Development of a p-Hydroxybenzyl-Alcohol-Linked Glutamate Prodrug for Activation by Pseudomonas Carboxypeptidase G2.

Directed enzyme-prodrug therapies used for targeted drug delivery require prodrugs that are chemically stable and processed efficiently by the activating enzyme. We recently reported the development of AMS-6-Glu (2), a glutamate-masked version of the cytotoxic natural product 5'-O-sulfamoyladenosine (AMS, 1) that can be activated by Pseudomonas carboxypeptidase G2 (CPG2). Herein, we report the development of a second-generation prodrug, AMS-5'-PHOBA-Glu (5), that undergoes cleavage by CPG2 with >160-fold higher efficiency. Use of a p-hydroxybenzyl alcohol (PHOBA) self-immolative linker overcame unexpected chemical instability observed with a conventional p-aminobenzyl alchohol (PABA) linker.

Host Interactions with Engineered T-cell Micropharmacies.

Genetically engineered, cytotoxic, adoptively transferred T cells localize to antigen-positive cancer cells inside patients, but tumor heterogeneity and multiple immune escape mechanisms have prevented the eradication of most solid tumor types. More effective, multifunctional engineered T cells are in development to overcome the barriers to the treatment of solid tumors, but the interactions of these highly modified cells with the host are poorly understood. We previously engineered prodrug-activating enzymatic functions into chimeric antigen receptor (CAR) T cells, endowing them with a killing mechanism orthogonal to conventional T-cell cytotoxicity. These drug-delivering cells, termed Synthetic Enzyme-Armed KillER (SEAKER) cells, demonstrated efficacy in mouse lymphoma xenograft models. However, the interactions of an immunocompromised xenograft with such complex engineered T cells are distinct from those in an immunocompetent host, precluding an understanding of how these physiologic processes may affect the therapy. Herein, we expanded the repertoire of SEAKER cells to target solid-tumor melanomas in syngeneic mouse models using specific targeting with T-cell receptor (TCR)-engineered T cells. We demonstrate that SEAKER cells localized specifically to tumors, and activated bioactive prodrugs, despite host immune responses. We additionally show that TCR-engineered SEAKER cells were efficacious in immunocompetent hosts, demonstrating that the SEAKER platform is applicable to many adoptive cell therapies.

Potentiating antibody-dependent killing of cancers with CAR T cells secreting CD47-SIRPα checkpoint blocker.

Chimeric antigen receptor (CAR) T-cell therapy has shown success in the treatment of hematopoietic malignancies; however, relapse remains a significant issue. To overcome this, we engineered "Orexi" CAR T cells to locally secrete a high-affinity CD47 blocker, CV1, at the tumor and treated tumors in combination with an orthogonally targeted monoclonal antibody. Traditional CAR T cells plus the antibody had an additive effect in xenograft models, and this effect was potentiated by CAR T-cell local CV1 secretion. Furthermore, OrexiCAR-secreted CV1 reversed the immunosuppression of myelomonocytoid cells both in vitro and within the tumor microenvironment. Local secretion of the CD47 inhibitor bypasses the CD47 sink found on all cells in the body and may prevent systemic toxicities. This combination of CAR T-cell therapy, local CD47 blockade, and orthogonal antibody may be a combinatorial strategy to overcome the limitations of each monotherapy.

Investigating N-arylpyrimidinamine (NAPA) compounds as early-stage inhibitors against human cytomegalovirus.

Human cytomegalovirus (CMV) is a ubiquitous ß-herpesvirus that establishes latent asymptomatic infections in healthy individuals but can cause serious infections in immunocompromised people, resulting in increased risk of morbidity and mortality. The current FDA-approved CMV drugs target late stages of the CMV life-cycle. While these drugs are effective in most cases, they have serious drawbacks, including poor oral bioavailability, dose-limiting toxicity, and a low barrier to resistance. Given the clinical relevance of CMV-associated diseases, novel therapies are needed. Thus, a novel class of compounds that inhibits the early stages of the CMV life-cycle was identified and found to block infection of different strains in physiologically relevant cell types. This class of compounds, N-arylpyrimidinamine (NAPA), demonstrated potent anti-CMV activity against ganciclovir-sensitive and -resistant strains in in vitro replication assays, a selectivity index >30, and favorable in vitro ADME properties. Mechanism of action studies demonstrated that NAPA compounds inhibit an early step of virus infection. NAPA compounds are specific inhibitors of cytomegaloviruses and exhibited limited anti-viral activity against other herpesviruses. Collectively, we have identified a novel class of CMV inhibitor that effectively limits viral infection and proliferation.

A TCR mimic CAR T cell specific for NDC80 is broadly reactive with solid tumors and hematologic malignancies.

Target identification for chimeric antigen receptor (CAR) T-cell therapies remains challenging due to the limited repertoire of tumor-specific surface proteins. Intracellular proteins presented in the context of cell surface HLA provide a wide pool of potential antigens targetable through T-cell receptor mimic antibodies. Mass spectrometry (MS) of HLA ligands from 8 hematologic and nonhematologic cancer cell lines identified a shared, non-immunogenic, HLA-A*02-restricted ligand (ALNEQIARL) derived from the kinetochore-associated NDC80 gene. CAR T cells directed against the ALNEQIARL:HLA-A*02 complex exhibited high sensitivity and specificity for recognition and killing of multiple cancer types, especially those of hematologic origin, and were efficacious in mouse models against a human leukemia and a solid tumor. In contrast, no toxicities toward resting or activated healthy leukocytes as well as hematopoietic stem cells were observed. This shows how MS can inform the design of broadly reactive therapeutic T-cell receptor mimic CAR T-cell therapies that can target multiple cancer types currently not druggable by small molecules, conventional CAR T cells, T cells, or antibodies.

Engineering CAR-T cells to activate small-molecule drugs in situ.

Chimeric antigen receptor (CAR)-T cells represent a major breakthrough in cancer therapy, wherein a patient's own T cells are engineered to recognize a tumor antigen, resulting in activation of a local cytotoxic immune response. However, CAR-T cell therapies are currently limited to the treatment of B cell cancers and their effectiveness is hindered by resistance from antigen-negative tumor cells, immunosuppression in the tumor microenvironment, eventual exhaustion of T cell immunologic functions and frequent severe toxicities. To overcome these problems, we have developed a novel class of CAR-T cells engineered to express an enzyme that activates a systemically administered small-molecule prodrug in situ at a tumor site. We show that these synthetic enzyme-armed killer (SEAKER) cells exhibit enhanced anticancer activity with small-molecule prodrugs, both in vitro and in vivo in mouse tumor models. This modular platform enables combined targeting of cellular and small-molecule therapies to treat cancers and potentially a variety of other diseases.

Targeted Cellular Micropharmacies: Cells Engineered for Localized Drug Delivery.

The recent emergence of engineered cellular therapies, such as Chimeric antigen receptor (CAR) CAR T and T cell receptor (TCR) engineered T cells, has shown great promise in the treatment of various cancers. These agents aggregate and expand exponentially at the tumor site, resulting in potent immune activation and tumor clearance. Moreover, the ability to elaborate these cells with therapeutic agents, such as antibodies, enzymes, and immunostimulatory molecules, presents an unprecedented opportunity to specifically modulate the tumor microenvironment through cell-mediated drug delivery. This unique pharmacology, combined with significant advances in synthetic biology and cell engineering, has established a new paradigm for cells as vectors for drug delivery. Targeted cellular micropharmacies (TCMs) are a revolutionary new class of living drugs, which we envision will play an important role in cancer medicine and beyond. Here, we review important advances and considerations underway in developing this promising advancement in biological therapeutics.

CD46 facilitates entry and dissemination of human cytomegalovirus.

Human cytomegalovirus (CMV) causes a wide array of disease to diverse populations of immune-compromised individuals. Thus, a more comprehensive understanding of how CMV enters numerous host cell types is necessary to further delineate the complex nature of CMV pathogenesis and to develop targeted therapeutics. To that end, we establish a vaccination strategy utilizing membrane vesicles derived from epithelial cells to generate a library of monoclonal antibodies (mAbs) targeting cell surface proteins in their native conformation. A high-throughput inhibition assay is employed to screen these antibodies for their ability to limit infection, and mAbs targeting CD46 are identified. In addition, a significant reduction of viral proliferation in CD46-KO epithelial cells confirms a role for CD46 function in viral dissemination. Further, we demonstrate a CD46-dependent entry pathway of virus infection in trophoblasts, but not in fibroblasts, highlighting the complexity of CMV entry and identifying CD46 as an entry factor in congenital infection.

Functional screening for anti-CMV biologics identifies a broadly neutralizing epitope of an essential envelope protein.

The prototypic ß-herpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. The CMV envelope consists of various protein complexes that enable wide viral tropism. More specifically, the glycoprotein complex gH/gL/gO (gH-trimer) is required for infection of all cell types, while the gH/gL/UL128/130/131a (gH-pentamer) complex imparts specificity in infecting epithelial, endothelial and myeloid cells. Here we utilize state-of-the-art robotics and a high-throughput neutralization assay to screen and identify monoclonal antibodies (mAbs) targeting the gH glycoproteins that display broad-spectrum properties to inhibit virus infection and dissemination. Subsequent biochemical characterization reveals that the mAbs bind to gH-trimer and gH-pentamer complexes and identify the antibodies' epitope as an 'antigenic hot spot' critical for virus entry. The mAbs inhibit CMV infection at a post-attachment step by interacting with a highly conserved central alpha helix-rich domain. The platform described here provides the framework for development of effective CMV biologics and vaccine design strategies.

The Microtubule Inhibitor Podofilox Inhibits an Early Entry Step of Human Cytomegalovirus.

Human cytomegalovirus is a ubiquitous ß-herpesvirus that infects many different cell types through an initial binding to cell surface receptors followed by a fusion event at the cell membrane or endocytic vesicle. A recent high-throughput screen to identify compounds that block a step prior to viral gene expression identified podofilox as a potent and nontoxic inhibitor. Time-of-addition studies in combination with quantitative-PCR analysis demonstrated that podofilox limits an early step of virus entry at the cell surface. Podofilox was also able to drastically reduce infection by herpes simplex 1, an α-herpesvirus with a very similar entry process to CMV. Podofilox caused a reduced maximal plateau inhibition of infection by viruses with single step binding processes prior to fusion-like Newcastle disease virus, Sendai virus, and influenza A virus or viruses that enter via endocytosis like vesicular stomatitis virus and a clinical-like strain of CMV. These results indicate that microtubules appear to be participating in the post-binding step of virus entry including the pre- and post-penetration events. Modulation of the plasma membrane is required to promote virus entry for herpesviruses, and that podofilox, unlike colchicine or nocodazole, is able to preferentially target microtubule networks at the plasma membrane.

Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway.

The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease.

ISG15 deficiency and increased viral resistance in humans but not mice.

ISG15 is an interferon (IFN)-α/ß-induced ubiquitin-like protein. It exists as a free molecule, intracellularly and extracellularly, and conjugated to target proteins. Studies in mice have demonstrated a role for Isg15 in antiviral immunity. By contrast, human ISG15 was shown to have critical immune functions, but not in antiviral immunity. Namely, free extracellular ISG15 is crucial in IFN-γ-dependent antimycobacterial immunity, while free intracellular ISG15 is crucial for USP18-mediated downregulation of IFN-α/ß signalling. Here we describe ISG15-deficient patients who display no enhanced susceptibility to viruses in vivo, in stark contrast to Isg15-deficient mice. Furthermore, fibroblasts derived from ISG15-deficient patients display enhanced antiviral protection, and expression of ISG15 attenuates viral resistance to WT control levels. The species-specific gain-of-function in antiviral immunity observed in ISG15 deficiency is explained by the requirement of ISG15 to sustain USP18 levels in humans, a mechanism not operating in mice.

Human cytomegalovirus gH stability and trafficking are regulated by ER-associated degradation and transmembrane architecture.

The prototypic betaherpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. While benign in healthy individuals, CMV poses a significant threat to the immune compromised, including transplant recipients and neonates. The CMV glycoprotein complex gH/gL/gO mediates infection of fibroblasts, and together with the gH/gL/UL128/130/131 a pentameric complex permits infection of epithelial, endothethial, and myeloid cells. Given the central role of the gH/gL complex during infection, we were interested in studying cellular trafficking of the gH/gL complex through generation of human cells that stably express gH and gL. When expressed alone, CMV gH and gL were degraded through the ER-associated degradation (ERAD) pathway. However, co-expression of these proteins stabilized the polypeptides and enhanced their cell-surface expression. To further define regulatory factors involved in gH/gL trafficking, a CMV gH chimera in which the gH transmembrane and cytoplasmic tail were replaced with that of human CD4 protein permitted cell surface gH expression in absence of gL. We thus demonstrate the ability of distinct cellular processes to regulate the trafficking of viral glycoproteins. Collectively, the data provide insight into the processing and trafficking requirements of CMV envelope protein complexes and provide an example of the co-opting of cellular processes by CMV.

Targeting Viral Proteostasis Limits Influenza Virus, HIV, and Dengue Virus Infection.

Viruses are obligate parasites and thus require the machinery of the host cell to replicate. Inhibition of host factors co-opted during active infection is a strategy hosts use to suppress viral replication and a potential pan-antiviral therapy. To define the cellular proteins and processes required for a virus during infection is thus crucial to understanding the mechanisms of virally induced disease. In this report, we generated fully infectious tagged influenza viruses and used infection-based proteomics to identify pivotal arms of cellular signaling required for influenza virus growth and infectivity. Using mathematical modeling and genetic and pharmacologic approaches, we revealed that modulation of Sec61-mediated cotranslational translocation selectively impaired glycoprotein proteostasis of influenza as well as HIV and dengue viruses and led to inhibition of viral growth and infectivity. Thus, by studying virus-human protein-protein interactions in the context of active replication, we have identified targetable host factors for broad-spectrum antiviral therapies.

ATPase4A Autoreactivity and Its Association With Autoimmune Phenotypes in the Type 1 Diabetes Genetics Consortium Study.

Autoantibodies targeting the H+/K+-ATPase proton pump of the gastric parietal cell (parietal cell antibodies [PCA]) are diagnostic of atrophic body gastritis (ABG) leading to pernicious anemia (PA). PCA, ABG, and PA occur in increased frequency in patients with type 1 diabetes and their relatives and are considered "minor" components of forms of autoimmune polyglandular syndrome (APS). A customized radioimmunoprecipitation assay was applied to 6,749 samples from the Type 1 Diabetes Genetics Consortium to measure ATP4A autoreactivity. Autoantibody prevalence was correlated with variants in HLA class II, PTPN22, and CTLA4 genes. With an ATP4A radioimmunoprecipitation assay, PCA were detected in sera from 20.9% of affected individuals. PCA prevalence increased with age and was greater in females (25.3%) than males (16.5%) and among Hispanics (36.3%) and blacks (26.2%) compared with non-Hispanic whites (20.8%) and Asians (16.7%). PCA and other organ-specific autoantibodies GAD65, IA-2, thyroid peroxidase (TPO), 21-hydroxylase (21-OH), and transglutaminase (TG) clustered within families with heritability estimates from 71 to 95%. PCA clustered with TPO, 21-OH, and persistent GAD65 autoantibodies but not with celiac (TG) or IA-2 autoantibodies. PCA-positive subjects showed an increased frequency of DRB1*0404, DPB1*0201, and PTPN22 R620W (rs2476601-T) and a decreased frequency of DRB1*0101, DPB1*0301, and CTLA4 CT60 (rs3087243-T). Genetic variants accounted for 4-5% of the heritable risk for PCA. The same alleles were associated with other autoantibody phenotypes in a consistent pattern. Whereas most of the heritable risk for PCA and other antibodies reflects genetic effects that are tissue specific, parietal cell autoimmunity is a major pathogenetic contributor in APS2.

Development of a high-content screen for the identification of inhibitors directed against the early steps of the cytomegalovirus infectious cycle.

Human cytomegalovirus (CMV) is a latent and persistent virus whose proliferation increases morbidity and mortality of immune-compromised individuals. The current anti-CMV therapeutics targeting the viral DNA polymerase or the major immediate-early (MIE) gene locus are somewhat effective at limiting CMV-associated disease. However, due to low bioavailability, severe toxicity, and the development of drug resistant CMV strains following prolonged treatment, current anti-CMV therapeutics are insufficient. To help address this shortfall, we established a high-content assay to identify inhibitors targeting CMV entry and the early steps of infection. The infection of primary human fibroblasts with a variant of the CMV laboratory strain AD169 expressing a chimeric IE2-yellow fluorescence protein (YFP) (AD169IE2-YFP) provided the basis for the high-content assay. The localization of IE2-YFP to the nucleus shortly following an AD169IE2-YFP infection induced a robust fluorescent signal that was quantified using confocal microscopy. The assay was optimized to achieve outstanding assay fitness and high Z' scores. We then screened a bioactive chemical library consisting of 2080 compounds and identified hit compounds based on the decrease of fluorescence signal from IE2-YFP nuclear expression. The hit compounds likely target various cellular processes involved in the early steps of infection including capsid transport, chromatin remodeling, and viral gene expression. Extensive secondary assays confirmed the ability of a hit compound, convallatoxin, to inhibit infection of both laboratory and clinical CMV strains and limit virus proliferation. Collectively, the data demonstrate that we have established a robust high-content screen to identify compounds that limit the early steps of the CMV life cycle, and that novel inhibitors of early infection events may serve as viable CMV therapeutics.

Human cytomegalovirus US28 facilitates cell-to-cell viral dissemination.

Human cytomegalovirus (HCMV) encodes a number of viral proteins with homology to cellular G protein-coupled receptors (GPCRs). These viral GPCRs, including US27, US28, UL33, and UL78, have been ascribed numerous functions during infection, including activating diverse cellular pathways, binding to immunomodulatory chemokines, and impacting virus dissemination. To investigate the role of US28 during virus infection, two variants of the clinical isolate TB40/E were generated: TB40/E-US28(YFP) expressing a C-terminal yellow fluorescent protein tag, and TB40/E-FLAG(YFP) in which a FLAG-YFP cassette replaces the US28 coding region. The TB40/E-US28(YFP) protein localized as large perinuclear fluorescent structures at late times post-infection in fibroblasts, endothelial, and epithelial cells. Interestingly, US28(YFP) is a non-glycosylated membrane protein throughout the course of infection. US28 appears to impact cell-to-cell spread of virus, as the DUS28 virus (TB40/E-FLAG(YFP)) generated a log-greater yield of extracellular progeny whose spread could be significantly neutralized in fibroblasts. Most strikingly, in epithelial cells, where dissemination of virus occurs exclusively by the cell-to-cell route, TB40/E-FLAG(YFP) (DUS28) displayed a significant growth defect. The data demonstrates that HCMV US28 may contribute at a late stage of the viral life cycle to cell-to-cell dissemination of virus.

Neutralizing antibodies against previously encountered influenza virus strains increase over time: a longitudinal analysis.

Antigenic diversity shapes immunity in distinct and unexpected ways. This is particularly true of the humoral response generated against influenza A viruses. Although it is known that immunological memory developed against previously encountered influenza A virus strains affects the outcome of subsequent infections, exactly how sequential exposures to antigenically variant viruses shape the humoral immune response in humans remains poorly understood. To address this important question, we performed a longitudinal analysis of antibody titers against various pandemic and seasonal strains of influenza virus spanning a 20-year period (1987 to 2008) with samples from 40 individuals (birth dates, 1917 to 1952) obtained from the Framingham Heart Study. Longitudinal increases in neutralizing antibody titers were observed against previously encountered pandemic H2N2, H3N2, and H1N1 influenza A virus strains. Antibody titers against seasonal strains encountered later in life also increased longitudinally at a rate similar to that against their pandemic predecessors. Titers of cross-reactive antibodies specific to the hemagglutinin stalk domain were also investigated because they are influenced by exposure to antigenically diverse influenza A viruses. These titers rose modestly over time, even in the absence of major antigenic shifts. No sustained increase in neutralizing antibody titers against an antigenically more stable virus (human cytomegalovirus) was observed. The results herein describe a role for antigenic variation in shaping the humoral immune compartment and provide a rational basis for the hierarchical nature of antibody titers against influenza A viruses in humans.

Development of a high-throughput assay to measure the neutralization capability of anti-cytomegalovirus antibodies.

Infection by human cytomegalovirus (CMV) elicits a strong humoral immune response and robust anti-CMV antibody production. Diagnosis of virus infection can be carried out by using a variety of serological assays; however, quantification of serum antibodies against CMV may not present an accurate measure of a patient's ability to control a virus infection. CMV strains that express green fluorescent protein (GFP) fusion proteins can be used as screening tools for evaluating characteristics of CMV infection in vitro. In this study, we employed a CMV virus strain, AD169, that ectopically expresses a yellow fluorescent protein (YFP) fused to the immediate-early 2 (IE2) protein product (AD169IE2-YFP) to quantify a CMV infection in human cells. We created a high-throughput cell-based assay that requires minimal amounts of material and provides a platform for rapid analysis of the initial phase of virus infection, including virus attachment, fusion, and immediate-early viral gene expression. The AD169IE2-YFP cell infection system was utilized to develop a neutralization assay with a monoclonal antibody against the viral surface glycoprotein gH. The high-throughput assay was extended to measure the neutralization capacity of serum from CMV-positive subjects. These findings describe a sensitive and specific assay for the quantification of a key immunological response that plays a role in limiting CMV dissemination and transmission. Collectively, we have demonstrated that a robust high-throughput infection assay can analyze the early steps of the CMV life cycle and quantify the potency of biological reagents to attenuate a virus infection.

Human cytomegalovirus US3 modulates destruction of MHC class I molecules.

Human cytomegalovirus (HCMV), a member of the Herpesviridae family, is proficient at establishing lifelong persistence within the host in part due to immune modulating genes that limit immune recognition. HCMV encodes at least five glycoproteins within its unique short (US) genomic region that interfere with MHC class I antigen presentation, thus hindering viral clearance by cytotoxic T lymphocytes (CTL). Specifically, US3 retains class I within the endoplasmic reticulum (ER), while US2 and US11 induce class I heavy chain destruction. A cooperative effect on class I down-regulation during stable expression of HCMV US2 and US3 has been established. To address the impact of US3 on US11-mediated MHC class I down-regulation, the fate of class I molecules was examined in US3/US11-expressing cells and virus infection studies. Co-expression of US3 and US11 resulted in a decrease of surface expression of class I molecules. However, the class I molecules in US3/US11 cells were mostly retained in the ER with an attenuated rate of proteasome destruction. Analysis of class I levels from virus-infected cells using HCMV variants either expressing US3 or US11 revealed efficient surface class I down-regulation upon expression of both viral proteins. Cells infected with both US3 and US11 expressing viruses demonstrate enhanced retention of MHC class I complexes within the ER. Collectively, the data suggests a paradigm where HCMV-induced surface class I down-regulation occurs by diverse mechanisms dependent on the expression of specific US genes. These results validate the commitment of HCMV to limiting the surface expression of class I levels during infection.