Efficient Killing Research Articles

Chiral Au nanostars for SERS sensing of enantiomers discrimination, multibacteria recognition and photothermal antibacterial application

The recognition and detection of chirality is of great significance in the fields of chemistry, pharmacy and biomedicine. In this work, we fabricated a chiral Au nanostars (C-AuNSs) with intense chiroptical responses and surface-enhanced Raman scattering (SERS) features by controlling chiral ligand D-/L-cysteine (Cys) concentration. The proposed chiral AuNSs can be applied for the enantiodiscrimination of amino acid based on SERS-chiral anisotropy (SERS-ChA) effect, in which homochiral amino acid has distinct affinities to the C-AuNSs of homogeneous handedness. By demonstrating selective affinity of D-AuNSs with D-glutamic acid (D-Glu) on bacterial peptidoglycan, we further realized effective identification of multiple bacteria according to SERS specific bacterial “fingerprints” by discriminant analysis (DA). More importantly, D-AuNSs can also be carried out to classify two bacterial pathogens (E. coli and S. aureus) in serum samples, as well as differentiate the infected patients from healthy subjects successfully with great accuracy. Benefiting from intrinsic photothermal capability, chiral AuNSs exhibited high efficiency in killing of bacteria and disrupting of biofilms through a synergistic chemotherapy and phototherapy (PTT) functions. Altogether, the chiral AuNSs provides a novel strategy for SERS discrimination of enantiomer and bacteria, as well as incorporation of photothermal bacterial elimination, contributing to further broaden the chiral nanomaterials-based application.

(TiZrNbTaMo)N nanocomposite coatings embedded with silver nanoparticles: imparting mechanical, osteogenic and antibacterial traits to dental implants

Designing highly osteogenic and effective antibacterial biomaterials with a prolonged service life for oral implants remains an ongoing challenge. In this work, the multi-element (TiZrTaNbMo)N high entropy nitride (HEN), with different concentrations of Ag, was co-deposited onto surgical grade commercially pure titanium by a reactive sputter-deposition technique using a double cathode glow discharge plasma system. The effects of Ag concentration on the microstructure and mechanical properties of the HEN-Ag nanocomposite coatings were investigated by various testing methods. Potentiodynamic polarization measurements were used to evaluate the corrosion resistance of the coatings in simulated body fluid. The antibacterial activity of the HEN-Ag nanocomposite coatings against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Candida albicans (C. albicans) strains was examined based on the bacterial inhibitory zones and bacterial killing efficiency. The cytocompatibility and osteogenesis activity of the HEN nanocomposite coatings using MC3T3-E1 cells were evaluated by a CCK-8 assay and an alkaline phosphatase quantitative assay. These studies demonstrate that the Ag-free HEN coating was composed of a face-centered-cubic (FCC) nitride phase, which displays good mechanical properties, anti-corrosion performance and osteogenic function, but no antibacterial properties were observed. In contrast, the HEN0.9Ag0.1 coating, composed of amorphous/nanocrystalline high-entropy nitride embedded with silver nanoparticles ∼10 nm in diameter, displays good mechanical and anti-corrosion performance, together with the excellent cell compatibility and antibacterial performance, which offer potential for providing outstanding protective efficiency for dental implants. Although the HEN0.8Ag0.2 nanocomposite coating exhibited superior antibacterial efficacy, potential for in vitro cytotoxicity was identified.

CRISPR-Cas9-Based Gene Knockout of Immune Checkpoints in Expanded NK Cells.

Natural killer (NK) cell immunotherapy has emerged as a novel treatment modality for various cancer types, including leukemia. The modulation of inhibitory signaling pathways in T cells and NK cells has been the subject of extensive investigation in both preclinical and clinical settings in recent years. Nonetheless, further research is imperative to optimize antileukemic activities, especially regarding NK-cell-based immunotherapies. The central scientific question of this study pertains to the potential for boosting cytotoxicity in expanded and activated NK cells through the inhibition of inhibitory receptors. To address this question, we employed the CRISPR-Cas9 system to target three distinct inhibitory signaling pathways in NK cells. Specifically, we examined the roles of A2AR within the metabolic purinergic signaling pathway, CBLB as an intracellular regulator in NK cells, and the surface receptors NKG2A and CD96 in enhancing the antileukemic efficacy of NK cells. Following the successful expansion of NK cells, they were transfected with Cas9+sgRNA RNP to knockout A2AR, CBLB, NKG2A, and CD96. The analysis of indel frequencies for all four targets revealed good knockout efficiencies in expanded NK cells, resulting in diminished protein expression as confirmed by flow cytometry and Western blot analysis. Our in vitro killing assays demonstrated that NKG2A and CBLB knockout led to only a marginal improvement in the cytotoxicity of NK cells against AML and B-ALL cells. Furthermore, the antileukemic activity of CD96 knockout NK cells did not yield significant enhancements, and the blockade of A2AR did not result in significant improvement in killing efficiency. In conclusion, our findings suggest that CRISPR-Cas9-based knockout strategies for immune checkpoints might not be sufficient to efficiently boost the antileukemic functions of expanded (and activated) NK cells and, at the same time, point to the need for strong cellular activating signals, as this can be achieved, for example, via transgenic chimeric antigen receptor expression.

NK cell-induced damage to P.falciparum-infected erythrocytes requires ligand-specific recognition and releases parasitophorous vacuoles that are phagocytosed by monocytes in the presence of immune IgG.

Natural killer (NK) cells lyse virus-infected cells and transformed cells through polarized delivery of lytic effector molecules into target cells. We have shown that NK cells lyse Plasmodium falciparum-infected red blood cells (iRBC) via antibody-dependent cellular cytotoxicity (ADCC). A high frequency of adaptive NK cells, with elevated intrinsic ADCC activity, in people chronically exposed to malaria transmission is associated with reduced parasitemia and resistance to disease. How NK cells bind to iRBC and the outcome of iRBC lysis by NK cells has not been investigated. We applied gene ablation in inducible erythrocyte precursors and antibody-blocking experiments with iRBC to demonstrate a central role of CD58 and ICAM-4 as ligands for adhesion by NK cells via CD2 and integrin αMβ2, respectively. Adhesion was dependent on opsonization of iRBC by IgG. Live imaging and quantitative flow cytometry of NK-mediated ADCC toward iRBC revealed that damage to the iRBC plasma membrane preceded damage to P. falciparum within parasitophorous vacuoles (PV). PV were identified and tracked with a P.falciparum strain that expresses the PV membrane-associated protein EXP2 tagged with GFP. After NK-mediated ADCC, PV were either found inside iRBC ghosts or released intact and devoid of RBC plasma membrane. Electron microscopy images of ADCC cultures revealed tight NK-iRBC synapses and free vesicles similar in size to GFP+ PV isolated from iRBC lysates by cell sorting. The titer of IgG in plasma of malaria-exposed individuals that bound PV was two orders of magnitude higher than IgG that bound iRBC. This immune IgG stimulated efficient phagocytosis of PV by primary monocytes. The selective NK-mediated damage to iRBC, resulting in release of PV, and subsequent phagocytosis of PV by monocytes may combine for efficient killing and removal of intra-erythrocytic P.falciparum parasite. This mechanism may mitigate the inflammation and malaria symptoms during blood-stage P. falciparum infection.

Periprosthetic joint infection and immunity: Current understanding of host-microbe interplay.

Periprosthetic joint infection (PJI) is a major complication of total joint arthroplasty. Even with current treatments, failure rates are unacceptably high with a 5-year mortality rate of 26%. Majority of the literature in the field has focused on development of better biomarkers for diagnostics and treatment strategies including innovate antibiotic delivery systems, antibiofilm agents, and bacteriophages. Nevertheless, the role of the immune system, our first line of defense during PJI, is not well understood. Evidence of infection in PJI patients is found within circulation, synovial fluid, and tissue and include numerous cytokines, metabolites, antimicrobial peptides, and soluble receptors that are part of the PJI diagnosis workup. Macrophages, neutrophils, and myeloid-derived suppressor cells (MDSCs)are initially recruited into the joint by chemokines and cytokines produced by immune cells and bacteria and are activated by pathogen-associated molecular patterns.While these cells are efficient killers of planktonic bacteria by phagocytosis, opsonization, degranulation, and recruitment of adaptive immune cells, biofilm-associated bacteria are troublesome. Biofilm is not only a physical barrier for the immune system but also elicits effector functions. Additionally, bacteria have developed mechanisms to evade the immune system by inactivating effector molecules, promoting killing or anti-inflammatory effector cell phenotypes, and intracellular persistence and dissemination. Understanding these shortcomings and the mechanisms by which bacteria can subvert the immune system may open new approaches to better prepare our own immune system to combat PJI. Furthermore, preoperative immune system assessment and screening for dysregulation may aid in developing preventative interventions to decrease PJI incidence.

The CD38/CD3xCD28 Trispecific Antibody (SAR442257) Potentially Represents a Novel Therapeutic Strategy for Peripheral T-Cell Lymphomas

Introduction Peripheral T-cell lymphomas (PTCL) remain an unmet medical need and novel therapeutic options inducing longstanding responses are needed. Immunotherapies with humanized monoclonal antibodies targeting cell surface receptors have dramatically improved the prognosis of many cancers. However, so far, only two antibodies targeting CCR4 and CD30 have been approved for the treatment of subsets of PTCL patients. Currently, several bi- and tri-specific antibodies are under pre-clinical or clinical evaluation to treat lymphomas. TSAR442257, a trispecific antibody (TriAb) which targets and binds CD38, CD3 and CD28 to engage T-cells against CD38-expressing tumor cells and enhance their CD3-triggered activation via CD28 co-stimulation. Here, we performed a preclinical investigation to evaluate the potential therapeutic value of the SAR442257 to treat PTCL, knowing that both CD28 and CD38 might represent targets in tumor T cells. Material and methodsCD28 and CD38 expression was studied in tumor cells of 268 PTCL patients. In situ semi-quantitative evaluation was done using immunohistochemistry on FFPE samples (n=250), and quantitative evaluation was performed using flow cytometry (FCM) on PBMCs (n=18) and PTCL lines (n=6). Multiplex immunofluorescence stainings with tumor cell marker were also performed on biopsies sections of selected cases. In vitro cytotoxic assays were performed by using 24h co-cultures of PTCL cell lines stained with a viability dye with allogeneic blood cells from healthy donors (HD) and SAR442257 or control TriAbs with null mutations in CD28 (D28), CD38 (D38) or both recognition domains at various concentrations (0-25000 pM) at 10:1 effector-to-target ratio. For primary PTCL cells, similar experiments were done with appropriate panels of surface markers aiming to identify tumor cells. Activation assays, measuring expression of CD25 and CD69, were done on PTCL lines and PBMCs from HD using the afore mentioned TriAbs at similar concentrations. ResultsWe found a recurrent but variable expression of CD28 and CD38 among the different PTCL entities (Figure 1). Overall, CD38 expression by tumor cells was found in 42% of PTCL samples in situ and 67% of PBMCs (ranging from 2.5% to 65%; data not shown). CD28 was found in neoplastic T cells in 56% of PTCL samples by IHC and 100% of PBMCs with a strong heterogeneity ranging from 30% to 99.4% (data not shown). CD38 and CD28 showed a range of expression among PTCL categories, with CD38 being more frequently expressed (Avg. IHC score: 250-300)) in PTCL diseases with a cytotoxic profile postulated to derive from innate immune/cytotoxic cells whereas CD28 was more frequently expressed (Avg. IHC score: 150-200) in PTCLs known to derive from helper T-cells (p<0.001, Figure 2). Co-expression of both targets was rare. Multiplex stainings confirmed co-expression of CD38 and/or CD28 targets in tumor cells but consistent with the FCM analysis, CD28 and CD38 were variably expressed in tumor T-cells of TFH PTCLs. The SAR442257 induced efficient killing of all CD28+/CD38+ and CD28-/CD38+ PTCL lines (with maximum killing reaching about 35%), while the D28 and D38 control TriAbs showed less efficient cytotoxic activities in six different cell lines. SAR442257 also induced CD25 and CD69 expression on normal T-cells suggesting efficient T-cell activation, (data not shown). Conclusion Altogether, this study shows that 1) most PTCL cells express at least CD28 or CD38, and 2) SAR442257 can efficiently kill malignant PTCL cells, while ensuring effective T-cell activation; In view of these results, clinical investigation of SAR442257 in PTCL is warranted.

Gamma-Delta (γδ) CAR-T Cells Lacking the CD3z Signaling Domain Enhance Targeted Killing of Tumor Cells and Preserve Healthy Tissues

Introduction: Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy against B cell malignancies for patients who have limited treatment options. Successful advancement of CAR-T therapy to myeloid hematopoietic and solid malignancies, however, has been limited by the potential of life-threatening on-target, off-tumor toxicities against healthy tissues, the natural tumor heterogeneity and consequent selection pressure leading to relapse. Addressing these limitations to widen the therapeutic index is crucial as we seek safer and more effective CAR-T therapies. In this regard, γδ T cells as first responders of immunity hold promise as they can directly identify and eliminate malignant cells through the recognition of multiple tumor-associated stress antigens not commonly expressed on normal tissues. We sought to combine the tumor-sensing capabilities of γδ T cells and the localization enhancement of CAR-T by excluding the CD3ζ domain in a non-signaling CAR (nsCAR) to allow the targeted killing against tumors, while sparing healthy tissues. Methods: Standard and nsCAR constructs against CD19, CD33 and CD123 were cloned into separate second-generation lentiviral vectors. The CAR constructs contained either a scFv targeting the respective antigen (anti-CD19/anti-CD33) or an IL-3 zetakine (IL-3z) targeting the CD123 followed by a flag-tag, a hinge, a transmembrane domain and a costimulatory domain + CD3ζ. IL-15 co-expression was incorporated to enhance γδ T cell fitness and persistence. CAR activation was determined by Jurkat T cells co-cultured with target ALL or AML cell lines followed by lentivector transduction of activated and expanded Vδ2 + γδ T cells. Cellular cytotoxicity was assessed at multiple E:T ratios against respective leukemia cell lines and healthy peripheral blood cells expressing the target antigen. Results: We observed significant upregulation of CD69 surface expression indicating activation of CD3ζ+ CD19CAR/CD33CAR/IL-3z Jurkat cells (3x, 20x, and 2x respectively) following culture with Nalm6 (CD19) and KG-1 (CD33 & IL-3z). CD3ζ- ns19CAR/ns33CAR/nsIL-3z Jurkat cells did not upregulate CD69 in parallel cultures. We also observed a time-dependent reduction of the CD3ζ+ CD33 CAR + population over 7 days (43%) in Jurkat cells following extended coculture with KG-1 cells, while the nsCAR + Jurkat population remained unchanged, indicating the nsCAR may mitigate activation induced cell death (AICD). Ex vivo activated γδ T cells from healthy donors (N=4), were transduced with nsCAR lentiviral vectors with high efficiency (up to 80%). The ns19CAR cells effectively killed CD19 + Nalm6 cells and demonstrated enhanced (>1.5x) cytotoxicity compared to untransduced γδ T cells (UTD). After 48hr co-culture (E:T=2:1), the ns19CAR cells killed 79.7%+6.6% Nalm6 cells compared to 46.1%+7.2%) with UTD. Minimal cytotoxicity was observed for ns19CAR and UTD γδ T cells against B cells from healthy donor PBMC 5.2%+6.6% for ns19CAR vs. to -4.6%+7.7% with UTD. No significant difference in cytotoxicity against the CD19- K562 cells between the UTD or ns19CAR cells (73.5% vs. 71.5%, E:T=2:1) suggesting the enhanced cytotoxicity is ns19CAR directed. Similarly, ns33CAR and nsIL-3z cells, although requiring higher E:T ratios for similar killing efficiency, continued to demonstrate enhanced (up to 2.0x) killing against AML cells (HL-60, KG-1 and MOLM13) and CML cells K-562 compared to UTD in cytotoxicity assay with 24hr co-culture. Meantime, minimal cytotoxicity (<10%) was observed for ns33CAR, nsIL-3z, or UTD γδ T cells against CD33+ cells isolated from healthy donor PBMC. Conclusions: Ex vivo activated nsCAR cells efficiently recognize and kill leukemia cell lines while sparing peripheral blood cells bearing the same target antigen. The nsCAR cells also show increased cytotoxicity against leukemias over unmodified activated γδ T cells suggesting improvement in tropism and/or binding efficiency. In summary, our findings showed that the combination of nsCAR on γδ T cells may increase the therapeutic index to allow expansion of CAR-T therapy to cancers with unacceptable target expression on critical healthy cell populations. Further optimization of the nsCAR constructs may potentially enhance both the efficacy and safety profile of the next generation adoptive cell therapies against wider selection of cancers.

Combining Allogeneic Gamma Delta T Cells with a Phosphoantigen Prodrug Promotes Apoptosis of Acute Myeloid Leukemia Stem and Progenitor Cells

Despite recent therapeutic advances, patients with relapsed/refractory acute myeloid leukemia (r/r AML) have limited treatment options and poor prognosis. Leukemic stem cells (LSCs) are considered as relapse-initiating cells and the “culprit” of treatment resistance. Allogeneic and autologous gamma delta (γδ) T cell-based therapies have been explored for a long time and proven to be safe in patients through multiple clinical trials. However, quite a few AML blasts obtained from r/r AML patients show resistance to γδ T cell-mediated cytotoxicity. Novel strategies targeted LSCs may improve efficacy of γδ T cell-mediated r/r AML therapy. Given that γδ T cells exert antitumor capacity via multiple pathways, to identify the way matters most in the resistance of γδ T cell-mediated cytotoxicity, we examined the median fluorescence intensity (MFI) of the ligands expressed in tumor cells by flow cytometry, including butyrophilin 3A (BTN3A), butyrophilin 2A1 (BTN2A1), the human MHC class I chain-related genes (MICA and MICB), the unique-long 16 binding proteins (ULBP1, ULBP2/5/6, ULBP3), Fas, the receptor of TNF-related apoptosis-inducing ligand (TRAIL) DR5, CD112 and CD155. The cytotoxicity activity of γδ T cells was measured by flow cytometry-based killing assay or bright-lite luciferase assay system after coculturing with AML cell lines and blasts at an appropriate effector to target (E:T) ratio. Then we employed a Support Vector Machine model to predict the killing efficiency and identified that BTN3A and BTN2A1 play the predominant role in γδ T cell-mediated cytotoxicity based on the Shapley additive explanation. BTN3A and BTN2A1 are the key ligands in the γδ TCR-phosphoantigen (pAg) recognition activity. PAgs, which are produced in tumor cells function as “molecular glues” to promote heteromeric association of BTN3A1 and BTN2A1 (Yuan L et al., BioRxiv 2022). To promote the γδ T cell-mediated cytotoxicity targeting r/r AML, here we synthesized a pAg prodrug, which show great plasma stability and membrane permeability. We anticipate this small molecule would lead pAg accumulation in tumor cells and cause increased γδ T cell activation. To identify the killing efficacy of pAg prodrug-induced γδ T cell activation, we select KG-1α (a human leukemia stem cell-like cell) as target cell. Zoledronate (ZOL), which is employed to stimulate γδ T cells in multiple γδ T cell-based clinical trials, is used in our killing assay as compared group. As expected, combining γδ T cells with pAg prodrug or ZOL both significantly improved the percent of specific killing when the E:T ratio is 1:1. The pAg prodrug showed more potent activity than ZOL group with EC50 value of 0.1 nM vs 17.93μM. And the specific killing percent of pAg prodrug group is 83.26±2.1 % (1 nM), comparing with negative control 15.17±3.4 % (only γδT cells). Futhermore, at relatively low E:T ratio (1:4), γδ T cells with pAg prodrug remain excellent cytotoxicity when targeting MV411 cell and OCI-AML3 cell, which are widely used in r/r AML research. After coculturing with primary leukemia stem and progenitor cells (CD34 +) obtained from r/r AML patients for 6~8 h, the apoptosis percentage of CD34 + leukemia cells was 42.34% comparing with negative control (only γδT cells) 2.53%. Then we evaluated the in vivo efficacy of combining γδT cells and pAg prodrug in NSG mice bearing MV411 cells. Compared with only γδT cells-treated group, combination with pAg prodrug presented better tumor clearing. To elucidate the pathway of pAg prodrug-mediated killing, we tested mitochondria apoptosis related events. After treated by γδ T cells with pAg prodrug, KG-1α cells showed loss of mitochondrial outer membrane potential and increasingly actived caspase 3/7. Meanwhile, pAg prodrug directly activates effector γδ T cells with increased secretion of CD107a and IFN-γ. Considering the possibility of toxicity towards normal hematopoietic stem and progenitor cells, we conducted colony-forming units (CFUs) assay. After treatment with γδT cells and pAg prodrug, the number of CFUs of CD34 + cells from cord blood was not effected compared with the CD34 + cells only group. Overall, we explored the predominant factor in γδ T cell-mediated killing and synthesized a pAg prodrug to improve the killing efficacy. We demonstrated that combining γδ T cells with pAg prodrug has effectively promoted apoptosis of AML stem and progenitor cells sparing normal hematopoietic stem and progenitor cells.

Engineering Off-the-Shelf Gamma Delta CAR T Cells for the Treatment of Acute Myeloid Leukemia

Chimeric antigen receptor T cell therapy (CART) therapy has shown remarkable success in the treatment of B cell acute lymphoblastic leukemias (B-ALL) and lymphomas. However, CART therapies for acute myeloid leukemia (AML), where 5-year survival rates are significantly lower than for B-ALL, are only in their infancy. CD33-CART have potent activity against AML in preclinical models and a first-in-child/first-in-human phase 1/2 CD33-CART clinical trial for AML is ongoing in the Pediatric Oncology Branch of the National Cancer Institute (NCT03971799). Nonetheless, published outcomes suggest a modest efficacy of approximately 50% (Shahzad et al., Front Immunol 2023), highlighting the critical need to develop new strategies to improve CART accessibility and a more robust anti-AML response. We hypothesized that off-the-shelf gamma delta (γδ) CD33 CART cells could potentially overcome current challenges for the treatment of AML. γδ lineage T cells are unconventional lymphocytes whose functions are not restricted to MHC-mediated antigen presentation; they are primed for immediate responses, including tumor killing. Furthermore, allogeneicγδ T cells have the potential to induce robust anti-tumor cytotoxicity without causing graft versus host disease (GVHD). Here, we generated γδ CAR T cells from healthy donor elutriated lymphocytes by activation with zoledronic acid and IL-2 for 7-14 days. Within 9 days post stimulation, the vast majority of lymphocytes were Vδ2+ and 30-40% were successfully transduced with a lentiviral CD33 CAR construct harboring the 4-1BB costimulatory domain. Importantly, and unlike conventional alpha beta (ab) T lymphocytes, >98% of these γδ CD33CAR T cells expressed IFNγ under basal conditions. This characteristic likely accounted for the efficient in vitro killing of AML cell lines by untransduced γδ T lymphocytes under conditions of high effector/target (E/T) ratios. While untransduced γδ T cells did not exhibit cytotoxicity following repeat AML stimulations, γδ CD33CAR T lymphocytes exhibited proficient in vitro cytotoxicity, with killing rates that were more rapid than those initiated by ab CD33 CART (Figure 1). These characteristics were associated with a prolonged metabolic activity of γδT cells; γδ CD33 CART expressed high levels of the GLUT1 glucose transporter for >14 days post activation whereas GLUT1 levels on ab CD33 CART returned to resting within 10 days. High GLUT1 levels were linked to efficient killing under conditions of basal glucose levels. Most notably, γδ CD33CAR T lymphocytes achieved high in vivo cytotoxicity, assessed using bioluminescent AML cell line xenografts in humanized NSG mice. Together, these data highlight the feasibility of generating allogeneic γδ CD33CART with a strong anti-AML cytotoxic response.

Improving Intracellular Delivery of an Antibody-Drug Conjugate Targeting Carcinoembryonic Antigen Increases Efficacy at Clinically Relevant Doses In Vivo.

Solid tumor antibody-drug conjugates (ADC) have experienced more clinical success in the last 5 years than the previous 18-year span since the first ADC approval in 2000. While recent advances in protein engineering, linker design, and payload variations have played a role in this success, high expression and readily internalized targets have also been crucial to solid tumor therapy. However, these factors are also paradoxically connected to poor tissue penetration and lower efficacy. Previous work shows that potent ADCs can benefit from slower internalization under subsaturating doses to improve tissue penetration and increase tumor response. In contrast, faster internalization is predicted to increase efficacy under higher, tumor saturating doses. In this work, the intracellular delivery of SN-38 conjugated to an anti-carcinoembryonic antigen (anti-CEA) antibody (Ab) is increased by coadministering a noncompeting (cross-linking) anti-CEA Ab to improve efficacy in a colorectal carcinoma animal model. The SN-38 payload enables broad tumor saturation with clinically-tolerable doses, and under these saturating conditions, using a second CEA receptor cross-linking Ab yields faster internalization, which increases tumor killing efficacy. Our spheroid results show indirect bystander killing can also occur, but the more efficient direct cell killing from targeted intracellular payload release drives a greater tumor response. These results provide a strategy to increase therapeutic effectiveness with improved intracellular delivery under tumor saturating doses with the potential to expand the ADC target repertoire.

Tumor-targeted metabolic inhibitor prodrug labelled with cyanine dyes enhances immunoprevention of lung cancer

Recent progress in targeted metabolic therapy of cancer has been limited by the considerable toxicity associated with such drugs. To address this challenge, we developed a smart theranostic prodrug system that combines a fluorophore and an anticancer drug, specifically 6-diazo-5-oxo-l-norleucine (DON), using a thioketal linkage (TK). This system enables imaging, chemotherapy, photodynamic therapy, and on-demand drug release upon radiation exposure. The optimized prodrug, DON-TK-BM3, incorporating cyanine dyes as the fluorophore, displayed potent reactive oxygen species release and efficient tumor cell killing. Unlike the parent drug DON, DON-TK-BM3 exhibited no toxicity toward normal cells. Moreover, DON-TK-BM3 demonstrated high tumor accumulation and reduced side effects, including gastrointestinal toxicity, in mice. This study provides a practical strategy for designing prodrugs of metabolic inhibitors with significant toxicity stemming from their lack of tissue selectivity.

Preclinical Synergistic Combination Therapy of Lurbinectedin with Irinotecan and 5-Fluorouracil in Pancreatic Cancer.

Pancreatic cancer is a devastating disease with a poor prognosis. Novel chemotherapeutics in pancreatic cancer have shown limited success, illustrating the urgent need for new treatments. Lurbinectedin (PM01183; LY-01017) received FDA approval in 2020 for metastatic small cell lung cancer on or after platinum-based chemotherapy and is currently undergoing clinical trials in a variety of tumor types. Lurbinectedin stalls and degrades RNA Polymerase II and introduces breaks in DNA, causing subsequent apoptosis. We now demonstrate lurbinectedin's highly efficient killing of human-derived pancreatic tumor cell lines PANC-1, BxPC-3, and HPAF-II as a single agent. We further demonstrate that a combination of lurbinectedin and irinotecan, a topoisomerase I inhibitor with FDA approval for advanced pancreatic cancer, results in the synergistic killing of pancreatic tumor cells. Western blot analysis of combination therapy indicates an upregulation of γH2AX, a DNA damage marker, and the Chk1/ATR pathway, which is involved in replicative stress and DNA damage response. We further demonstrate that the triple combination between lurbinectedin, irinotecan, and 5-fluorouracil (5-FU) results in a highly efficient killing of tumor cells. Our results are developing insights regarding molecular mechanisms underlying the therapeutic efficacy of a novel combination drug treatment for pancreatic cancer.

Polybenzoxazines as new photothermal therapy agents

To make photothermal therapy an attractive alternative to conventional cancer treatments, the development of novel photothermal agents with enhanced properties is demanded. To tackle this challenge, herein we explored the use of polybenzoxazines, a well-known class of phenolic resins. Two different types of polybenzoxazine materials were prepared as potential photothermal agents: water-soluble polybenzoxazines as well as nanoparticles of hydrophobic polybenzoxazines, which can be generated in situ by polymer addition to cell culture medium. For both systems, irradiation with visible light led to significant photothermal effects, whose application to photoinduce human breast cancer cell’s death was investigated in vitro. The best results were obtained for polybenzoxazine nanostructures, which exhibited successful cell internalization, minimal cytotoxicity in the dark and efficient photothermal killing of cancer cells upon illumination with high-penetrating red light. In combination with their low cost and facile preparation, this makes these polymer nanomaterials highly promising for photothermal therapy applications.

Metallic-Polyphenolic Nanoparticles Reinforced Cationic Guar Gum Hydrogel for Effectively Treating Burn Wound.

The development of injectable hydrogel dressings which are long-term moisturizing, easy-to-apply, and effectively inhibiting infection and inflammatory is essential to promote burn wound repairing. Herein, an injectable hydrogel with moisturizing, antibacterial, and anti-inflammation abilities via multiple reversible interactions between cation guar gum (CG) and metallic-polyphenolic nanoparticles (PA-ZnII NPs) is developed. Specifically, PA-ZnII NPs is formed by synergistic complexation of protocatechualdehyde (PA) and zinc ion (Zn2+ ), provides CGPZ hydrogel with plentiful reversible interactions to inhibit the loss of moist. By interacting with PA-ZnII NPs, the CGPZ hydrogel can provide enhanced moisturization for more than 3 days. Moreover, the CGPZ hydrogel can maintain good adhesion for a period of time with injection and self-healing capabilities due to reversible interactions between CG and PA-ZnII NPs. In addition, CGPZ hydrogel exhibits outstanding broad spectrum antibacterial performance, as its killing efficiency against Escherichia coli and Staphylococcus aureus is all greater than 99.99%. Importantly, compared with commercial dressing, the CGPZ hydrogel can promote wound healing faster by inhibiting tissue damage from dysregulated inflammation and accelerating neovascularization. It is believed that the moisturizing CGPZ hydrogel with antibacterial and anti-inflammation performance can serve as a promising dressing for the effective treatment of burn wound.

Utilizing Ceftazidime/Avibactam Therapeutic Drug Monitoring in the Treatment of Neurosurgical Meningitis Caused by Difficult-to-Treat Resistant Pseudomonas aeruginosa and KPC-Producing Enterobacterales.

Central nervous system (CNS) infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales and difficult-to-treat resistant (DTR) Pseudomonas aeruginosa represent a formidable clinical challenge. Antimicrobial regimens that efficiently penetrate the cerebrospinal fluid (CSF) and achieve sufficient concentrations associated with microbiologic and clinical cure are limited. We evaluated therapy with ceftazidime-avibactam (CAZ-AVI) in order to guide precise dosing in the treatment of CNS infections. Therapeutic drug monitoring (TDM) was performed in 3 patients with health care-associated ventriculitis and meningitis (HAVM) using CAZ-AVI 2.5 g infused intravenously every 8 hours as standard and extended infusion. Simultaneous CSF and plasma samples were obtained throughout the dosing interval in each patient. Concentrations of CAZ and AVI were determined by liquid chromatography/mass spectrometry. Bacterial identification revealed KPC-producing Klebsiella pneumoniae (KPC-Kp), DTR Pseudomonas aeruginosa, and KPC-producing Enterobacter cloacae (KPC-Ent.c). All isolates were resistant to carbapenems. The minimum inhibitory concentrations (MICs) of CAZ-AVI were 0.25/4, 4/4, and 0.25/4 μg/mL, respectively. CAZ and AVI concentrations were determined in CSF samples ranging from 29.0 to 15.0 µg/mL (CAZ component) and 4.20 to 0.92 µg/mL (AVI component), respectively. AVI achieved concentrations ≥1 µg/mL in 11 out of 12 CSF samples collected throughout the dosing interval. Clinical and microbiologic cure were attained in all patients. Postinfusion concentrations of CAZ-AVI were measured in plasma and CSF samples obtained from 3 patients with complicated CNS infections caused by antimicrobial-resistant isolates. The measured concentrations revealed that standard CAZ and AVI exposures sufficiently attained values correlating to 50% fT > MIC, which are associated with efficient bacterial killing.

GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy.

Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary treatment that has become a mainstay of advanced cancer treatment. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes in advanced hepatocellular carcinoma (HCC), and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7-19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects. We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces co-expression of IL-7 at a moderate level (500pg/mL) and CCL19 at a high level (15000pg /mL) and transduced it into human T cells. In vitro, cell killing efficacy was validated by the xCELLigence RTCA system, LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Finally, we initiated a phase I clinical trial to evaluate the safety and effect of GPC3-7-19-CAR-T cells in the clinic. GPC3-7-19-CAR-T cells had 1.5-2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in GPC3-7-19-CAR-T cells treated model were reduced (3/5vs.5/5), and the average tumor volumes were 0.74 cm3 ± 1.17 vs. 0.34 cm3 ± 0.25. Of note, increased proportion of CD4+ TEM and CD8+ TCM cells was infiltrated in GPC3-7-19-CAR-T cells group. GPC3-7-19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues. In one patient with advanced HCC, GPC3-7-19-CAR-T-cell treatment resulted in tumor reduction 56days after intravenous infusion. In conclusion, GPC3-7-19-CAR-T cells achieved antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4+ TEM and CD8+ TCM cell subsets. Most importantly, GPC3-7-19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic. ► Novel GPC3-7-19-CAR-T cells designed with mediate level of IL-7 secretion and high level of CCL19 secretion, which could recruit more mature DCs to assist killing on GPC3+HCCs. ►DC cells recruited by CCL19 could interact with CD4+ T cells and promote the differentiation of CD4+TEFF cells into CD4+TEM and CD8+TCM subsets, leading a better anti-tumor effect on GPC3+HCCs. ►Compared with conventional GPC3-CAR-T, GPC3-7-CCL19-CAR-T cells could reverse tumor immunosuppressive microenvironment by reducing PMN-MDSC and Treg cell infiltration.

Macrophage targeted iron oxide nanodecoys augment innate immunological and drug killings for more effective Mycobacterium Tuberculosis clearance

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) infection, is still one of the top killers worldwide among infectious diseases. The escape of Mtb from immunological clearance and the low targeting effects of anti-TB drugs remain the substantial challenges for TB control. Iron is particularly required for Mtb growth but also toxic for Mtb in high dosages, which makes iron an ideal toxic decoy for the ‘iron-tropic’ Mtb. Here, a macrophage-targeted iron oxide nanoparticles (IONPs)-derived IONPs-PAA-PEG-MAN nanodecoy is designed to augment innate immunological and drug killings against intracellular Mtb. IONPs-PAA-PEG-MAN nanodecoy exhibits preferential uptake in macrophages to significantly increase drug uptake with sustained high drug contents in host cells. Moreover, it can serve as a specific nanodecoy for the ‘iron-tropic’ Mtb to realize the localization of Mtb contained phagosomes surrounding the drug encapsulated nanodecoys and co-localization of Mtb with the drug encapsulated nanodecoys in lysosomes, where the incorporated rifampicin (Rif) can be readily released under acidic lysosomal condition for enhanced Mtb killing. This drug encapsulated nanodecoy can also polarize Mtb infected macrophages into anti-mycobacterial M1 phenotype and enhance M1 macrophage associated pro-inflammatory cytokine (TNF-α) production to trigger innate immunological responses against Mtb. Collectively, Rif@IONPs-PAA-PEG-MAN nanodecoy can synergistically enhance the killing efficiency of intracellular Mtb in in vitro macrophages and ex vivo monocyte-derived macrophages, and also significantly reduce the mycobacterial burdens in the lung of infected mice with alleviated pathology. These results indicate that Rif@IONPs-PAA-PEG-MAN nanodecoy may have a potential for the development of more effective therapeutic strategy against TB by manipulating augmented innate immunity and drug killings.Graphic

Antibody-drug conjugates: Recent advances in payloads.

Antibody‒drug conjugates (ADCs), which combine the advantages of monoclonal antibodies with precise targeting and payloads with efficient killing, show great clinical therapeutic value. The ADCs' payloads play a key role in determining the efficacy of ADC drugs and thus have attracted great attention in the field. An ideal ADC payload should possess sufficient toxicity, low immunogenicity, high stability, and modifiable functional groups. Common ADC payloads include tubulin inhibitors and DNA damaging agents, with tubulin inhibitors accounting for more than half of the ADC drugs in clinical development. However, due to clinical limitations of traditional ADC payloads, such as inadequate efficacy and the development of acquired drug resistance, novel highly efficient payloads with diverse targets and reduced side effects are being developed. This perspective summarizes the recent research advances of traditional and novel ADC payloads with main focuses on the structure-activity relationship studies, co-crystal structures, and designing strategies, and further discusses the future research directions of ADC payloads. This review also aims to provide valuable references and future directions for the development of novel ADC payloads that will have high efficacy, low toxicity, adequate stability, and abilities to overcome drug resistance.

Engineering Hepatitis B Virus (HBV) Protein Particles for Therapeutic Delivery.

Nature provides an abundance of proteins whose structures and reactivity have been perfected through evolution to perform specific tasks necessary for biological function. The structural and functional properties of many natural proteins are quite valuable for the construction and customization of drug delivery vehicles. Self-assembling protein nanoparticle platforms are particularly useful scaffolds, as their multi-subunit designs allow the attachment of a high density of modifying molecules such as cell-binding ligands that provide avidity for targeting and facilitate encapsulation of large quantities of therapeutic payload. We explored SpyCatcher/SpyTag conjugation as a system to modify hepatitis B virus (HBV)-like particles (HBV VLPs). Using this simple decoration strategy, we demonstrated efficient and cell-selective killing of inflammatory breast cancer cells via delivery of yeast cytosine deaminase suicide enzymes combined with 5-fluoro-cytosine prodrugs.

Upregulation of the NKG2D Ligand ULBP2 by JC Polyomavirus Infection Promotes Immune Recognition by Natural Killer Cells.

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a potentially fatal complication of severe immune suppression with no effective treatment. Natural killer (NK) cells play critical roles in defense against viral infections; however, NK-cell response to JCPyV infection remains unexplored. NK- and T-cell responses against the JCPyV VP1 were compared using intracellular cytokine staining upon stimulation with peptide pools. A novel flow cytometry-based assay was developed to determine NK-cell killing efficiency of JCPyV-infected astrocyte-derived SVG-A cells. Blocking antibodies were used to evaluate the contribution of NK-cell receptors in immune recognition of JCPyV-infected cells. In about 40% of healthy donors, we detected robust CD107a upregulation and IFN-γ production by NK cells, extending beyond T-cell responses. Next, using the NK-cell-mediated killing assay, we showed that coculture of NK cells and JCPyV-infected SVG-A cells leads to a 60% reduction in infection, on average. JCPyV-infected cells had enhanced expression of ULBP2-a ligand for the activating NK-cell receptor NKG2D, and addition of NKG2D blocking antibodies decreased NK-cell degranulation. NKG2D-mediated activation of NK cells plays a key role in controlling JCPyV replication and may be a promising immunotherapeutic target to boost NK-cell anti-JCPyV activity.