-(3-carboxypropyl)triphenylphosphine bromide) polymeric ligands (HA-c-mPEG-Deta-g-(Ce6-DHLA-TPP)) and NaErF4:Tm@NaYF4 core-shell UCNPs (termed CMPNs). The CMPNs presented ideal physiological stability, a good drug loading capacity and an improved capacity for the generation of singlet oxygen (1O2) based on the FRET mechanism. Significantly, confocal images revealed that CMPNs not only facilitated cellular uptake through CD44-receptor-targeted endocytosis, subsequently enabling rapid evasion from endo-lysosomal sequestration, but also specifically targeted mitochondria, ultimately inducing a profound disruption of mitochondrial membrane potential, which triggered apoptosis upon laser irradiation, thereby significantly enhancing the therapeutic effect. Furthermore, in vitro antitumor experiments further confirmed the substantial enhancement in cancer cell killing efficiency achieved by treating with CMPNs upon near-infrared (NIR) laser irradiation. This innovative approach holds promise for the development of NIR-laser-activated photodynamic nanoagents specifically designed for mitochondria-targeted PDT, thus addressing the limitations of the current PDT treatments.

Efficacy of photodynamic therapy using 5-aminolevulinic acid-induced photosensitization is enhanced in pancreatic cancer cells with acquired drug resistance

The use of 5-aminolevulinic acid (ALA) as a precursor for protoporphyrin IX (PpIX) is an established photosensitization strategy for photodynamic therapy (PDT) and fluorescence guided surgery. Ongoing studies are focused on identifying approaches to enhance PpIX accumulation as well as to identify tumor sub-types associated with high PpIX accumulation. In this study, we investigated PpIX accumulation and PDT treatment response with respect to nodule size in 3D cultures of pancreatic cancer cells (Panc1) and a derivative subline (Panc1OR), which has acquired drug resistance and exhibits increased epithelial mesenchymal transition. In monolayer and 3D culture dose response studies the Panc1OR cells exhibit significantly higher cell killing at lower light doses than the drug naïve cells. Panc1OR also exhibits increased PpIX accumulation. Further analysis of cell killing efficiency per molecule of intracellular PpIX indicates that the drug resistant cells are intrinsically more responsive to PDT. Additional investigation using exogenous delivery of PpIX also shows higher cell killing in drug resistant cells, under conditions which achieve approximately the same intracellular PpIX. Overall these results are significant as they demonstrate that this example of drug-resistant cells associated with aggressive disease progression and poor clinical outcomes, show increased sensitivity to ALA-PDT.

Lead-212/Bismuth-212 InVivo Generator Based on Ultrasmall Silver Telluride Nanoparticles.

Radionuclide therapy employing alpha emitters holds great potential for personalized cancer treatment. However, certain challenges remain when designing alpha radiopharmaceuticals, including the lack of stability of used radioconjugates due to nuclear decay events. In this work, ultrasmall silver telluride nanoparticles with a core diameter of 2.1 nm were prepared and radiolabeled with lead-212 using a chelator-free method with a radiolabeling efficiency of 75%. The results from the invitro radiochemical stability assay indicated a very high retention of bismuth-212 despite the internal conversion effects originating from the decay of 212Pb. To further evaluate the potential of the nanoparticles, they were radiolabeled with indium-111, and their cell uptake and subcellular distribution were determined in 2D U87 cells, showing accumulation in the nucleus. Although not intentional, it was observed that the indium-111-radiolabeled nanoparticles induced efficient tumor cell killing, which was attributed to the Auger electrons emitted by indium-111. Combining the results obtained in this work with other favorable properties such as fast renal clearance and the possibility to attach targeting vectors on the surface of the nanoparticles, all well-known from the literature, these ultra-small silver telluride nanoparticles provide exciting opportunities for the design of theragnostic radiopharmaceuticals.

Smart nanozymes coupled with dynamic magnet field and laser exposures for cancer therapy

Developing nanozymes for cancer therapy has attracted great attention from researchers. However, enzymes-loaded magnetic particles triggered by both a low-frequency vibrating magnetic field (VMF) and laser for inhibiting tumor growth have never been reported. Herein, we developed a magnetic nanozyme with 3D flower-like nanostructures for cancer therapy. Specifically, the flower-like nanozymes exposed to a VMF could efficiently damage the mitochondrial membrane and cell structure, and inhibit tumor growth through magneto-mechanical force. In parallel, magnetic nanozymes in a weak acid environment containing glucose could generate abundant hydrogen peroxide through glucose oxidase-catalyzed oxidation of glucose, and further significantly promote the Fenton reaction. Interestingly, both glucose oxidase- and Fenton-based catalytic reactions were significantly promoted by the VMF exposure. Flower-like magnetic nanospheres upon a near-infrared laser irradiation could also damage cancer cells and tumor tissues through photothermal effect. The cell-killing efficiency of magnetic nanozymes triggered by the VMF or laser significantly increased in comparison with that of nanozymes without exposures. Mouse tumors grown after injection with magnetic nanozymes was inhibited in a significant way or the tumors disappeared after exposure to a VMF and laser due to the synergistic effect of four major stimuli, viz., magneto-mechanical force, photothermal conversion, improved Fenton reaction, and intratumoral glucose consumption-based starvation effect. This is a great platform that may be suitable for treating many solid tumors.

Remotely Controlled Surface Charge Modulation of Magnetoelectric Nanogenerators for Swift and Efficient Drug Delivery.

We have developed a highly efficient technique of magnetically controlled swift loading and release of doxorubicin (DOX) drug using a magnetoelectric nanogenerator (MENG). Core-shell nanostructured MENG with a magnetostrictive core and piezoelectric shell act as field-responsive nanocarriers and possess the capability of field-triggered drug release in a cancerous environment. MENGs generate a surface electric dipole when subjected to a magnetic field due to the strain-mediated magnetoelectric effect. The capability of directional magnetic field-assisted modulation of the surface electrical dipole of MENG provides a mechanism to create/break ionic bonds with DOX molecules, which facilitates efficient drug attachment and on-demand swift detachment of the drug at a targeted site. The magnetic field-assisted drug-loading mechanism was minutely analyzed using spectrophotometry and Raman spectroscopy. The detailed time-dependent analysis of controlled drug release by the MENG under unidirectional and rotating magnetic field excitation was conducted using field-emission scanning electron microscopy, energy-dispersive X-ray, and atomic force microscopic measurements. In vitro, experiments validate the cytocompatibility and magnetically assisted on-demand and swift DOX drug delivery by the MENG near MCF-7 breast cancer cells, which results in a significant enhancement of cancer cell killing efficiency. A state-of-the-art experiment was performed to visualize the nanoscale magnetoelectric effect of MENG using off-axis electron holography under Lorentz conditions.

Development of an arenavirus-based immunotherapy for treatment of KRAS mutant cancer.

e14672 Background: Oncogenic mutations in KRAS are prevalent in more than 80% of pancreatic ductal adenocarcinomas (PDAC), approximately 30% of colorectal cancer (CRC) and 15-20% of lung adenocarcinoma (LUAD). It has been demonstrated previously that CD8+ T cell responses specific for KRAS mutations can lead to tumor control and regression of metastasis. Replicating arenavirus vectors are currently evaluated in multiple clinical trials in infectious diseases and oncology and have demonstrated potent induction of target antigen specific CD8+ T cell responses of up to almost 50% of the total CD8+ T cell compartment in peripheral blood. To augment KRAS mutation specific CD8+ T cell responses in patients, we are using replicating arenavirus vectors targeting five prevalent KRAS mutations which are found in approximately 95% (PDAC), 74% (CRC), and 83% (LUAD) of the KRAS mutated tumors, representing a promising off-the-shelf immunotherapy for these indications. Here, we systematically investigate immune responses induced by mutant KRAS targeted arenavirus-vector-based cancer vaccines in HLA transgenic mice and characterize the efficiency of target cell killing of activated KRAS mutation specific CD8+ T cells. Methods: A transgene cassette consisting of peptide stretches including KRAS mutations G12D, G12V, G12C, G12R and G13D was generated by in silico aided antigen design. Vectors encoding peptide stretches of single KRAS mutations as well as the corresponding wildtype KRAS sequence served as controls in preclinical experiments. KRAS mutation specific CD8+ T cell expansion was evaluated in HLA transgenic mice and functionality of induced CD8+ T cells was evaluated by assessing CD8+ T cell mediated killing of mutant KRAS peptide loaded target cells in vivo. Results: All treatment regimens were well tolerated, and no mortalities or major adverse events were observed. Following vector administration, HLA I restricted antigen-specific CD8+ T cell responses were detected against 4 encoded mutant KRAS epitopes in HLA-A*11:01 (KRAS G12D and G12V), or HLA-B*07:02 (KRAS G12C and G12R) transgenic mice. These cells did not cross-react with wildtype KRAS epitopes presented in the same HLA I molecules. Specific cytotoxicity against KRAS G12D/V or KRAS G12C/R pulsed target cells was observed in vivo in HLA transgenic animals, indicating functionality of the induced KRAS mutation specific T cells. No specific cytotoxicity towards target cells pulsed with KRAS WT peptides was observed in any of the groups. Conclusions: This study indicates efficient induction of KRAS mutation specific T cell responses in HLA transgenic mice using an arenavirus vector based vaccine. Expanded CD8+ T cells were capable of killing cells loaded with KRAS mutation specific epitopes in vivo indicating functionality of the induced T cell responses. Based on these results, initiation of a Phase I study for the treatment of KRAS mutated cancers is planned.

Preparation of a Novel Multifunctional Cationic Liposome Drug-carrying System and its Functional Study on Lung Cancer.

Low-dose chemotherapy is a promising treatment strategy that may be improved by controlled delivery. This study aimed to design polyethylene glycol-stabilized bilayer-decorated magnetic Cationic Liposomes (CLs) as a drug delivery system for integrated functional studies of lung cancer cell therapy and imaging. A novel multifunctional folic acid targeting magnetic CLs docetaxel drug-loading system (FA-CLs-Fe- DOC) was prepared and tested for its physical properties, encapsulation rate and drug release performance. The feasibility of FA-CLs-Fe-DOC ability to inhibit tumor cells and act as an MRI contrast agent was investigated in vitro, and the target recognition and therapeutic ability of FA-CLs-Fe-DOC was studied in vivo. FA-CLs-Fe-DOC had a particle size of 221.54 ± 6.42 nm and a potential of 28.64 ± 3.56 mv, with superparamagnetic properties and better stability. The encapsulation rate was 95.36 ± 1.63%, and the drug loading capacity was 9.52 ± 0.22%, which possessed the drug slow-release performance and low cytotoxicity and could effectively inhibit the proliferation of lung cancer cells, promoting apoptosis of lung cancer cells. MRI showed that it had the function of tracking and localization of lung cancer cells. In vivo experiments confirmed the targeted recognition property and therapeutic function of lung cancer cells. In this study, we successfully prepared an FA-CLs-Fe-DOC capable of specifically targeting lung cancer cells with integrated functions of efficient lung cancer cell killing and imaging localization. This targeted drug packaging technology may provide a new strategy for the design of integrated carriers for targeted cancer therapy and imaging.

A Unique Approach: Biomimetic Graphdiyne-Based Nanoplatform to Treat Prostate Cancer by Combining Cuproptosis and Enhanced Chemodynamic Therapy.

Current treatment approaches for Prostate cancer (PCa) often come with debilitating side effects and limited therapeutic outcomes. There is urgent need for an alternative effective and safe treatment for PCa. We developed a nanoplatform to target prostate cancer cells based on graphdiyne (GDY) and a copper-based metal-organic framework (GDY-CuMOF), that carries the chemotherapy drug doxorubicin (DOX) for cancer treatment. Moreover, to provide GDY-CuMOF@DOX with homotypic targeting capability, we coated the PCa cell membrane (DU145 cell membrane, DCM) onto the surface of GDY-CuMOF@DOX, thus obtaining a biomimetic nanoplatform (DCM@GDY-CuMOF@DOX). The nanoplatform was characterized by using transmission electron microscope, atomic force microscope, X-ray diffraction, etc. Drug release behavior, antitumor effects in vivo and in vitro, and biosafety of the nanoplatform were evaluated. We found that GDY-CuMOF exhibited a remarkable capability to load DOX mainly through π-conjugation and pore adsorption, and it responsively released DOX and generated Cu+ in the presence of glutathione (GSH). In vivo experiments demonstrated that this nanoplatform exhibits remarkable cell-killing efficiency by generating lethal reactive oxygen species (ROS) and mediating cuproptosis. In addition, DCM@GDY-CuMOF@DOX effectively suppresses tumor growth in vivo without causing any apparent side effects. The constructed DCM@GDY-CuMOF@DOX nanoplatform integrates tumor targeting, drug-responsive release and combination with cuproptosis and chemodynamic therapy, offering insights for further biomedical research on efficient PCa treatment.

Near-infrared photoimmunotherapy targeting PD-L1: Improved efficacy by preconditioning the tumor microenvironment.

Near-infrared photoimmunotherapy (NIR-PIT) is a new type of cancer therapy that employs antibody-IRDye700DX conjugates (AbPCs) and near-infrared (NIR) light at a wavelength of 689 nm, the excitation wavelength of IR700. Administered intravenously, injected AbPCs bind specifically to cells expressing the target antigen, whereupon NIR light exposure causes rapid, selective killing. This process induces an anticancer T cell response, leading to sustained anticancer host immune response. Programmed cell death ligand-1 (PD-L1) is a major inhibitory immune checkpoint molecule expressed in various cancers. In this study, we first assessed the efficacy of PD-L1-targeted NIR-PIT (αPD-L1-PIT) in immune-competent tumor mouse models. αPD-L1-PIT showed a significant therapeutic effect on the tumor models with high PD-L1 expression. Furthermore, αPD-L1-PIT induced an abscopal effect on distant tumors and long-term immunological memory. In contrast, αPD-L1-PIT was not as effective for tumor models with low PD-L1 expression. To improve the efficacy of PD-L1-targeted NIR-PIT, PEGylated interferon-gamma (IFNγ) was administered with αPD-L1-PIT. The combination therapy improved the treatment efficacy by increasing PD-L1 expression leading to more efficient cell killing by αPD-L1-PIT. Furthermore, the PEGylated IFNγ led to a CD8+ T cell-dominant tumor microenvironment (TME) with an enhanced anticancer T cell response after αPD-L1-PIT. As a result, even so-called cold tumors exhibited complete responses after αPD-L1-PIT. Thus, combination therapy of PEGylated IFNγ and PD-L1-targeted NIR-PIT has the potential to be an important future strategy for cancer immunotherapy.

Potential of Ruthenium as a photosensitizer in radiation-activated Photodynamic Therapy (radioPDT)

In Photodynamic Therapy (PDT), light is used to activate photosensitizers (PS) to generate reactive oxygen species (ROS) as a cell-killing mechanism. However, PDT's application is limited to the surface and small size tumors because of the inefficiency of light in penetrating larger and deep-seated tumors. We have developed novel nanoparticles (NPs) that can be activated by radiation, a process known as the radiation-activated PDT (radioPDT). In our studies we developed a new variant of pegylated poly-lactic-co-glycolic (PEG-PLGA) encapsulated nanoscintillators (NSC) and ruthenium-based photosensitizer (Ru/rPDT) that exhibits minimal cell toxicity while having significant cell killing effect upon radiation activation. Our data suggest that radioPDT with Ru/rPDT can be a more effective strategy than previous protoporphyrin-based iterations in treating many deep-seated tumors.Introduction and Background: Toxicity and normal tissue damage are limiting factors for the application of radiotherapy (RT) where higher doses of radiation is required to cure deep-seated tumor such as prostate cancer. In Photodynamic Therapy (PDT), photosensitizers (PS) are activated by light to generate reactive oxygen species (ROS) to kill tumor cells. However, the main limitation of the current PDT is the limited tissue penetration of PS-activating light. Thus, PDT cannot be effectively used to treat deep-seated and larger size tumors. RT has higher tissue penetration depth, making the radiation-activated PDT (radioPDT) advantageous in treating deep seated tumors.We aim to develop novel radioPDT nanoparticles (NP) with more efficient anti-cancer therapeutic effect in vitro and in vivo to enhance targeted radiotherapy.Methods: LaF3:Ce3+ nanoscintillators (NSCs) were synthesized in a single step wet chemistry technique. The NSCs along with Ruthenium-based PS (Ru) with matching excitation/emission spectra were successfully encapsulated in PEG-PLGA. UV-Vis spectrometry and inductively coupled plasma mass spectrometry (ICP-MS) were used to confirm the presence of NSCs and Ru, and dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to determine the size of NP. Singlet oxygen generation was measured by Singlet Oxygen Sensor Green (SOSG) and direct singlet oxygen fluorescence. RadioPDT therapeutic potential was evaluated in PC3 prostate cancer cell under radiation by Alamar Blue cell viability assay.Results: UV-Vis spectrometry and ICP-MS determined the successful PEG-PLGA encapsulation of NSC and Ru. The TEM imaging confirmed the encapsulation NSC inside the nanocarrier PEG-PLGA. TEM and DLS measurements showed the size of radioNP ranged from 90nm to 150nm with polydispersity index of <0.3. Singlet oxygen generation by Ru/rPDT NPs was greater than PPIX based radioPDT (PPIX/rPDT) NPs. Furthermore, the direct measurement of singlet oxygen generation with fluorescence spectroscopy of Ru/rPDT showed preserved singlet oxygen yield and no evidence of quenching. The Ru/rPDT and PPIX/rPDT showed minimal PC3 cell cytotoxicity in dark conditions. The PC3 cell killing efficiency was comparable between Ru/rPDT and PPIX/rPDT either under light or radiation activation.Conclusion: Our in vitro studies show Ru/rPDT significantly increases anti-cancer therapeutic effect of RT, with minimal toxicity. Future studies will refine in vivo NP biodistribution, dosing, and RT dose/fractionation schemes.

Core-Shell Structure of Photopolymer-Grafted Polyurethane as a Controlled Drug Delivery Vehicle for Biomedical Application.

Functionalized ultraviolet photocurable bisphenol A-glycerolate dimethacrylates with tailorable size have been synthesized as the core, which have further been grafted using the diisocyanate chain end of polyurethane (PU) as the shell to create a core-shell structure of tunable size for a controlled drug delivery vehicle. The core-shell structure has been elucidated through spectroscopic techniques like 1H NMR, FTIR, and UV-vis and their relative shape and size through TEM and AFM morphology. The greater cross-link density of the core is reflected in the higher glass transition temperature, and the improved thermal stability of the graft copolymer is proven from its thermogravimetric analyses. The flow behavior and enhanced strength of the graft copolymers have been revealed from rheological measurements. The graft copolymer exhibits sustained release of the drug, as compared to pure polyurethane and photopolymer, arising from its core-shell structure and strong interaction between the copolymer and drug, as observed through a significant shifting of absorption peaks in FTIR and UV-vis measurements. Biocompatibility has been tested for the real application of the novel graft copolymer in medical fields, as revealed from MTT assay, cell imaging, and cell adhesion studies. The efficacy of controlled release from a graft copolymer has been verified from the gradual cell killing and ∼70% killing in 3 days vs meager cell killing of ∼25% very quickly in 1 day, followed by the increased cell viability of the system treated with the pure drug. The mechanism of slow and controlled drug release from the core-shell structure has been explored. The fluorescence images support the higher cell-killing efficiency as opposed to a pure drug or a drug embedded in polyurethane. Cells seeded on 3D scaffolds have been developed by embedding a graft copolymer, and fluorescence imaging confirms the successful growth of cells within the scaffold, realizing the potential of the core-shell graft copolymer in the biomedical arena.

Abstract 1689: CRISPR functional genetic screening for immune enhancers of inhibitor receptors in human primary CD8 T cells

Abstract The restrained efficacy of T cell therapies in cancer treatment is associated with T cell exhaustion (TEX), an impaired effector function state that is marked by distinct epigenetic landscape. These unique epigenetic features promoted the progression of TEX, hindered reinvigoration of TEX state and controlled the abnormal expression of inhibitor receptors (IRs), which could serve as target sites in cancer treatment. Thus, deciphering the regulatory elements in primary human T cells will deeply reveal the mechanism controlling TEX and provide novel therapeutic strategies to promote clinical response. However, how enhancers can be effectively applied in anti-cancer treatment is still poorly understood because high throughput identifying the functional enhancers of inhibitor receptors at scale is technically limited in human T cells. Here, we first developed a pooled screening platform that employs a compact CRISPRi system and enabled efficient one-step delivery of pooled screening libraries to primary human T cells. Leveraging this platform, we screened large genomic regions (~300kb) at saturation around key TEX genes, two IRs—PDCD1, HAVCR2 and one transcription factor (TF)—TBX21 to interrogate functional enhancers in human primary CD8 T cells. We identified two enhancers for PDCD1, three enhancers for HAVCR2 and three enhancers for TBX21, all of which are located over 12kb away from each gene’s transcription start sites. Then, to parse the essential sequences that fulfill enhancer function, we employed Cas9-based in-situ saturation mutagenesis screening to the identified enhancers of PDCD1 and HAVCR2. We found disrupt essential sequences impaired the entire enhancer function, quantitatively phenocopied the effect of targeting the entire enhancer with CRISPRi. Transcription factor motifs analyses of these sequences revealed potential regulation of ZFX for controlling PDCD1 expression and STAT for HAVCR2 expression. Finally, we explored the functional consequences of applying enhancers to anti-cancer treatment. Indeed, targeting the identified enhancers of PDCD1 and HAVCR2 in CAR T cells lead to enhanced cancer cell killing efficiency in vitro. Together, our study provides a platform and paradigm to interrogate functional enhancers in human T cells, illuminated the great potential of manipulating enhancers in T cell therapies. Citation Format: Ping Wang, Josephine R. Giles, Hua Huang, Junwei Shi, E.John Wherry. CRISPR functional genetic screening for immune enhancers of inhibitor receptors in human primary CD8 T cells [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 1689.

Bioreducible Amphiphilic Hyperbranched Polymer-Drug Conjugate for Intracellular Drug Delivery.

This paper reports synthesis of a bioreducible hyperbranched (HB) polymer by A2+B3 approach from commercially available dithiothreitol (DTT) (A2) and an easily accessible trifunctional monomer (B3) containing three reactive pyridyl-disulfide groups. Highly efficient thiol-activated disulfide exchange reaction leads to the formation of the HB polymer (Mw = 21000; Đ = 2.3) with bioreducible disulfide linkages in the backbone and two different functional groups, namely, hydroxyl and pyridyl-disulfide in the core and periphery, respectively, of the HB-polymer. Postpolymerization functionalization of the hydroxyl-groups with camptothecin (CPT), a topoisomerase inhibitor and known anticancer drug, followed by replacing the terminal pyridyl-disulfide groups with oligo-oxyethylene-thiol resulted in easy access to an amphiphilic HB polydisulfide-CPT conjugate (P1) with a very high drug loading content of ∼40%. P1 aggregated in water (above ∼10 μg/mL) producing drug-loaded nanoparticles (Dh ∼ 135 nm), which showed highly efficient glutathione (GSH)-triggered release of the active CPT. Mass spectrometry analysis of the GSH-treated P1 showed the presence of the active CPT drug as well as a cyclic monothiocarbonate product, which underpins the cascade-degradation mechanism involving GSH-triggered cleavage of the labile disulfide linkage, followed by intramolecular nucleophilic attack by the in situ generated thiol to the neighboring carbonate linkage, resulting in release of the active CPT drug. The P1 nanoparticle showed excellent cellular uptake as tested by confocal fluorescence microscopy in HeLa cells by predominantly endocytosis mechanism, resulting in highly efficient cell killing (IC50 ∼ 0.6 μg/mL) as evident from the results of the MTT assay, as well as the apoptosis assay. Comparative studies with an analogous linear polymer-CPT conjugate showed much superior intracellular drug delivery potency of the hyperbranched polymer.

Chalcogen Atom-Modulated Croconaine for Efficient NIR-II Photothermal Theranostics.

Organic dye-based agents with near-infrared (NIR)-II absorption have great potential for cancer theranostics because of the deeper tissue penetration and good biocompatibility. However, proper design is required to develop NIR-II-absorbing dyes with good optical properties. We proposed to construct chalcogen atom-modulated croconaine for NIR-II light-triggered photothermal theranostics. By introducing different chalcogen atoms (O, S, Se, or Te) into the structure of croconaine, the light absorption of croconaine can be precisely regulated from the NIR-I to the NIR-II range due to the heavy-atom effect. Especially, Te-substituted croconaine (CRTe) and its nanoformulations exhibit superior NIR-II responsiveness, a high photothermal conversion efficiency (70.6%), and good photostability. With their favorable tumor accumulation, CRTe-NPs from tumor regions can be visualized by NIR-II optoacoustic systems with high resolution and high contrast; meanwhile, their superior photothermal performance also contributes to efficient cell killing and tumor elimination upon 1064 nm laser irradiation. Therefore, this work provides an efficient strategy for the molecular design of NIR-II organic photothermal agents.

A 3D hanging spheroid-filter plate for high-throughput drug testing and CAR T cell cytotoxicity assay.

Tumour spheroids are widely used in immune cell cytotoxicity assays and anticancer drug testing, providing a physiologically relevant model replicating the tumour microenvironment. However, co-culture of immune and tumour cells complicates quantification of immune cell killing efficiency. We present a novel 3D hanging spheroid-filter plate that efficiently facilitates spheroid formation and separates unbound/dead cells during cytotoxicity assays. Optical imaging directly measures the cytotoxic effects of anti-cancer drugs on tumour spheroids, eliminating the need for live/dead fluorescent staining. This approach enables cost-effective evaluation of T-cell cytotoxicity with specific chimeric antigen receptors (CARs), enhancing immune cell-based assays and drug testing in three-dimensional tumour models.

Novel rod‐like carbon nanomaterials as near infrared‐responsive drug delivery system for potential anticancer applications

AbstractThe combination of chemotherapy and photothermal therapy shows great potential in cancer treatment, and the carbon nanomaterials have been attracted great attention in biomedical technology. However, it is still a great challenge to design stimuli‐responsive nano‐carbon‐based drug release systems with integrated functions. In this paper, rod‐like carbon nanomaterials (RCNs) were prepared by the soft template hydrothermal method with glucose as raw materials, which were proved to have high photothermal efficiency (∼23.7%), as well as good biocompatibility and excellent drug‐loading capacity. After that, RCNs were used to load doxorubicin (DOX) as DOX@RCNs for integrated photothermal/chemotherapy towards cancer, which demonstrated 52.2% higher cancer cell killing efficiency than that of RCNs under near infrared (NIR) irradiation in vitro. The authors’ approach provided a novel NIR‐responsive nano platform for combined photothermal/chemotherapy towards cancer, which was considered to be of great potential in anticancer applications.

Computational simulations of bispecific T cell engagers by a multiscale model

The use of bispecific antibodies as T cell engagers can bypass the normal T cell receptor-major histocompatibility class interaction, redirect the cytotoxic activity of T cells, and lead to highly efficient tumor cell killing. However, this immunotherapy also causes significant on-target off-tumor toxicologic effects, especially when it is used to treat solid tumors. To avoid these adverse events, it is necessary to understand the fundamental mechanisms involved in the physical process of T cell engagement. We developed a multiscale computational framework to reach this goal. The framework combines simulations on the intercellular and multicellular levels. On the intercellular level, we simulated the spatial-temporal dynamics of three-body interactions among bispecific antibodies, CD3 and tumor-associated antigens (TAAs). The derived number of intercellular bonds formed between CD3 and TAAs was further transferred to the multicellular simulations as the input parameter of adhesive density between cells. Through the simulations under various molecular and cellular conditions, we were able to gain new insights into how to adopt the most appropriate strategy to maximize the drug efficacy and avoid the off-target effect. For instance, we discovered that the low antibody-binding affinity resulted in the formation of large clusters at the cell-cell interface, which could be important to control the downstream signaling pathways. We also tested different molecular architectures of the bispecific antibody and suggested the existence of an optimal length in regulating the T cell engagement. Overall, the current multiscale simulations serve as a proof-of-concept study to help in the future design of new biological therapeutics.

Targeted Alpha Therapy: All We Need to Know about 225Ac's Physical Characteristics and Production as a Potential Theranostic Radionuclide.

The high energy of α emitters, and the strong linear energy transfer that goes along with it, lead to very efficient cell killing through DNA damage. Moreover, the degree of oxygenation and the cell cycle state have no impact on these effects. Therefore, α radioisotopes can offer a treatment choice to individuals who are not responding to β- or gamma-radiation therapy or chemotherapy drugs. Only a few α-particle emitters are suitable for targeted alpha therapy (TAT) and clinical applications. The majority of available clinical research involves 225Ac and its daughter nuclide 213Bi. Additionally, the 225Ac disintegration cascade generates γ decays that can be used in single-photon emission computed tomography (SPECT) imaging, expanding the potential theranostic applications in nuclear medicine. Despite the growing interest in applying 225Ac, the restricted global accessibility of this radioisotope makes it difficult to conduct extensive clinical trials for many radiopharmaceutical candidates. To boost the availability of 225Ac, along with its clinical and potential theranostic applications, this review attempts to highlight the fundamental physical properties of this α-particle-emitting isotope, as well as its existing and possible production methods.

Abstract LB_A01: Rapidly cleared MCL1 inhibitor TTX-810 showing robust efficacy in preclinical tumor models with no effects on cardiomyocytes in vitro and in vivo

Abstract MCL1, an anti-apoptotic protein frequently amplified in cancer, confers resistance to standard of care, thus highlighting its importance as a therapeutic target. However, the involvement of MCL1 in cardiomyocyte function raises concerns for safely targeting the protein in cancer patients. MCL1 inhibitors currently in clinical trials exhibit shortcomings, either due to insufficient potency or extended half-lives that heighten the risk of cardiotoxicity. This study systematically explores MCL1 inhibitor-induced cell dynamics in cancer cells and healthy cardiomyocytes. TTX-810, a potent, selective, and rapidly-cleared MCL1 inhibitor, was used to identify a treatment regimen that induces efficacy, but spares healthy cardiomyocytes. TTX-810 demonstrates potent and reversible binding to the BH3-binding groove of human MCL1 (KD = 0.3 nM). It inhibits the interaction of MCL1 with the pro-apoptotic effector protein BAK or BH3-only protein BIM in cells in a dose- and time-dependent manner, leading to activation of the intrinsic apoptotic cascade and cancer cell death. At suprapharmacologic concentrations and extended exposure durations TTX-810 does induce apoptosis in human iPSC-derived cardiomyocytes, aligning with the recognized mechanism of MCL1 inhibitor-related cardiotoxicity. In contrast, short-term TTX-810 exposure induces efficient cancer cell killing in vitro, and has no impact on cell viability, apoptosis induction, or troponin I release in human iPSC-derived cardiomyocytes, even at high concentration. Similarly, TTX-810 induces strong monotherapy efficacy with once weekly in vivo dosing in an aggressive AML cancer model that is resistant to current standard of care agents like Venetoclax or Azacytidine. Repeated once weekly dosing in dogs over 4-weeks had no impact on troponin I levels in blood, electrocardiogram parameters (PR, QRS, QT and calculated QTcV intervals), heart rate, nor cardiac tissue morphology. In conclusion, this study identifies an optimal TTX-810 treatment regimen that effectively targets cancer cells, while preserving healthy cardiomyocytes. The study also demonstrates that a minimum exposure time is sufficient for efficacy, and this effect is even more pronounced when combined with standard-of-care agents. Moreover, limiting the exposure time expands the TTX-810 therapeutic window with regard to cardiotoxicity, which was validated in GLP toxicology studies in dogs and positions TTX-810 for future clinical development. Citation Format: Ulrike Rauh, Virendar Kaushik, Sabine Pilari, Georgios Kosmidis, Michael Serrano-Wu, Guo Wei, Weiqun Zhang, Stefan Kaulfuss, Kai Thede, Guillaume Poncet-Montange, Douglas Daniels, Christopher T Lemke, Brian Hubbard, Todd R. Golub. Rapidly cleared MCL1 inhibitor TTX-810 showing robust efficacy in preclinical tumor models with no effects on cardiomyocytes in vitro and in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A01.