Nonspecific Mechanisms Research Articles

Review of Lipid Nanoparticles for Breast Cancer Management: Targeting Strategies and Clinical Perspective

Abstract: Chemotherapy, a conventional breast cancer treatment, has limitations due to its nonspecific mechanisms and undesirable side effects. Lipid-based nanoparticles (LNPs) have emerged as a versatile and superior platform for targeted breast cancer treatment, offering several distinct advantages over traditional therapies. This class of nanocarriers, which includes solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), and liposomes, enhances the bioavailability and stability of therapeutic agents while minimizing systemic toxicity. LNPs can be engineered to precisely target breast cancer cells by exploiting tumor-specific markers, enabling selective delivery of chemotherapeutic agents and reducing off-target effects. SLNs provide controlled drug release and improved drug retention within the tumor, while NLCs offer higher drug-loading capacities and better stability. Liposomes, with their flexible design, allow for the encapsulation of both hydrophilic and hydrophobic drugs and can be modified for active targeting through surface functionalization. These lipid-based carriers also address the challenges of multi-drug resistance (MDR) by enhancing intracellular drug concentration and overcoming efflux mechanisms. In this review, we focus on lipid-based nanoparticles- a subtype of nanoparticles, and their potential advantages as drug delivery systems for breast cancer treatment. Results revealed that lipidbased nanoparticles have shown potential in breast cancer management, these include improved drug efficacy, enhanced ability to overcome cancer therapy resistance, and effective drug carrier for co-loaded drugs.

DsAMP and dsAMPGAN: Deep Learning Networks for Antimicrobial Peptides Recognition and Generation.

Antibiotic resistance is a growing public health challenge. Antimicrobial peptides (AMPs) effectively target microorganisms through non-specific mechanisms, limiting their ability to develop resistance. Therefore, the prediction and design of new AMPs is crucial. Recently, deep learning has spurred interest in computational approaches to peptide drug discovery. This study presents a novel deep learning framework for AMP classification, function prediction, and generation. We developed discoverAMP (dsAMP), a robust AMP predictor using CNN Attention BiLSTM and transfer learning, which outperforms existing classifiers. In addition, dsAMPGAN, a Generative Adversarial Network (GAN)-based model, generates new AMP candidates. Our results demonstrate the superior performance of dsAMP in terms of sensitivity, specificity, Matthew correlation coefficient, accuracy, precision, F1 score, and area under the ROC curve, achieving >95% classification accuracy with transfer learning on a small dataset. Furthermore, dsAMPGAN successfully synthesizes AMPs similar to natural ones, as confirmed by comparisons of physical and chemical properties. This model serves as a reliable tool for the identification of novel AMPs in clinical settings and supports the development of AMPs to effectively combat antibiotic resistance.

Effects of Arnebia benthamii Extract on Growth Performance, Immunological Parameters and Disease Resistance against Flexibacter columnare in Spotted Snakehead Channa punctata (Bloch)

Herbal immunostimulants are known to improve disease resistance in fish by enhancing specific and non-specific defensive mechanisms. The present study was conducted to evaluate the effects of Arnebia benthamii extract (AE) on growth rate, immune status, and disease resistance towards Flexibacter columnare in spotted snakehead Channa punctata. Each experimental group of healthy C. punctata was fed on one of the four experimental diets viz. basal diet (without any AE supplementation), AE10 (supplemented with 10 mg AE/kg basal diet), AE20 (supplemented with 20 mg AE/kg basal diet) and AE40 (supplemented with 40 mg AE/kg basal diet) for 42 days. When the concentration of Arnebia benthamii was raised from 0 to 40 mg/kg, the weight gain (WG) and specific growth rate (SGR) were considerably enhanced. The lysozyme activity, C3, C4, and serum globulin levels were also significantly increased in all Arnebia benthamii supplemented groups. IgM concentration in fish fed on AE40 diet was significantly higher than in fish fed on basal diet. All A. benthamii treatment groups exhibited significantly higher survival rates after being challenged with F. columnare. These findings showed that supplementing diet of C. punctata with A. benthamii improved growth, immunological response, and disease resistance against F. columnare, with the greatest benefits in fish fed on AE40 diet for 42 days.

Response of Native and Non-Native Subarctic Plant Species to Continuous Illumination by Natural and Artificial Light.

This study addressed the following questions: How does continuous lighting (CL) impact plant physiology, and photosynthetic and stress responses? Does the impact of CL depend on the source of the light and other environmental factors (natural vs. artificial)? Do responses to CL differ for native and non-native plant species in the subarctic region and, if differences exist, what physiological reasons might they be associated with them? Experiments were conducted with three plants native to the subarctic region (Geranium sylvaticum L., Geum rivale L., Potentilla erecta (L.) Raeusch.) and three non-native plant species (Geranium himalayense Klotzsch, Geum coccineum Sibth. and Sm., Potentilla atrosanguinea Loddiges ex D. Don) introduced in the Polar-Alpine Botanic Garden (KPABG, 67°38' N). The experimental groups included three species pairs exposed to (1) a natural 16 h photoperiod, (2) natural CL, (3) an artificial 16 h photoperiod and (4) artificial CL. In the natural environment, measurements of physiological and biochemical parameters were carried out at the peak of the polar day (at the end of June), when the plants were illuminated continuously, and in the second week of August, when the day length was about 16 h. Th experiments with artificial lighting were conducted in climate chambers where plants were exposed to 16 h or 24 h photoperiods for two weeks. Other parameters (light intensity, spectrum composition, temperature and air humidity) were held constant. The obtained results have shown that plants lack specific mechanisms of tolerance to CL. The protective responses are non-specific and induced by developing photo-oxidative stress. In climate chambers, under constant environmental conditions artificial CL causes leaf injuries due to oxidative stress, the main cause of which is circadian asynchrony. In nature, plants are not photodamaged during the polar day, as endogenous rhythms are maintained due to daily fluctuations of several environmental factors (light intensity, spectral distribution, temperature and air humidity). The obtained data show that among possible non-specific protective mechanisms, plants use flavonoids to neutralize the excess ROS generated under CL. In local subarctic plants, their photoprotective role is significantly higher than in non-native introduced plant species.

THE EFFECTIVENESS OF THE IMMUNE-BOOSTING PREPARATIONS IN PREVENTION AND TREATMENT OF RESPIRATORY INFECTIONS

Respiratory infections are undoubtedly one of the most common diseases that both primary care physicians and specialists working in hospitals have to deal with. The percentage of these diseases is particularly high in children and, depending on the child's age, may range from 6% to even 25%. A major clinical problem is the recurrent nature of these infections and their complications. This results in receiving an increased number of medications that can cause numerous side effects. Overprescription of antibiotics and consequently, increase of antibiotic resistance are also observed. Looking forward, the prevention of the recurrent infections is the key problem. The aim of our work was to evaluate the efficacy of the most popular preparations available on the pharmaceutical market that can improve immunity and complement conventional methods of treating infections or completely replace them. These include: elderberry, β-Glucans, lactoferrin, colostrum, black cumin preparations, echinacea, propolis, Streptococcus salivarius K12, inosine pranobex, ginseng, OM-85 bacterial lysate. We reviewed the literature from 2012 to 2024 using Pubmed and Google Scholar. The assesment of the effectiveness of preparations for immunity was based primarily on the systematic reviews, meta-analyses and randomised controlled trials. The above-mentioned preparations are largely characterized by a positive impact on improving the immunity of people supplementing them by activating non-specific immunity mechanisms. Thanks to this, some of the respondents can observe the regulation of the body's inflammatory response and, consequently, a decrease in the number of infections and their course. Due to the still insufficient number of studies on these preparations and the discrepancies in the results obtained among authors, further researches are needed to take a clear stance on their effectiveness.

Dried Chlorella powder supplementation: Impact on broiler chicken growth, health, and intestinal microflora

The use of dried Chlorella as an immune and growth stimulant to enhance nonspecific host defense mechanisms or as an antimicrobial to inhibit bacterial growth has been reported. This study aimed to assess the effects of dried Chlorella powder (DCP) supplementation on the growth, health, and intestinal microflora of commercial broiler chicks, comparing a diet containing DCP with an antibiotic-based diet. A total of 120 pieces day-old Cobb 500 broiler chicks were reared at Sher-e-Bangla Agricultural University Poultry Farm, Dhaka, and randomly divided into four experimental groups of three replicates each, with 10 chicks per replication. One group was fed a control diet, while the remaining three groups were fed diets with 0.5% and 1.0% DCP, and antibiotics, respectively. Results indicated significant (P<0.05) improvements in body weight and dressing percentage with DCP inclusion compared to control-fed broilers. A linear increase in body weight was observed with higher DCP levels, with birds on the 1% DCP diet achieving superior body weights (1665.13±8.82) compared to the control and antibiotic groups. Feed Conversion Ratio (FCR) and feed consumption were also significantly (P<0.05) improved, with the best FCR at 1% DCP (1.37±0.01) and the highest FCR in the control group (1.45±0.00). The highest feed consumption was noted in the control group. No significant (P>0.05) differences were observed in the relative weight of spleen and bursa among the groups. DCP had no significant (P>0.05) effects on liver, gizzard, intestine, and heart weights. Hematological studies revealed no significant (P>0.05) differences, except for Hemoglobin and Red Blood Cells (RBC), which were significantly (P<0.05) increased by DCP compared to control and antibiotic groups. DCP supplementation significantly (P<0.05) reduced E. coli and Salmonella sp. counts while increasing Lactobacillus sp. counts. Additionally, treatments with DCP significantly (P<0.05) boosted Newcastle disease (ND) titre levels compared to the control group. The study showed that DCP can be effectively replaced antibiotics in broiler diets, enhancing growth, health, and immune response, thereby promoting sustainable and safer poultry production practices.

Carbon dots-based dual-mode sensor for highly selective detection of nitrite in food substrates through diazo coupling reaction

As an antioxidant and preservative agent, nitrite (NO2−) plays an essential role in the food industry to maintain freshness or inhibit microbial growth. However, excessive addition of NO2− is detrimental to health, so accurate and portable detection of NO2− is critical for food quality control. Notably, the selectivity of most carbon dots (CDs)-based fluorescence sensors was not enough due to the nonspecific interaction mechanism of hydrogen bond, electrostatic interaction and inner filter effect etc. Herein, a novel fluorescence/UV–vis absorption (FL/UV–vis) dual-mode sensor was developed on basis of mC-CDs, which were prepared by simple solvothermal treatment of m-Phenylenediamine (m-PDA) and cyanidin cation (CC). The fluorescence of these mC-CDs could be selectively responded by NO2− through the specific diazo coupling reaction between NO2− and amino groups on the surface of mC-CDs, thus effectively improving the selectivity of NO2− detection. The CDs-based fluorescence sensor possessed a low detection limit of 0.091 μM and 0.143 μM for FL and UV–vis methods and the excellent linear range of 0.0–60.0 μM. Furthermore, the mC-CDs sensor was employed to detect NO2− in real samples with a recovery rate of 97.11 %–104.15 % for quantitative addition. Moreover, the smartphone-assisted fluorescence sensing platform developed could identify the subtle color changes that could not be distinguished by the naked eye, and had the advantages of fast detection speed and intelligence. More importantly, the portable solid phase sensor based on mC-CDs had been successfully applied to the specific fluorescence identification and concentration monitoring of NO2−. Accordingly, the designed sensor provided a new strategy for the highly selective and convenient sensing of NO2− in food substrates, and paved the way for the wide application of CDs-based nanomaterials in the detection of food safety.

Research Advances on Natural Killer Cells in Infections

Natural killer (NK) cells play a crucial role in the human immune system, swiftly recognizing and eliminating infected cells through non-specific immune mechanisms. This paper reviews the mechanisms by which NK cells act in various infections, including those caused by viruses, bacteria, parasites, and fungi, and explores their biological characteristics and functional regulation in the context of infection. By thoroughly analyzing the regulatory mechanisms of cytokines, receptor-ligand interactions, and the microenvironment on NK cell function, this paper further elucidates the response mechanisms of NK cells under different infection scenarios. Finally, it discusses the clinical application prospects of NK cells in treating infectious diseases and outlines future research directions, providing new ideas and possibilities for NK cell-based immunotherapy strategies.

Lineage Mapping of Extracellular Vesicles: What Cells Do They Come from And Where Do They Go?

Significance: Release of extracellular vesicles (EVs) by various cell types has been shown to mediate the delivery of biologically active payloads from donor cells to recipient cells; however, it remains unclear what cell types these EVs come from. With a focus on fluorescent reporters to monitor the release of EVs, especially those under the control of cell type-specific promoters, we address the translational relevance of genetic tools in cultured cells, normal tissues, and in models of development, injury, cancer, and wound healing. Recent Advances: It is well established that EVs are released by many cell types in the body via fusion and release processes at the plasma membrane. Since there remains debate about what fraction of EVs are released through regulated endosomal trafficking pathways versus nonspecific mechanisms, the development and validation of novel molecular tools are important to address the cellular source of EVs. Critical Issues: There is a need to develop and characterize new cell type-specific reporter mouse models that build upon the examples detailed here to identify the cellular source of EVs with genetic approaches being useful in addressing these critical limitations. Future Directions: Advances in reporter systems will drive a better understanding of EV subsets to identify compartment-specific EV localization to guide the development of more translationally relevant models for the wound healing field.

Gentiopicroside and swertiamarin induce non-selective oxidative stress-mediated cytotoxic effects in human peripheral blood mononuclear cells

Gentiopicroside (Gp) and swertiamarin (Sm), secoiridoid glycosides commonly found in plants of the Gentianaceae family, differ in one functional group. They exhibit promising cytotoxic effects in cancer cell lines and overall protective outcomes, marking them as promising molecules for developing novel pharmaceuticals. To investigate potential variations in cellular sensitivity to compounds of similar molecular structures, we analyzed the mode of Gp and Sm induced cell death in human peripheral blood mononuclear cells (PBMCs) after 48 h of treatment. The lowest tested concentration that significantly reduces cell viability, 50 μM, was applied. Oxidative stress parameters were estimated by measuring the levels of prooxidative/antioxidative balance, lipid peroxidation products, and 8-oxo-7,8-dihydro-2-deoxyguanosine, while gene expression of DNA repair enzymes was evaluated by employing quantitative real-time PCR. Cellular morphology was analyzed by fluorescent microscopy, and immunoblot analysis of apoptosis and necroptosis-related proteins was used to assess the type of cell death induced by the treatments. The discriminatory impact of Gp/Sm treatments on apoptosis and necroptosis-induced cell death was evaluated by monitoring the cell survival in co-treatment with specific cell death inhibitors. Obtained results show greater cytotoxicity of Gp than Sm suggesting that variations in the molecular structures of the tested compounds can substantially affect their biological effects. Gp/Sm co-treatment with apoptosis and necroptosis inhibitors revealed a distinct, albeit non-specific mechanism of PBMCs cell death. Although the therapeutic may not directly cause a specific type of cell death, its extent can be pivotal in assessing the safety of therapeutic application and developing phytopharmaceuticals with improved features. Since phytopharmaceuticals affect all exposed cells, identification of cytotoxic mechanisms on PBMCs after Gp and Sm treatment is important for addressing the formulation and dosage of potential phytopharmaceuticals.

Innate and germline immune memory: specificity and heritability of the ancient immune mechanisms for adaptation and survival.

The immune memory is one of the defensive strategies developed by both unicellular and multicellular organisms for ensuring their integrity and functionality. While the immune memory of the vertebrate adaptive immune system (based on somatic recombination) is antigen-specific, encompassing the generation of memory T and B cells that only recognize/react to a specific antigen epitope, the capacity of vertebrate innate cells to remember past events is a mostly non-specific mechanism of adaptation. This "innate memory" can be considered as germline-encoded because its effector tools (such as innate receptors) do not need somatic recombination for being active. Also, in several organisms the memory-related information is integrated in the genome of germline cells and can be transmitted to the progeny for several generations, but it can also be erased depending on the environmental conditions. Overall, depending on the organism, its environment and its living habits, innate immune memory appears to be a mechanism for achieving better protection and survival against repeated exposure to microbes/stressful agents present in the same environment or occurring in the same anatomical district, able to adapt to changes in the environmental cues. The anatomical and functional complexity of the organism and its lifespan drive the generation of different immune memory mechanisms, for optimal adaptation to changes in the living/environmental conditions. The concept of innate immunity being non-specific needs to be revisited, as a wealth of evidence suggests a significant degree of specificity both in the primary immune reaction and in the ensuing memory-like responses. This is clearly evident in invertebrate metazoans, in which distinct scenarios can be observed, with both non-specific (immune enhancement) or specific (immune priming) memory-like responses. In the case of mammals, there is evidence that some degree of specificity can be attained in different situations, for instance as organ-specific protection rather than microorganism-specific reaction. Thus, depending on the challenges and conditions, innate memory can be non-specific or specific, can be integrated in the germline and transmitted to the progeny or be short-lived, thereby representing an exceptionally plastic mechanism of defensive adaptation for ensuring individual and species survival.

Assessment of formed elements, vitamins B12, folic acid and iron in the blood of men with mental work

Changes in the components of the hemogram respond to the influence of living and working conditions. The goal is to assess blood parameters, vitamins B12 and B9 in men of mental work. A random sample of blood was collected from men permanently residing in Krasnoyarsk (n = 58). We assessed the degree of anthropogenic load and working conditions according to the degree of harmfulness and danger. A clinical blood test was performed, and the state of the body’s nonspecific adaptive reactions was assessed using lymphocytes. Vitamins B12, B9, and iron were determined. In terms of intellectual, emotional stress and work schedule, the work is harmful (3.3). Signs of anemia were revealed (decrease in erythrocytes in 12.1 %, hemoglobin in 10.3 %, hematocrit in 10.3 %), macrocytic (average erythrocyte volume increased in 10.3 %), hypochromic (reduced average hemoglobin concentration in erythrocyte in 22.3 %), developing due to deficiency of vitamin B12 (in 67.2 %) and B9 (in 32.8 %), iron (below the median in 51.7 %). The proportion of rod-nuclear neutrophils was reduced, the proportion of eosinophils (in 13.8 %) and monocytes (in 25.6 %) was increased, indicating sensitization, allergization, a decrease in nonspecific defense mechanisms and the presence of an inflammatory reaction in the body. The presence of stress in the body was evidenced by indicators of nonspecific adaptive reactions of the body (reactivation in 27.6 %), an increase in the proportion of monocytes in 25.6 % of those examined. It can be assumed that an environmentally unfavorable living environment and hard work in conditions of inadequate provision of the body with cyanocobalamin, folic acid and iron negatively affect the composition of the blood, which, in turn, can affect health.

Nanobody-liposomes as novel cancer vaccine platform to efficiently stimulate T cell immunity

Cancer vaccines can be utilized in combination with checkpoint inhibitors to optimally stimulate the anti-tumor immune response. Uptake of vaccine antigen by antigen presenting cells (APCs) is a prerequisite for T cell priming, but often relies on non-specific mechanisms. Here, we have developed a novel vaccination strategy consisting of cancer antigen-containing liposomes conjugated with CD169- or DC-SIGN-specific nanobodies (single domain antibodies) to achieve specific uptake by APCs. Our studies demonstrate efficient nanobody liposome uptake by human and murine CD169+ and DC-SIGN+ APCs in vitro and in vivo when compared to control liposomes or liposomes with natural ligands for CD169 and DC-SIGN. Uptake of CD169 nanobody liposomes resulted in increased T cell activation by human APCs and stimulated naive T cell priming in mouse models. In conclusion, while nanobody liposomes have previously been utilized to direct drugs to tumors, here we show that nanobody liposomes can be applied as vaccination strategy that can be extended to other receptors on APCs in order to elicit a potent immune response against tumor antigens.

Arbutus unedo L. Fractions Exhibit Chemotherapeutic Properties for the Treatment of Gastrointestinal Stromal Tumors.

Novel treatments in gastrointestinal stromal tumors (GISTs) are essential due to imatinib resistance and the modest results obtained with multi-target tyrosine kinase inhibitors. We investigated the possibility that the hydroalcoholic extract from the leaves of Arbutus unedo L. (AUN) could harbor novel chemotherapeutics. The bio-guided fractionation of AUN led to a subfraction, FR2-A, that affected the viability of both imatinib-sensitive and -resistant GIST cells. Cells treated with FR2-A were positive for Annexin V staining, a marker of apoptosis. A rapid PARP-1 downregulation was observed, although without the traditional caspase-dependent cleavage. The fractionation of FR2-A produced nine further active subfractions (FRs), indicating that different molecules contributed to the effect promoted by FR2-A. NMR analysis revealed that pyrogallol-bearing compounds, such as gallic acid, gallic acid hexoside, gallocatechin, myricetin hexoside, and trigalloyl-glucose, are the main components of active FRs. Notably, FRs similarly impaired the viability of GIST cells and peripheral blood mononuclear cells (PBMCs), suggesting a non-specific mechanism of action. Nevertheless, despite the lack of specificity, the established FRs showed promising chemotherapeutic properties to broadly affect the viability of GIST cells, including those that are imatinib-resistant, encouraging further studies to investigate whether pyrogallol-bearing compounds could represent an alternative avenue in GISTs.

Adsorption of DNA and Aptamers to Sodium Urate Crystals and Inhibition of Crystal Growth.

An elevated level of blood uric acid (UA) can cause the formation of kidney stones, gout, and other diseases. We recently isolated a few DNA aptamers that can selectively bind to UA. In this work, we investigated the adsorption of a UA aptamer and random sequence DNA onto sodium urate crystals. Both DNA strands adsorbed similarly to urate crystals. In addition, both the UA aptamer and random DNA can inhibit the growth of urate crystals, suggesting a nonspecific adsorption mechanism rather than specific aptamer binding. In the presence of 500 nM DNA, the growth of needle-like sodium urate crystals was inhibited, and the crystals appeared granular after 6 h. To understand the mechanism of DNA adsorption, a few chemicals were added to desorb DNA. DNA bases contributed more to the adsorption than the phosphate backbone. Surfactants induced significant DNA desorption. Finally, DNA could also be adsorbed onto real UA kidney stones. This study provides essential insights into the interactions between DNA oligonucleotides and urate crystals, including the inhibition of growth and interface effects of DNA on sodium urate crystals.

The complement system: a potential target for the comorbidity of chronic pain and depression.

The mechanisms of the chronic pain and depression comorbidity have gained significant attention in recent years. The complement system, widely involved in central nervous system diseases and mediating non-specific immune mechanisms in the body, remains incompletely understood in its involvement in the comorbidity mechanisms of chronic pain and depression. This review aims to consolidate the findings from recent studies on the complement system in chronic pain and depression, proposing that it may serve as a promising shared therapeutic target for both conditions. Complement proteins C1q, C3, C5, as well as their cleavage products C3a and C5a, along with the associated receptors C3aR, CR3, and C5aR, are believed to have significant implications in the comorbid mechanism. The primary potential mechanisms encompass the involvement of the complement cascade C1q/C3-CR3 in the activation of microglia and synaptic pruning in the amygdala and hippocampus, the role of complement cascade C3/C3a-C3aR in the interaction between astrocytes and microglia, leading to synaptic pruning, and the C3a-C3aR axis and C5a-C5aR axis to trigger inflammation within the central nervous system. We focus on studies on the role of the complement system in the comorbid mechanisms of chronic pain and depression.

Older adults preserve audiovisual integration through enhanced cortical activations, not by recruiting new regions.

Effective interactions with the environment rely on the integration of multisensory signals: Our brains must efficiently combine signals that share a common source, and segregate those that do not. Healthy ageing can change or impair this process. This functional magnetic resonance imaging study assessed the neural mechanisms underlying age differences in the integration of auditory and visual spatial cues. Participants were presented with synchronous audiovisual signals at various degrees of spatial disparity and indicated their perceived sound location. Behaviourally, older adults were able to maintain localisation accuracy. At the neural level, they integrated auditory and visual cues into spatial representations along dorsal auditory and visual processing pathways similarly to their younger counterparts but showed greater activations in a widespread system of frontal, temporal, and parietal areas. According to multivariate Bayesian decoding, these areas encoded critical stimulus information beyond that which was encoded in the brain areas commonly activated by both groups. Surprisingly, however, the boost in information provided by these areas with age-related activation increases was comparable across the 2 age groups. This dissociation-between comparable information encoded in brain activation patterns across the 2 age groups, but age-related increases in regional blood-oxygen-level-dependent responses-contradicts the widespread notion that older adults recruit new regions as a compensatory mechanism to encode task-relevant information. Instead, our findings suggest that activation increases in older adults reflect nonspecific or modulatory mechanisms related to less efficient or slower processing, or greater demands on attentional resources.

Impact of tissue penetration and albumin binding on design of T cell targeted bispecific agents

Bispecific agents are a rapidly growing class of cancer therapeutics, and immune targeted bispecific agents have the potential to expand functionality well beyond monoclonal antibody agents. Humabodies⁎⁎Humabody® is a registered trademark of Crescendo Biologics Ltd. are fully human single domain antibodies that can be linked in a modular fashion to form multispecific therapeutics. However, the effect of heterogeneous delivery on the efficacy of crosslinking bispecific agents is currently unclear. In this work, we utilize a PSMA-CD137 Humabody with an albumin binding half-life extension (HLE) domain to determine the impact of tissue penetration on T cell activating bispecific agents. Using heterotypic spheroids, we demonstrate that increased tissue penetration results in higher T cell activation at sub-saturating concentrations. Next, we tested the effect of two different albumin binding moieties on tissue distribution using albumin-specific HLE domains with varying affinities for albumin and a non-specific lipophilic dye. The results show that a specific binding mechanism to albumin does not influence tissue penetration, but a non-specific mechanism reduced both spheroid uptake and distribution in the presence of albumin. These results highlight the potential importance of tissue penetration on bispecific agent efficacy and describe how the design parameters including albumin-binding domains can be selected to maximize the efficacy of bispecific agents.

Endogenous bystander killing mechanisms enhance the activity of novel FAP-specific CAR-Tcells against glioblastoma.

CAR-T cells are being investigated as a novel immunotherapy for glioblastoma, but clinical success has been limited. We recently described fibroblast activation protein (FAP) as an ideal target antigen for glioblastoma immunotherapy, with expression on both tumor cells and tumor blood vessels. However, CAR-T cells targeting FAP have never been investigated as a therapy for glioblastoma. We generated a novel FAP targeting CAR with CD3ζ and CD28 signalling domains and tested the resulting CAR-T cells for their lytic activity and cytokine secretion function invitro (using real-time impedance, flow cytometry, imaging and bead-based cytokine assays), and in vivo (using a xenograft mimicking the natural heterogeneity of human glioblastoma). FAP-CAR-T cells exhibited target specificity against model cell lines and potent cytotoxicity against patient-derived glioma neural stem cells, even when only a subpopulation expressed FAP, indicating a bystander killing mechanism. Using co-culture assays, we confirmed FAP-CAR-T cells mediate bystander killing of antigen-negative tumor cells, but only after activation by FAP-positive target cells. This bystander killing was at least partially mediated by soluble factors and amplified by IL-2 which activated the non-transduced fraction of the CAR-T product. Finally, a low dose of intravenously administered FAP-CAR-T cells controlled, without overt toxicity, the growth of subcutaneous tumors created using a mixture of antigen-negative and antigen-positive glioblastoma cells. Our findings advance FAP as a leading candidate for clinical CAR-T therapy of glioblastoma and highlight under-recognised antigen nonspecific mechanisms that may contribute meaningfully to the antitumor activity of CAR-T cells.

The Role of Amino Acids in Non-Enzymatic Antioxidant Mechanisms in Cancer: A Review.

Currently, the antioxidant properties of amino acids and their role in the physicochemical processes accompanying oxidative stress in cancer remain unclear. Cancer cells are known to extensively uptake amino acids, which are used as an energy source, antioxidant precursors that reduce oxidative stress in cancer, and as regulators of inhibiting or inducing tumor cell-associated gene expression. This review examines nine amino acids (Cys, His, Phe, Met, Trp, Tyr, Pro, Arg, Lys), which play a key role in the non-enzymatic oxidative process in various cancers. Conventionally, these amino acids can be divided into two groups, in one of which the activity increases (Cys, Phe, Met, Pro, Arg, Lys) in cancer, and in the other, it decreases (His, Trp, Tyr). The review examines changes in the metabolism of nine amino acids in eleven types of oncology. We have identified the main nonspecific mechanisms of changes in the metabolic activity of amino acids, and described direct and indirect effects on the redox homeostasis of cells. In the future, this will help to understand better the nature of life of a cancer cell and identify therapeutic targets more effectively.