Chimeric antigen receptor (CAR) T cells have robust antitumor activity against hematologic malignancies and have the potential to benefit patients with solid tumors. Immune recognition of murine proteins expressed in adoptively transferred T cells and the lack of homeostatic cytokines in the tumor microenvironment can limit the expansion and persistence of CAR T cells. CARs generated only from human sequences could reduce the risk of immune-mediated rejection, and interleukin 15 (IL15), which promotes T-cell survival and fitness, may improve the expansion and persistence of CAR T cells. In this study, we report a CAR construct (ABBz) assembled from human sequences including a single-chain variable fragment (scFv) specific to alkaline phosphatase, placental-like 2 (ALPPL2). This binder was selected through an unbiased, high-throughput screen of a human antibody-derived, phage-displayed scFv library based on binding specificity, stringency, and low dissociation constant. We demonstrated specificity to the antigen, effective cytolytic function, and cytokine production in ABBz T cells. We showed NK-like effector differentiation with sustained proliferative capacity specific to secreted IL15 coexpression in ABBz T cells. Lastly, we demonstrated that ABBz CAR T cells had robust antitumor activity, which was further enhanced through IL15 coexpression, resulting in NK-like effector differentiation with increased cytotoxicity and superior expansion capacity due to reduced apoptosis of CAR T cells. These results demonstrate that IL15 coexpression can promote effector differentiation while maintaining the proliferative capacity of huALPPL2-CAR T cells and provide a foundation for further clinical development of IL15-coexpressing huALPPL2-CAR T cells in patients.

Background: Atherosclerosis (AS) is a primary driver of cardiovascular disease. This study aimed to investigate the effects of a high-cholesterol diet (HD) on body weight, lipid profile, inflammatory cytokines, plaque stability, and histopathological changes in Apolipoprotein E (ApoE)(-/-) and wild-type (WT) mice, to clarify the underlying mechanisms of AS. Methods: ApoE(-/-) and wild-type mice were fed HD or normal diet (ND) for 4 weeks. Body weight was monitored weekly to assess the development of obesity. The serum levels of blood lipids and inflammatory factors were examined using the corresponding kits. The expression of plaque stability markers was confirmed using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot. Besides, the structure and fat deposition of coronary artery tissues were assessed using hematoxylin and eosin (H&E), Masson trichrome, oil red O, Prussian blue, and immunohistochemical staining. Results: Relative to the ND group, the body weight of mice notably increased in the HD group (p < 0.01). HD feeding elevated serum lipids and pro-inflammatory markers in ApoE(-/-) and wild-type mice (p < 0.01). Expression of plaque instability markers was significantly upregulated in HD-fed groups (p < 0.05). Histological analysis revealed structural disorganization, increased lipid and iron deposition, and greater collagen accumulation in coronary tissues, particularly in the ApoE(-/-) mice. Conclusion: This study demonstrated that HD might accelerate AS by promoting obesity, dyslipidemia, inflammation, plaque instability, and vascular remodeling, especially in genetically susceptible ApoE(-/-) mice. These results may provide new insights for AS prevention and therapy.

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy, largely due to the paucity of reliable biomarkers for early detection and therapeutic targeting. Existing blood protein biomarkers for PDAC often suffer from replicability issues, arising from inherent limitations such as unmeasured confounding factors in conventional epidemiologic study designs. To circumvent these limitations, we use genetic instruments to identify proteins with genetically predicted levels to be associated with PDAC risk. Leveraging genome and plasma proteome data from the INTERVAL study, we established and validated models to predict protein levels using genetic variants. By examining 8,275 PDAC cases and 6,723 controls, we identified 40 associated proteins, of which 16 are novel. Functionally validating these candidates by focusing on 2 selected novel protein-encoding genes, GOLM1 and B4GALT1, we demonstrated their pivotal roles in driving PDAC cell proliferation, migration, and invasion. Furthermore, we also identified potential drug repurposing opportunities for treating PDAC. SIGNIFICANCE:PDAC is a notoriously difficult-to-treat malignancy, and our limited understanding of causal protein markers hampers progress in developing effective early detection strategies and treatments. Our study identifies novel causal proteins using genetic instruments and subsequently functionally validates selected novel proteins. This dual approach enhances our understanding of PDAC etiology and potentially opens new avenues for therapeutic interventions.

Supplementary Figure 7 - PDF file 166K, Real-time RT-PCR shows the mRNA levels of all the factors in the proposed network and Linear regression analyses depict the correlations between each factor and its counterparts

Supplementary Results and Methods - PDF file 255K, Additional information on results and methods

Pancreatic ductal adenocarcinoma (PDAC) remains an extremely aggressive disease characterized by rapidly acquired multi-drug resistance, including to first-line chemotherapeutic agent gemcitabine. Autophagy is a process that is often exploited by cancer and is one of several intrinsic factors associated with resistance to gemcitabine. We have previously found that miR-198 acts as a tumor suppressor in PDAC through the targeting of factors including Valosin-containing protein (VCP). VCP has been reported to play an important role in autophagic flux. In this study, we investigated whether the repression of VCP through miR-198 administration disrupts the autophagy process and sensitizes PDAC cells to gemcitabine treatment in vitro. Moreover, we used LGA-PEI (LPNP) nanoparticles to effectively administer miR-198 to tumors in vivo, inducing tumor sensitization to gemcitabine and leading to a significant reduction in tumor burden and metastases and a concomitant downregulation of VCP expression and autophagy maturation. Our results indicate a potential therapeutic strategy for targeting gemcitabine resistant PDAC and establishes the use of LPNPs for effective therapeutic delivery of nucleic acids in vitro and in vivo.

Supplementary Figure 1 - PDF file 147K, Additional evidence that miR-198 is regulated by MSLN in PC cells

Supplementary Figure 2 - PDF file 207K, PBX-1 and VCP are predicted targets for miR-198

Supplementary Figure 6 - PDF file 388K, miR-198 modulation reduces the tumorigenic functions of mesothelin-overexpressing pancreatic cancer cells in vitro and in vivo

Supplementary Figure 5 - PDF file 212K, Construct details for miR-198 target analysis of MSLN CDS

Supplementary Figure 3 - PDF file 142K, miR-198 modulation in stable cell lines

OBJECTIVES: Disseminated fibrin-rich microthrombi have been reported in patients who died from COVID-19. Our objective is to determine whether the fibrin clot structure and function differ between critically ill patients with or without COVID-19 and to correlate the structure with clinical coagulation biomarkers. DESIGN: A cross-sectional observational study. Platelet poor plasma was used to analyze fibrin clot structure; the functional implications were determined by quantifying clot turbidity and porosity. SETTING: ICU at an academic medical center and an academic laboratory. PATIENTS: Patients admitted from July 1 to August 1, 2020, to the ICU with severe acute respiratory syndrome coronavirus 2 infection confirmed by reverse transcription-polymerase chain reaction or patients admitted to the ICU with sepsis. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood was collected from 36 patients including 26 ICU patients with COVID-19 and 10 ICU patients with sepsis but without COVID-19 at a median of 11 days after ICU admission (interquartile range, 3–16). The cohorts were similar in age, gender, body mass index, comorbidities, Sequential Organ Failure Assessment (SOFA) score, and mortality. More patients with COVID-19 (100% vs 70%; p = 0.003) required anticoagulation. Ex vivo fibrin clots formed from patients with COVID-19 appeared to be denser and to have smaller pores than those from patients with sepsis but without COVID-19 (percent area of fluorescent fibrin 48.1% [SD, 16%] vs 24.9% [SD, 18.8%]; p = 0.049). The turbidity and flow-through assays corroborated these data; fibrin clots had a higher maximum turbidity in patients with COVID-19 compared with patients without COVID-19 (0.168 vs 0.089 OD units; p = 0.003), and it took longer for buffer to flow through these clots (216 vs 103 min; p = 0.003). In patients with COVID-19, d -dimer levels were positively correlated with percent area of fluorescent fibrin ( ρ = 0.714, p = 0.047). Denser clots (assessed by turbidity and thromboelastography) and higher SOFA scores were independently associated with delayed clot lysis. CONCLUSIONS: We found aberrant fibrin clot structure and function in critically ill patients with COVID-19. These findings may contribute to the poor outcomes observed in COVID-19 patients with widespread fibrin deposition.

Most cancers harbor a diverse collection of cell types including a typically heterogeneous cancer cell fraction. To reconstruct cell-intrinsic and heterotypic interactions driving tumor progression, we combine the XDec deconvolution method with cell-type-specific gene expression correlation analysis into the XDec-CHI method. XDec-CHI identifies intra-and inter-cellular pathways using correlation and places them in the context of specific tumor subtypes, as defined by the state of constituent cancer cells. We make the method web-accessible for analysis of publicly accessible pancreatic ductal adenocarcinoma, breast, head and neck, glioblastoma, and glioma tumors. We apply the method to TCGA and ICGC datasets to identify immune-suppressive interactions within PDAC tumors that are relevant for immunotherapies targeting PD-L1. Subtype-specific interactions derived from correlative analyses validated in co-culture experiments suggest PDAC subtypes have distinct therapeutic weaknesses, with Basal-like and MSLN-high Classical B tumors most likely to respond to therapies targeting PD-L1.

We previously reported a new polymer, lactic-co-glycolic acid-polyethylenimine (LGA-PEI), as an improved nanoparticle (NP) delivery for therapeutic nucleic acids (TNAs). Here, we further developed two antibody (Ab)-conjugated LGA-PEI NP technologies for active-targeting delivery of TNAs. LGA-PEI was covalently conjugated with a single-chain variable fragment antibody (scFv) against mesothelin (MSLN), a biomarker for pancreatic cancer (PC), or a special Ab fragment crystallizable region-binding peptide (FcBP), which binds to any full Ab (IgG). TNAs used in the current study included tumor suppressor microRNA mimics (miR-198 and miR-520h) and non-coding RNA X-inactive specific transcript (XIST) fragments; green fluorescence protein gene (GFP plasmid DNA) was also used as an example of plasmid DNA. MSLN scFv-LGA-PEI NPs with TNAs significantly improved their binding and internalization in PC cells with high expression of MSLN in vitro and in vivo. Anti-epidermal growth factor receptor (EGFR) monoclonal Ab (Cetuximab) binding to FcBP-LGA-PEI showed active-targeting delivery of TNAs to EGFR-expressing PC cells.

A highly cytotoxic T cell memory subset demonstrates intrinsic advantages when used as a CAR T cell platform in a solid tumor model.

Abstract T cell priming against tumor antigens (tAgs) is essential for checkpoint blockade immunotherapy (CBI). When these therapies are delivered systemically (intravenously, i.v), Ag-indiscriminate T cell priming and proliferation can occur, reducing anti-tumor immunity and increasing the potential for immune related adverse events (irAEs). Herein, we use lymphatic delivery (intradermally, i.d) of αCTLA4 combined with virus-like particles (VLPs) to increase tAg functionality of tumor infiltrating lymphocytes (TILs) in mono- and combinational CBI regimens. Beginning at 7 days post implant (p.i) of B16F10 in the right hindlimb, αCTLA4 alone or in combination with αPD1 was administered i.v or i.d at the right base of the tail to reach popliteal or inguinal tumor draining lymph nodes (tdLNs) at intervals of 2 days for three doses. Near-infrared fluorescence imaging confirmed delivery to tdLNs. VLP presenting Melan-A as tAg was administered i.d at days 7 and 14 p.i. Tumor volumes were measured daily and at sacrifice on day 17 p.i. Animals with exponential tumor growth at time of sacrifice were deemed to be non-responders. PBMCs and tumor tissues were collected and analyzed via flow cytometry for CD4+ and CD8+ cells as well as for functionality to tAg as reflected from INFγ and TNFα production. αCTLA4 delivered i.d (N=5) exhibited significantly (p<0.05) reduced growth rates compared to when αCTLA4 was delivered i.v (N=5). VLP administration further depressed growth rates (N=20, p<0.05). Additionally, dosing of αCTLA4 i.d and VLP i.d resulted in 50% more responders than when αCTLA4 was dosed i.v. Dose of αCTLA4, αPD1, and VLP i.d further reduced tumor growth compared to animals receiving VLP i.d and combinational CBI i.v (N=20, p<0.05). Finally, there were no responders in animals receiving αCTLA4 i.v alone (N=5) or in combination with αPD1 i.v and VLP i.d, highlighting the importance of lymphatic delivery on immune priming. We found increased tAg specific CD8+ TNFα+ TILs when αCTLA4 was administered i.d than i.v in both mono- and combinational CBIs (p<0.05). TAg specific CD8+TNFα+ T-cells in PBMCs were also higher in responders than non-responders who received αCTLA4 i.d. In non-responders of αCTLA4 monotherapy, IFNγ production was higher in CD4+ and CD8+ TILs than in responders. Taken together these data support the hypothesis that regional lymphatic delivery of αCTLA4 alone or in combination with αPD1 in tdLNs that are recipient of tAgs enhances anti-tumor immunity. Lymphatic delivery of αCTLA4 and VLP induces robust antitumor activity. Future work involves evaluating how manipulation of tdLNs and non-tdLNs can shape the immune status of the tumor microenvironment. Whether irAEs can be alleviated with lymphatic delivery remains to be evaluated in our pre-clinical models. Supported by CPRIT RP19009. Citation Format: Carolina Mantilla Rojas, Dongliang Liu, Fred Christian Velasquez, Janelle Morton, Wen-Jen Hwu, Qizhi Yao, Eva Sevick-Muraca. Lymphatic delivery of virus-like particles in combination with mono- or combinational checkpoint blockade immunotherapy enhances tumor response and antigen-specific functionality of T cells in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1688.

Development of a vaccine that can elicit robust HIV specific antibody responses in the mucosal compartments is desired for effective prevention of HIV via sexual transmission. However, the current mucosal vaccines have either poor immunogenicity when administered orally or invite safety concerns when administered intranasally. Sublingual immunization has received more attention in recent years based on its efficiency in inducing systemic and mucosal immune responses in both mucosal and extra-mucosal tissues. To facilitate the transport of the immunogen across the sub-mucosal epithelial barrier, we found that CD91, the receptor of C1q, is prevalently expressed in the sublingual mucosal lining, and thus, a modified chimeric C1q surface conjugated CD40L/HIV VLP was generated. The ability of this chimeric C1q/CD40L/HIV VLP to bind, cross the epithelial layer, access and activate the sub-mucosal layer dendritic cells (DCs), and ultimately induce enhanced mucosal and systemic immune responses against HIV is evaluated in this study. We found that C1q/CD40L/HIV VLPs have enhanced binding, increased transport across the epithelial layer, and upregulate DC activation markers as compared to CD40L/HIV VLPs alone. Mice immunized with C1q/CD40L/HIV VLPs by sublingual administration showed higher levels of IgA salivary antibodies against both HIV Gag and Env than mice immunized with CD40L/HIV VLPs. Moreover, sublingual immunization with C1q/CD40L/HIV VLPs induced more Env- and Gag-specific IFN-γ producing T cells than the CD40L/HIV VLPs group. Interestingly, C1q/CD40L/HIV VLP immunization can also induce more mucosal homing T cells than that in CD40L/HIV VLP group. Our data suggest that incorporation of C1q to CD40L/HIV VLPs is a promising novel strategy and that the sublingual immunization can be a favorite immunization route for HIV mucosal vaccines.

Abstract With recent advances in distinguishing Pancreatic Ductal Adenocarcinoma (PDAC) subtypes based on transcriptional profiles, it is becoming clear that different subtypes have distinct therapeutic responses– e.g. Basal-like tumors tend to show less response to chemotherapy than Classical tumors. Here, we asked if the Basal-like, Classical A, and Classical B PDAC subtypes show distinct immuno-suppressive molecular profiles of relevance for targeted immunotherapy of PDAC. Toward this goal, we leveraged two large, public sequencing cohorts – TCGA and ICGC. To accommodate the large variation in cell type composition among the tumors across these cohorts, we applied the novel expression deconvolution (XDec) method to computationally deconvolute the bulk RNA-seq profiles. The method identified cell-type composition of each tumor and identified distinct Basal-like, Classical A, and Classical B subtypes, thus allowing classification of each tumor based on the state of its constituent cancer cell fraction. We further extended the method to also infer cell-type specific gene expression for each tumor sample and to identify correlation of cell-type specific gene expression levels across cohorts. Using the extended method, we determined that Basal-like cancer cells are CD274 high, Classical B cancer cell CD274 expression correlated strongly with MSLN and RELA (a recently proposed mechanism controlling PD-L1), and Classical A cancer cells had low CD274 expression. Because heterotypic interactions may also contribute toward an immuno-suppressive microenvironment, we next correlated expression levels between cancer and stromal cell types. Cancer cell MSLN expression correlated strongly with stromal CD274 expression which also correlated with stromal IFNGR1 and IFNγ. Using co-culture experiments we validated the causative relationship between cancer cell MSLN and stromal INFGR1 and CD274. Taken together, our results suggest that PDAC subtypes have distinct therapeutic vulnerabilities, with Basal-like and MSLN-high Classical B patients more likely to respond to PD-L1 targeting therapies. Moreover, we provided a methodological advance by extending the XDec method and by making it web-accessible, thus allowing the community to derive hypotheses testable in cell line models by performing correlations of cell-type specific gene expression levels deconvoluted from bulk RNA-seq profiles of PDAC patient tumors. Citation Format: Emily L. LaPlante, Dongliang Liu, Aleksandar Milosavljevic, Qizhi Yao. Basal-like, Classical A, and Classical B subtypes of pancreatic cancer show distinct immuno-suppressive molecular profiles [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-004.

Intracellular microRNAs (miRNAs) can be released into extracellular compartments, thus entering into body fluids such as blood circulation. Circulating miRNAs are highly stable and considered as novel biomarkers for the diagnosis and/or prognosis of cardiovascular diseases (CVDs). There is a growing body of evidence supporting circulating miRNAs as prognosis and/or diagnosis biomarkers for CVDs. Using the signatures of circulating miRNAs could improve sensitivity and specificity as biomarkers for CVDs, including unstable angina, myocardial infarction, in-stent restenosis after coronary drug-eluting stents implantation, atrial fibrillation, and heart failure. Circulating miRNAs are associated with other traditional CVD biomarkers such as cardiac-specific troponin, myoglobin and creatine kinase-MB, and other CVD risk factors such as diabetes mellitus and hyperlipidemia. The present review is different with other review articles in that we provide detailed information for individual miRNAs, including both clinical and basic science data. We have selected the 8 miRNAs (miR-1, miR-21, miR-126, miR-133, miR-145, miR-208, miR-223, and miR-499) that are most studied and have shown major clinical value as biomarkers for CADs. The purpose of this review is to provide a full-spectrum overview of these miRNAs and to help clinicians/researchers to understand the clinical value of major circulating miRNAs and help them to design or participate in further investigations of these miRNAs, developing new technologies to improve health care for patients with CVDs.

Studies have shown that blockade of CTLA-4 promoted the expansion of germinal center B-cells in viral infection or immunization with model antigens. Few studies have evaluated the immunological consequences of CTLA-4 blockade during immunization against relevant vaccine candidates. Here, we investigated the effects of CTLA-4 blockade on HIV virus-like particles (VLPs) vaccination in a C57BL/6J mouse model. We found that CTLA-4 blockade during HIV VLP immunization resulted in increased CD4+ T-cell activation, promoted the expansion of HIV envelope (Env)-specific follicular helper T cell (Tfh) cells, and significantly increased HIV Gag- and Env-specific IgG with higher avidity and antibody-dependent cellular cytotoxicity (ADCC) capabilities. Furthermore, after only a single immunization, CTLA-4 blockade accelerated T-cell dependent IgG class switching and the induction of significantly high serum levels of the B-cell survival factor, A proliferation-inducing ligand (APRIL). Although no significant increase in neutralizing antibodies was observed, increased levels of class-switched Env- and Gag-specific IgG are indicative of increased polyclonal B-cell activation, which demonstrated the ability to mediate and enhance ADCC in this study. Altogether, our findings show that CTLA-4 blockade can increase the levels of HIV antigen-specific B-cell and antigen-specific Tfh cell activity and impact humoral immune responses when combined with a clinically relevant HIV VLP-based vaccine.