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AbstractBackgroundPatients with head and neck squamous cell carcinoma (HNSCC),particularly the human papillomavirus negative (HPV−) subset,have a dismal prognosis. Furthermore,patients with Fanconi anemia (FA) have a genetic predisposition with a 500-fold to 700-fold higher incidence of HNSCC. Thus,novel and more efficacious therapies are needed. As current immunotherapies often fail due to suppressive elements in the tumor microenvironment (TME),we developed a trispecific killer engager (TriKE) to direct multiple signals to natural killer (NK) cells to overcome the hypoxic TME. This TriKE is comprised of a camelid nanobody that binds to CD16 on NK cells,an interleukin (IL)-15 moiety,and another novel camelid nanobody that binds to the B7-H3 antigen,which is highly and specifically expressed on the tumor cell surface.MethodsThe B7H3 TriKE was generated using a mammalian expression system. Its functionality was evaluated using flow cytometry-based NK cell degranulation,cytokine production,proliferation and live cell imaging cytotoxicity assays. Models of acute and prolonged hypoxia (1% oxygen) were carried out to assess tumor killing. Tumor progression,NK cell persistence,and survival differences between IL-15-treated and TriKE-treated mice were studied using NOD-scidIL2Rgnull (NSG) mice engrafted with human HNSCC.ResultsHigh B7-H3 expression was found in HPV− HNSCC cell lines,even when the FA gene was knocked out,and The Cancer Genome Atlas patient data showed that high B7-H3 expression predicted poor survival in patients with HPV− HNSCC. Similar to the NK cell activity seen with healthy donors,the B7H3 TriKE enhanced activation,expansion and cytotoxicity of NK cells from patients with HPV− HNSCC,a target population for this therapeutic. Additionally,the B7H3 TriKE improved NK cell cytotoxicity in a three-dimensional spheroid model of HNSCC. In both acute and prolonged hypoxia (1% oxygen),the B7H3 TriKE mediated enhanced tumor killing,mitigating impairment of NK cell cytotoxicity in hypoxia. In vivo,the B7H3 TriKE-treated mice demonstrated substantial antitumor activity and prolonged survival.ConclusionsThe B7H3 TriKE is a novel immunotherapeutic approach that can overcome hypoxic suppression of NK cells in the HNSCC TME. These highly translational studies present an innovative therapy for patients with HNSCC and will be developed further for clinical application. View Publication

SummaryInterleukin-33 (IL-33) is an immunoregulatory cytokine that moderately suppresses experimental autoimmune encephalomyelitis (EAE),a murine model of multiple sclerosis (MS). However,poor pharmacokinetics and toxicity hinder its clinical translation. To address these limitations,we develop an activity-attenuated IL-33 by recombinant fusion to serum albumin (SA). SA-IL-33 exhibits reduced toxicity and prolonged residence in the secondary lymphoid organs (SLOs),sites of T cell priming in autoimmunity,compared to wild-type (WT) IL-33. Prophylactic SA-IL-33 administration prevents EAE with superior efficacy to WT IL-33 and comparable efficacy to fingolimod (FTY720),a Food and Drug Administration (FDA)-approved MS drug. Therapeutic SA-IL-33 treatment also reduces disease severity in both chronic and relapsing-remitting EAE. SA-IL-33 modulates immunity in EAE by suppressing CD45+ cell infiltration (including myelin-reactive T helper 17 [TH17] cells) in the spinal cord,while expanding type 2 immune cells (including type 2 innate lymphoid cells [ILC2s],ST2+ regulatory T cells [Tregs],T helper 2 [TH2] cells,and M2-polarized macrophages) in the SLOs. These findings suggest that SA-IL-33 is a promising therapeutic for neuroinflammatory diseases. Graphical abstract Highlights•Fusion of serum albumin (SA) to interleukin-33 (IL-33) attenuates its activity and toxicity•Engineered SA-IL-33 exhibits prolonged residence in the secondary lymphoid organs (SLOs)•SA-IL-33 treatment both prevents the onset of and reduces established neuroinflammation in mice•Cytokine therapy suppresses TH17 cells in the CNS and promotes immunoregulation in the SLOs The clinical utility of interleukin-33 is hindered by poor pharmacokinetics and toxicity. Budina et al. develop a fusion of serum albumin and interleukin-33 (SA-IL-33) with reduced toxicity and prolonged lymph node residence. SA-IL-33 prevents the onset of and suppresses established inflammation-mediated paralysis in mice,demonstrating promise as a therapeutic for neuroinflammatory diseases. View Publication

SummaryAlthough chimeric antigen receptor (CAR) T cells have demonstrated therapeutic activity in hematopoietic malignancies,tumor heterogeneity has impeded the efficacy of CAR T cells and their extension into successful solid tumor treatment. To address these challenges,induced pluripotent stem cell (iPSC)-derived T (iT) cells are engineered to uniformly express CAR and T cell receptor (TCR),enabling targeting of both surface and intracellular antigens,respectively,along with a high-affinity,non-cleavable variant of CD16a (hnCD16) to support antibody-dependent cellular cytotoxicity (ADCC) when combined with therapeutic antibodies. Co-expression of each antitumor strategy on engineered iT cells enables independent and antigen-specific targeting across a diverse set of liquid and solid tumors. In heterogeneous tumor models,coactivation of these modalities is required for measurable antitumor efficacy,with activation of all three modalities displaying maximal efficacy. These data highlight the therapeutic potential of an off-the-shelf engineered iPSC-derived trimodal T cell expressing CAR,TCR,and hnCD16 to combat difficult-to-treat heterogeneous tumors. Graphical abstract Highlights•CAR,TCR,and hnCD16 can be uniformly co-expressed and can function in iT cells•hnCD16 signals through CD3ζ and arms iT cells with targeting flexibility through ADCC•Concurring CAR,TCR,and hnCD16 activation demonstrates a cooperative effect•Multi-targeting with trimodal iT cells can control heterogeneous tumors in vivo Yang et al. show that (1) trimodal iPSC cells expressing CAR,TCR,and hnCD16 can commit to T cell lineage,(2) hnCD16 signals through CD3ζ in iT cells and arms iT cells with ADCC targeting flexibility,and (3) trimodal iT cells control antigen-heterogeneous tumors in vivo through multi-modal targeting. View Publication

SummarymRNA-based in vivo chimeric antigen receptor (CAR)-T cell engineering offers advantages over ex vivo therapies,including streamlined manufacturing and transient expression. However,current delivery methods require antibody-modified vehicles with manufacturing challenges. In this study,inspired by cardiolipin,we identify cardiolipin-like di-phosphoramide lipids that improve T cell transfection without targeting ligands,both in vitro and in vivo. The T cell-favored tropism is likely due to the lipid’s packing,shape,and rigidity. Encapsulating circular RNA further prolongs mRNA expression in the spleen and T cells. Using PL40 lipid nanoparticles,we deliver mRNA encoding a CAR targeting the senolytic and inflammatory antigen urokinase-type plasminogen activator receptor (uPAR),alleviating uPAR-related liver fibrosis and rheumatoid arthritis (RA). Single-cell sequencing in humans confirms uPAR’s relevance to senescence and inflammation in RA. To facilitate clinical translation,we screen and humanize single-chain variable fragments (scFvs) against uPAR,establishing a PL40 mRNA-encoded humanized uPAR CAR with potential for treating aging-inflamed disorders. Graphical abstract Highlights•Cardiolipin-mimic phosphoramide (CAMP) LNPs transfect T cells without antibody modification•Circular mRNA prolongs mRNA expression•Senolytic in vivo CAR-T treats inflamm-aging disease (liver fibrosis and rheumatoid arthritis)•Develop humanized anti-human uPAR scFv Zhang et al. develop Cardiolipin-mimic phosphoramide (CAMP) lipids,which enable T cell transfection without antibody modification. Using CAMP-based LNPs,they generate senolytic CAR-T cells in vivo to target inflamm-aging diseases. Additionally,they employ circular mRNA to prolong transgene expression. The authors also engineer a humanized anti-human uPAR scFv for clinically relevant applications. View Publication

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles composed of hyperphosphorylated tau protein. The combination of biomarkers is crucial for AD diagnosis. The triggering receptor expressed on myeloid cells 2 (TREM2),a receptor expressed on microglia,is important in AD pathogenesis. Impairment of TREM2 function aggravates the toxic effects of amyloid plaques,and its activation has been shown to reduce Aβ burden and memory deficits. Increased levels of soluble TREM2 (sTREM2) in blood and cerebrospinal fluid is associated with AD. Therefore,TREM2 could serve as a non-invasive biomarker for AD. In this study,we developed a preclinical immunoassay to detect TREM2 for AD diagnosis. Highly sensitive and specific TREM2 antibodies were produced using the hybridoma technique. The three optimized immunoassays exhibited lower limit of quantitation (LLOQ) of 0.474,0.807,and 0.415 ng/mL,respectively. These preclinical immunoassays showed high sensitivity and specificity. The sandwich enzyme-linked immunosorbent assay (ELISA) could potentially be used for AD diagnosis. View Publication

Macrophages play vital roles in innate and adaptive immunity,and their functions are mediated via phagocytosis and antigen presentation. Despite the effort to identify phagocytic checkpoints and explore their mechanism of action,current checkpoint-scanning strategies cannot provide a complete and systematic list of such immune checkpoints. Here,we perform in vitro phagocytosis assays using primary healthy donor macrophages co-cultured with breast cancer cells followed by ribosome profiling of sorted macrophages,to identify immune system-specific checkpoints. We observe a downregulation of CD37 in phagocytic macrophages and demonstrate that targeting CD37 with a specific antibody promotes the phagocytosis of multiple cancer cells in vitro. Mechanistically,tumorous macrophage migration inhibitory factor (MIF) directly binds to CD37,promoting the phosphorylation of CD37Y13 and activating a transduction cascade that involves the recruitment of SHP1 and inhibition of AKT signaling,ultimately impairing phagocytosis. In vivo,targeting CD37 promotes tumor clearance in multiple preclinical mouse models and synergizes with anti-CD47 therapy. Thus,our study identifies a previously unidentified phagocytic checkpoint and provides new potential for precise therapy. Cancer cells evade the immune system by disrupting phagocytic clearance. Here,the authors identify CD37 as a potential checkpoint molecule expressed on non-phagocytes and propose that binding to tumor-derived MIF reduces the phagocytic ability via inhibiting the AKT pathway. In preclinical mouse models,anti-CD37-based therapy enhances phagocytosis by macrophages,facilitating tumor clearance. View Publication

AbstractBackgroundThe pro-inflammatory cytokine,interleukin-18 (IL-18),plays an instrumental role in bolstering anti-tumor immunity. However,the therapeutic application of IL-18 has been limited due to its susceptibility to neutralization by IL-18 binding protein (IL-18BP),short in vivo half-life,and unfavorable physicochemical properties.MethodsIn order to overcome the poor drug-like properties of IL-18,we installed an artificial disulfide bond,removed the native,unpaired cysteines,and fused the stabilized cytokine to an IgG Fc domain. The stability,potency,pharmacokinetic and pharmacodynamic properties as well as efficacy of disulfide-stabilized IL-18 Fc-fusion (dsIL-18-Fc) were assessed via in vitro and in vivo studies.ResultsThe stability and mammalian host cell production yields of dsIL-18-Fc were improved,compared to the wild-type (WT) cytokine,while maintaining its biological potency and interactions with IL-18 receptor α (IL-18Rα) and IL-18BP. Recombinant fusion of the cytokine to an IgG Fc domain provided extended half-life. Notably,despite maintaining sensitivity to IL-18BP,dsIL-18-Fc was effective at activating both T and natural killer (NK) cells,and elicited a strong anti-tumor response,either as a single agent,or in conjunction with anti-programmed cell death-ligand 1 (anti-PD-L1) therapy.ConclusionsWe engineered IL-18 for reinforced stability,extended half-life,and improved manufacturability. The therapeutic benefit of dsIL-18-Fc,coupled with a more favorable manufacturability profile and enhanced drug-like properties,underscores the potential utility of this engineered cytokine in cancer immunotherapy. View Publication

Menopause not only affects fertility but also has widespread impact on systemic health. Yet,the molecular mechanisms underlying this process are not fully understood,partly due to the absence of robust,age-relevant preclinical models with comprehensive molecular and phenotypic characterization. To address this,we systematically compared three candidate mouse models of menopause: (1) intact aging,(2) chemical ovarian follicle depletion using 4-vinylcyclohexene diepoxide (VCD) administered at multiple ages,and (3) Foxl2 haploinsufficiency,a genetic model based on a transcription factor linked to human premature ovarian failure. Through histology,serum hormone profiling,single-cell transcriptomics and machine-learning approaches,we uncovered both shared and model-specific features of follicle loss,endocrine disruption,and transcriptional remodeling. The VCD and Foxl2 haploinsufficiency models revealed distinct patterns of hormonal and immune alterations not captured by intact aging alone. This comparative framework enables informed selection of context-appropriate preclinical rodent models to study menopause and the broader physiological consequences of ovarian aging. View Publication

Memory T cells are a highly heterogeneous collection of antigen-experienced cells that undergo dynamic adaptations upon antigen re-encounter and environmental signals. This heterogeneity hinders studies on memory T cell durability and age-related dysfunction. Using chronic Epstein-Barr virus (EBV) infection and barcode-enabled antigen tracing,we assess the influence of age on memory states at the level of single antigen-specific CD8+ T cells. In young adults (<40 years),EBV-specific CD8+ T cells recognizing different antigenic peptides assume divergent preferred differentiation phenotypes. In older adults (>65-years),antigen-specific cells show largely distinct phenotypic and transcriptomic aging trajectories. Common to many albeit not all antigen-specific populations are maintained TCR diversity,gained natural killer cell-like,innate signatures and lost stem-like features while no evidence is seen for cellular senescence or exhaustion. TCR avidity contributes to these phenotypic differences and aging-related changes. Collectively,our data uncover divergent antigen-guided aging shifts in memory T cell phenotypes,which are informative for antigen selection in optimizing vaccine design and adoptive T cell therapy. Homeostasis of memory T cells is modulated by each antigen encounter,thereby creating a heterogeneous population preventing precise tracking. Here,the authors use barcode-assisted tracing of Epstein-Barr virus-specific CD8+ memory T cells of young and older individuals to find antigen-guided,clonally divergent aging trajectories. View Publication

Imaging macrophage trafficking in solid tumors has major implications for cancer diagnosis,prognosis,and therapy. Here,we show that macrophage labeling with lipid-shelled microbubbles enables ultrasound imaging at single-cell level. Crucially,microbubble labeling and sonication at low mechanical indexes do not affect macrophage viability,migration,phenotype,and cytokine secretion profile,supporting the notion that ultrasound imaging can be used for nondestructive macrophage imaging. Despite the damping exerted on the microbubble oscillations by the cellular compartments,the microbubbles exhibit highly nonlinear behavior upon sonication,allowing for high specificity nonlinear US imaging under in vitro and in vivo conditions. Subsequently,we demonstrate that nonlinear ultrasound imaging can selectively monitor macrophage accumulation and extravasation in solid tumors in rodents for at least 8 h after intravenous administration. These findings establish ultrasound as a noninvasive platform for immune cell trafficking in solid tumors and highlight its potential to advance cancer diagnosis,monitoring,and therapy. Imaging macrophage trafficking in solid tumors has implications for cancer diagnosis and prognosis. by labeling macrophages with lipid-shelled microbubbles in combination with ultrasound,the authors here achieve nondestructive in vivo intravenously administrated macrophage imaging at single cell level with 100 µm resolution till 8 h in solid tumors in rodents. View Publication