Cell Killing Research Articles

A Novel B7-H4xCD3 Bispecific T Cell Engager (PF-07260437) Synergizes with Breast Cancer Standard of Care and Immune-Checkpoint Therapies.

Immune checkpoint inhibitors have shown limited success in breast cancer, the most common and deadly cancer in women worldwide. Novel immune therapies like CD3 engaging bispecific antibodies have shown clinical promise in hematologic malignancies. However, developing CD3 bispecifics for solid tumors has been challenging due to difficulty in identifying tumor-specific antigens. B7-H4 is proposed as an attractive tumor-associated antigen for breast cancer therapeutics with comprehensive coverage regardless of breast cancer molecular subtype. We designed a B7-H4 targeting CD3 bispecific molecule, PF-07260437, and demonstrated B7-H4-dependent pharmacology in vitro by directing cytotoxic T cell killing to breast cancer cell lines. Treatment of cell line-derived xenograft and patient-derived xenograft in vivo models of human breast cancer with PF-07260437 induced substantial tumoricidal activity, often resulting in complete responses. Mechanistically, PF-07260437 increased T cell number and activation, leading to efficient tumor killing. Additionally, combining PF-07260437 with standard of care (palbociclib plus fulvestrant) and a checkpoint inhibitor (anti-PD-1) showed combinatorial benefits in an immune competent in vivo model. Clinically relevant non-invasive PET/CT imaging with a CD8-targeting tracer demonstrated PF-07260437-mediated increases in intratumoral CD8 T cells highlighting the utility of CD8-PET technology to potentially assess biomarker changes in the clinic. Finally, the manageable toxicity profile of PF-07260437 was highlighted in an exploratory toxicology study in cynomolgus monkeys. These data support the clinical testing of PF-07260437 for treating B7-H4 expressing solid tumors, including breast cancer.

Inhibition of ENT1 relieves intracellular adenosine-mediated T cell suppression in cancer.

The benefit of immune checkpoint blockade for cancer therapy is limited to subsets of patients because of factors including the accumulation of immunosuppressive metabolites, such as adenosine, within tumors. Pharmacological inhibition of adenosine generation and signaling is an active area of clinical investigation, but only limited clinical benefit has been reported. Here, we show that adenosine suppresses anti-cancer T cell responses following uptake into activated T cells by equilibrative nucleoside transporter 1 (ENT1) and inhibition of de novo pyrimidine nucleotide synthesis. We identify EOS301984 as a potent ENT1 antagonist that restores pyrimidine levels in activated T cells in adenosine-rich environments, resulting in enhanced tumor cell killing by memory T cells and increased ex vivo expansion of functional human tumor-infiltrating lymphocytes. A combination of EOS301984 with anti-PD-1 led to synergistic control of tumor growth in a humanized mouse model of triple-negative breast cancer. ENT1 inhibition, therefore, augments anti-cancer immune responses through the restoration of pyrimidine nucleotide synthesis in T cells suppressed by adenosine.

Comparison of Tumor Cell Responses to Different Radiotherapy Techniques: Three-Dimensional Conformal Radiotherapy (3D-CRT), Volumetric Modulated Arc Therapy (VMAT), and Helical Tomotherapy (HT)

Currently, advanced RT techniques such as VMAT and HT are being developed to optimize tumor coverage while minimizing radiation exposure to the surrounding organs that are at risk. Despite their growing clinical use, comparative studies evaluating the dosimetric and radiobiological effects of these modalities remain limited. In this study, A549, HeLa, and HepG2 cells were exposed to a single 2 Gy dose, using three RT techniques (3D-CRT, dual arc VMAT, and HT). Treatment plans were generated using a water phantom to ensure consistent target coverage and comparable dosimetric parameters across the techniques. Multiple radiobiological endpoints were assessed to evaluate the cellular responses. Although all three techniques yielded similar dosimetric parameters without statistically significant differences, the biological responses varied among the cell lines. Notably, VMAT and HT demonstrated superior tumor cell suppression compared to 3D-CRT. This was likely due to their enhanced dose conformity and modulation precision, which potentially led to improved tumor cell killing. These findings highlight the importance of integrating radiobiological assessments with physical dose metrics to inform the clinical application of advanced RT technologies. However, this study had several limitations. The use of a single radiation dose limited its clinical relevance, and the immediate post-irradiation assessments may not have captured delayed biological responses. Additionally, the small number of replicates may have reduced the study’s statistical power. Future studies incorporating dose fractionation schemes, time course analyses, and larger sample sizes are warranted to better simulate clinical conditions and further elucidate the radiobiological effects of advanced RT techniques.

Effects of albumin-bound paclitaxel combined with Sophora subprostrate polysaccharide on inflammatory factors and immune function in breast cancer rats

BackgroundTumor occurrence and growth are highly correlated with the degree of inflammation and immunological activity. Reducing the level of inflammation in tumor-bearing body to relieve immune suppression and enhance anti-tumor immune function has become an important strategy for tumor treatment.ObjectiveTo investigate the effect of albumin-bound paclitaxel combined with Sophora subprostrate polysaccharide (SSP) on inhibiting inflammation, reducing immunosuppression, enhancing anti-tumor immune function and slowing the progression of tumor in tumor-bearing rats, and to provide certain scientific basis for the clinical application of combined drugs in tumor.MethodsThe rats were put into three groups at random: normal control, model group, and drug treatment group. After the end of drug intervention, the tumor was taken out and weighed to observe the tumor growth of the rats. Tumor necrosis factor (TNF-α), interleukin (IL) 1β, IL-10, perforin, and granzyme B were found by Western blot in the local tumor tissues of experimental rats. The protein expression levels of Arginase-1 (Arg-1) and Cyclooxygenase 2 (COX-2) were determined. HE staining was used to observe the inflammatory infiltration of the tumor. Using flow cytometry, the proportions of anti-tumor immune cells—CD8 + T cells, NK cells, and immunosuppressive cells—in local tumor tissues were evaluated. In addition, spleen T cells isolated from normal rats were co-cultured with spleen myeloid derived suppressor cells (MDSC) from tumor-bearing rats in the model group and the combined treatment group. Cell Trace Far Red was used to identify T cell proliferation, flow cytometry was used to determine the level of T cell activation from CD25 expression, and in vivo immunosuppression in tumor-bearing rats was examined.ResultsThe combined therapy group experienced a considerable decrease in tumor weight as compared to the model group. TNF-α and IL-1p levels in the vicinity of the tumor tissues reduced following intervention, although IL-10 levels, which are anti-inflammatory cytokines, did not significantly change. The results of the HE staining revealed that the intervention group's tumor had less inflammatory infiltration than the model group did. After intervention, the percentages of CD8 + T cells and NK cells in local tumor tissues increased. Additionally, the intervention group's levels of protein expression for perforin and granzyme B were considerably higher than those of the model group. In the nearby tumor tissues, there were lots of MDSC. Following the intervention, the proportion of MDSC in the local tumor tissues was significantly reduced, and the expansion of MDSC was reduced. Additionally, the intervention group's COX-2 and Arg-1 protein expression levels in the tumor-specific tissues were significantly lower than those of the model group. The outcomes of in vitro co-culture demonstrated that rats in the combination group had higher levels of T cell proliferation and activation than animals in the model group.ConclusionsAlbumin-bound paclitaxel combined with Sophora subprostrate polysaccharide can reduce the local inflammation level, promote the proportion of CDB + T cells and NK cells and cell killing function, reduce the proportion of MDSC and immunosuppressive level, enhance the anti-tumor immune function of tumor-bearing mice, and slow the growth of tumors.

Small Molecule-mediated Photothermal Therapy Induces Apoptosis in Cancer Cells.

Cancer remains as one of the most life-threatening diseases in the whole world. Most of the therapeutic strategies to eradicate cancer are highly invasive leading to severe injury and trauma to the patients. In recent times, phototherapy emerged as one of the non-invasive therapeutic strategies for cancer treatment. However, development of novel small molecule photothermal agents remained a major challenge. To address this, herein, we have designed and synthesized a small molecule library having aromatic substituted-3-methoxy-pyrrole and 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-ylidene) malononitrile in a concise synthetic strategy. One of the library members (7H), self-assembled into spherical-like nanoparticles having <100 nm size in water and was found to exhibit remarkable increase in temperature under 740 nm NIR light. Interestingly, compound 7H homed into the lysosomal compartments and the lipid droplets (LDs) in the HCT-116 colon cancer cells within 3h and induced photothermal effect (PTT) followed by generation of reactive oxygen species (ROS) while irradiating under 740 nm NIR light for 10 min. Moreover, 7H triggered programmed cell death (apoptosis) to induce remarkable HCT-116 cell killing. This small molecule-mediated photothermal effect showed potential to be an interesting tool for the next-generation non-invasive cancer phototherapy.

Highly sensitive live-cell imaging-based cytotoxicity assay enables functional validation of rare epitope-specific CTLs

Many immunotherapeutic approaches aim to induce epitope-specific T-cell cytotoxicity. However, the identification—and especially the functional validation—of suitable epitopes by in vitro cytotoxicity assays can be challenging, particularly when the number of available epitope-specific cytotoxic T cells (CTLs) is limited. Here, we present a highly sensitive image-based cytotoxicity assay that allows the functional analysis of rare epitope-specific T cells. The live-cell imaging-based setup combines transient red labeling of target cells with a green caspase 3/7 probe, allowing reliable measurement of the fraction of apoptotic target cells. Time-course analysis enables the monitoring of subtle differences. This highly flexible assay can be applied to assess the killing of either target cells with endogenous epitope presentation or those artificially loaded with the epitope of interest. Analysis of assay sensitivity demonstrated that cytotoxicity mediated by as few as 0.1% epitope-specific CTLs in a T-cell culture can still be detected. The epitope-specificity of the assay was additionally validated by specific upregulation of PD-1 and LAG-3 on epitope-specific T cells, as well as the epitope-specific induction of interferon-γ release. Finally, the assay was successfully applied to functionally validate human papillomavirus (HPV)16 epitopes, by detecting epitope-specific killing of established patient-derived tumor cell lines by rare T-cell populations expanded from peripheral blood. Overall, this cytotoxicity assay setup provides a straightforward approach to assess the cytotoxic capacity of rare epitope-specific T cells and enables the analysis of T-cell responses against endogenously presented epitopes.

Beneficial infections of the enterovirus genus in patients with liver cancer.

Hepatocellular carcinoma (HCC) is a significant global cancer burden, with rising incidence and lacking a unified prevention strategy due to complex aetiologies. Viral exposures may shape host immunity via specific reactive viral antigens that could induce immune responses against hepatocarcinogenesis. We aimed to characterise viral exposure differences between HCC patients and healthy individuals and identify potentially protective viral antigens against HCC. We profiled pan-viral serological antibody repertoires using a microbial phage library among 2647 study subjects and examined the biological activities of selective viral antigens on blood-derived immune cells from both healthy individuals and HCC patients. We identified 153 viral antigens with a significantly reduced serological response in HCC patients compared with healthy individuals. We also observed that a higher serological response to 153 viral antigens is associated with better clinical outcomes of patients with chronic liver diseases and HCC. These findings are consistent across different populations across sex, ethnicity and aetiology. We identified a common epitope (CE1) shared among 39% of reactive viral antigens that belong to the rhinovirus and enterovirus families. We demonstrated that CE1 could induce both CD4+ and CD8+ T-cell activation and CD8+ T-cell-mediated HCC cell killing. Our results suggest that past exposures to members of the Enterovirus genus may be advantageous for cancer patients, highlighting the potential for a viral peptide-based HCC vaccine.

Comparison of antibody-scTRAIL Fc fusion proteins with varying valency for EGFR and TRAIL receptors

Fusion proteins combining TNF-related apoptosis inducing ligand (TRAIL) and antibody building blocks have emerged as a strategy for the targeted treatment of cancer cells. Using a single-chain derivative of homotrimeric TRAIL (scTRAIL), several targeted and non-targeted scTRAIL fusion proteins of varying geometries and valencies for TRAIL receptors and target antigens, all comprising an Fc region, were generated. These fusion proteins comprised either 1 or 2 scTRAIL units, i.e. are tri- or hexavalent for TRAIL receptors and in the targeted versions, 1 or 2 binding sites for EGFR. These fusion proteins were analyzed for cell binding and cell death induction using the EGFR-expressing colorectal cancer cell lines Colo205 and HCT116. In line with previous findings, all fusion proteins that were hexavalent for TRAIL receptors exhibited a strongly increased cell killing activity compared to the trivalent ones. Interestingly, the fusion proteins comprising one scTRAIL unit, did not benefit from targeting to EGFR. In contrast, the hexavalent scTRAIL fusion proteins further benefited from EGFR targeting, resulting in an approximately 6- to 30-fold increase in cell killing. In summary, this study shed further light on the influence of geometry and valency of TRAIL fusion proteins and confirmed IgG-scTRAIL fusion proteins as highly potent cell death inducers.

Macrocyclic Peptide Probes for Immunomodulatory Protein CD59: Potent Modulators of Bacterial Toxin Activity and Antibody‐Dependent Cytotoxicity

AbstractCD59 is an immunomodulatory cell surface receptor associated with human disease. Despite its importance in complement regulation and bacterial pathogenesis, CD59 remains a challenging therapeutic target. Research to date has focused on antibody or protein‐based strategies. Here we present a new approach to target CD59 using macrocyclic peptides with low nanomolar affinity for CD59. Through X‐ray crystallographic studies and structure‐activity relationship (SAR) studies, we identify key interactions that are essential for binding and activity. We find that the macrocyclic peptide CP‐06 adopts a beta‐hairpin structure and binds CD59 through an intermolecular beta‐sheet, mimicking protein–protein interactions of biologically relevant CD59 interaction partners. We create dimeric and lipidated macrocyclic peptide conjugates as enhanced cell‐active CD59 inhibitors and show that these probes can be used to modulate both complement‐mediated killing of human cells and lytic activity of bacterial virulence factors. Together, our data provide a starting point for future development of macrocyclic peptides to target CD59 activity in diverse cellular contexts.

Spatiotemporal Blockade of Autophagy and Macropinocytosis in Pancreatic Cancer via Lysosomal‐Activated Nanoreactors

AbstractAutophagy blockade has emerged as a promising strategy to enhance the antitumor efficacy of pancreatic ductal adenocarcinoma (PDAC). However, autophagy inhibition in PDAC may induce compensatory macropinocytosis to maintain cellular homeostasis by upregulating nuclear factor erythroid 2‐related factor 2 (NRF2). In this study, lysosomal‐activated nanoreactors loaded with the NRF2 inhibitor ML385 (CPZI‐Ms) are developed to spatiotemporally block autophagy and inhibit macropinocytosis. The nanoreactors, which preferentially accumulate in lysosomes, undergo degradation in the acidic lysosomal environment, triggering a Fenton‐like reaction between the released Cu2⁺ ions and H₂O₂ to generate hydroxyl radicals. This results in lysosomal membrane permeabilization, which further leads to not only lysosomal autophagy blockade, but also the leakage of Cu2⁺ and ML385 into the cytoplasm. Cu2⁺ induces extensive oxidative stress throughout the whole cell, while ML385 suppresses compensatory macropinocytosis response activated by autophagy blocking. As a result, the Panc02 cells become a “besieged fortress”, with impaired autophagic clearance and restricted nutrient uptake. In vivo, CPZI‐Ms effectively suppress tumor growth in Panc02‐bearing mice, offering a promising nanoplatform for enhancing cancer cell killing. Taken together, the present work provides an innovative anti‐tumor insight by spatiotemporally blocking autophagic flux and inhibiting compensatory macropinocytosis.

Early Development of an Innovative Nanoparticle-Based Multimodal Tool for Targeted Drug Delivery: A Step-by-Step Approach.

Prostate cancer is the most common tumor in men in developed countries and it often responds poorly to conventional treatments. Monoclonal antibody (MoAb) therapy, for this pathology, has grown tremendously in the past decades, exploiting naked and conjugated antibodies to cytotoxic payloads to form antibody drug conjugates (ADCs). Several studies have been carried out conjugating biomolecules against prostate-specific membrane antigen (PSMA), highly expressed in this tumor, to cytotoxic drugs. Nano-based formulations show high potential in targeted drug delivery to enhance the bioavailability of drugs. Our research aimed to evaluate the feasibility of setting up a nanoparticle-based multimodal tool for targeted drug delivery, describing the step-by-step approach and to perform a first screening of these fluorescent PEGylated silica nanoparticles employed in selective cancer cell targeting and killing. These nanoparticles featured a core-shell structure to contemporarily conjugate the antibody and the cytotoxic payload monomethyl auristatin E (MMAE) using a step-by-step approach. We compared the cytotoxic effect of this multimodal nanotool near the antibody-MMAE and free MMAE. We found a lower cytotoxicity effect of the nanoparticle-based construct compared to free drugs, likely because of the preservation of the previously observed receptor-mediated endocytosis. Nanomedicine is confirmed as a powerful alternative to organic drug delivery systems, even if some aspects, such as drug loading efficacy, release, scalable manufacturing and long-term stability, need to be deepened.

The Application of Glycolipid-Type Microbial Biosurfactants as Active Pharmaceutical Ingredients for the Treatment and Prevention of Cancer

Pharmaceutical scientists have researched the potential of secondary metabolites biosynthesized by microorganisms as active pharmaceutical ingredients (APIs) for the treatment of cancer. Ideally, these APIs should possess anticancer bioactivity that specifically targets tumor cells while having little cytotoxic effect on healthy tissue. Biosurfactants are microbial secondary metabolites with surface-active properties and individual bioactivities that have the potential to either destroy cancer cells in a targeted fashion or prevent tumor cell formation. Currently, the best-studied class of microbial biosurfactants for the purpose of anticancer bioactivity is glycolipids, which contain a hydrophilic sugar moiety bonded to a hydrophobic fatty acid. Anticancer investigations are mainly carried out using in vitro models that show that compounds belonging to each of the four sub-classes of microbial glycolipid have significant anticancer bioactivity. The targeted action of this activity appears to be highly dependent on a specific congener molecular structure with nuanced alterations in structure leading to the killing of both tumor and healthy cells. This review compiles the current literature relating to glycolipid anticancer activity and provides a critical appraisal of exploiting the bioactivity of these compounds as novel anticancer agents. Finally, we propose several suggestions on how this research could be improved moving forward via method standardization.

CD73+CD8+ T cells define a subset with anti-tumor potential in DLBCL patients

IntroductionCD73, a recently discovered immune checkpoint, catalyzes the conversion of AMP to adenosine, thereby suppressing anti-tumor immune responses.CD8+ T cells play a critical role in the immune response against cancer, yet their functionality can be modulated by various factors within the tumor microenvironment. In this study, we focus on identifying and characterizing CD73+CD8+ T cells in the peripheral blood of patients with diffuse large B-cell lymphoma (DLBCL), aiming to elucidate their functional and phenotypic roles in tumor immunity.MethodsUsing flow cytometry, we analyzed the expression of inhibitory receptors (e.g., PD-1, TIM-3) and activating markers (e.g., CD25, CD69) on CD73+CD8+ T cells compared to CD73−CD8+ T cells. In vitro functional assays were conducted to assess their cytotoxic activity against tumor cells, including cytokine production and tumor cell killing capacity.ResultsCD73+CD8+ T cells exhibited a distinct immunophenotypic profile, characterized by reduced expression of inhibitory receptors and enhanced cytotoxic activity compared to their CD73− counterparts. These cells demonstrated higher levels of effector molecules (e.g., IFN-γ, TNF-α) and lower exhausted markers. The findings suggest that CD73+CD8+ T cells may retain stronger anti-tumor potential.DiscussionThis study highlights CD73+CD8+ T cells as a unique functional subset with potential therapeutic relevance in DLBCL. Their reduced exhaustion and heightened cytotoxicity position them as promising targets for immunotherapy strategies. However, the dual role of CD73 in adenosine-mediated immunosuppression warrants further investigation to reconcile its pro-tumorigenic effects with the observed anti-tumor activity of CD73+CD8+ T cells. Our findings deepen the understanding of CD8+ T cell heterogeneity in DLBCL and emphasize the need for mechanistic studies to explore CD73’s context-dependent functions.

Cell cycle-dependent radiosensitivity of CHO DNA repair mutants exposed to accelerated charged particles.

Cell cycle-dependent radiosensitivity of CHO DNA repair mutants exposed to accelerated charged particles.

Folate receptor-targeted and pH-tunable dextran modified geraniol-protein nano-scaffolds stir up oxidative assault and apoptotic killing of HCT-116 colorectal cancer cells.

Folate receptor-targeted and pH-tunable dextran modified geraniol-protein nano-scaffolds stir up oxidative assault and apoptotic killing of HCT-116 colorectal cancer cells.

Insulin Analogs and the Mode of Insulin Delivery: Recent Advances and Challenges.

Diabetes is a medical condition associated with impaired glucose regulation caused either due to insufficient insulin production or resistance to insulin (Type 2 diabetes, gestational diabetes) or the absence of insulin through the selective killing of beta cells in the pancreas (Type 1 diabetes). Irregular insulin production leads to various health complications. To prevent such complications, patients must adhere to medical recommendations before availing of any advanced insulin therapy(ies), considered productive for the treatment. Natural insulin, although highly effective in controlling blood glucose levels, patients are often at risk of developing hypoglycemia and many other complications. This has led to the development of insulin analogs, the modified variants of natural insulin having a minimal risk of causing hypoglycemia. Besides the development of analogs, the mode of insulin delivery is also considered critical in achieving better glycemic control in diabetic patients. Until recently, various exogenous insulin delivery methods were practiced, but effective glycemic control without any associated risk and ease of delivery remains a subject of paramount concern. It countered attenuation or delayed onset of diabetes-associated complications without a permanent cure, raising an unmet demand for insulin formulations and delivery methods that offer stability, biocompatibility, reproducibility, precision dosing, non-immunogenicity, and safety. The current practice utilizes non-physiological delivery methods with less invasive administration routes, offering glycemic stability and therapeutic effectiveness. This review focuses on the recent advances made and future perspectives envisioned about newer insulin therapies and delivery methods that tend to improve the management of diabetes by inculcating ideas to reduce the disease's severity and improve the quality of life.

Resistance to PD-1/PD-L1 immune checkpoint blockade in advanced non-small cell lung cancer.

Resistance to PD-1/PD-L1 immune checkpoint blockade in advanced non-small cell lung cancer.

Structural characterization of the exopolysaccharide produced by Bacillus amyloliquefaciens JM033 and evaluation of its ability to regulate immunity and intestinal flora.

Structural characterization of the exopolysaccharide produced by Bacillus amyloliquefaciens JM033 and evaluation of its ability to regulate immunity and intestinal flora.

Mechanoregulation of lymphocyte cytotoxicity.

Cytotoxic lymphocytes counter intracellular pathogens and cancer by recognizing and destroying infected or transformed target cells. The basis for their function is the cytolytic immune synapse, a structurally stereotyped cell-cell interface through which lymphocytes deliver toxic proteins to target cells. The immune synapse is a highly dynamic contact capable of exerting nanonewton-scale forces against the target cell. In recent years, it has become clear that the interplay between these forces and the biophysical properties of the target influences the entirety of the cytotoxic response, from the initial activation of cytotoxic lymphocytes to the release of dying target cells. As a result, cellular cytotoxicity has become an exemplar of the ways in which biomechanics can regulate immune cell activation and effector function. This Review covers recent progress in this area, which has prompted a reconsideration of target cell killing from a more mechanobiological perspective.

Catumaxomab: First Approval.

Catumaxomab (Korjuny®) is a first-in-class bispecific trifunctional rat-mouse hybrid monoclonal antibody currently under development with Lindis Biotech for malignant ascites, and bladder, gastric and ovarian cancers. It binds epithelial cell adhesion molecule (EpCAM) on tumour cells and CD3 on T cells, while its Fc domain engages Fcγ receptor-positive accessory cells, bringing immune and tumour cells into close proximity to enhance tumour cell killing through multiple immunological mechanisms. Initially approved in the EU on 20 April 2009 for malignant ascites in adults with EpCAM+ carcinomas when standard therapy was unavailable or no longer feasible, catumaxomab was marketed by Fresenius Biotech GmbH (later Neovii Biotech GmbH) before being withdrawn on 2 June 2017 for commercial reasons. Lindis Biotech later acquired the rights and pursued reapproval. On 11 February 2025, catumaxomab was approved in the EU for the intraperitoneal treatment of malignant ascites in adults with EpCAM+ carcinomas who are not eligible for further systemic anticancer therapy. This article summarizes the milestones in the development of catumaxomab leading to this new approval.