Generating Shigella that internalize into glioblastoma cells.

Introduction: The use of microorganisms as drug delivery systems to treat cancer has expanded recently, including FDA approval of certain viruses as oncolytics. Microorganisms have several unique benefits compared to traditional pharmacologic agents including dose independence, the ability to produce therapeutic proteins locally within the tumor, and simplicity of administration. However, current microbial delivery systems such as AAV9 and herpes virus have limited cassette sizes, minimal cancer cell selectivity, and low innate cytotoxicity. To address these issues, we sought to generate a strain of Shigella flexneri to selectively internalize into glioblastoma (GBM) brain tumor cells as an initial step to generating a bacterial-based drug delivery system. Methods: We generated S. flexneri that selectively internalize into GBM cells using iterative co-cultured assays. Results: After 50 rounds of co-culture, the new strain infected 95 percent of GBM cells in 2 hours. GBM-infecting Shigella demonstrate a 124-fold preference for internalizing in nine different GBM cell lines compared to Normal Astrocytes (NA) controls. Additionally, we developed an in-cell western to identify GBM-infecting Shigella clones that preferentially internalize in patient samples without iterative co-culture. Finally, we demonstrate internalization into GBM cells is mediated via a factor modified by myristoylation. Discussion: In conclusion, here we present a novel bacterial platform that preferentially internalizes in brain tumor cells. This system provides numerous potential benefits over current interventions and other microbial strategies for treating brain tumors.

A novel strategy to increase the therapeutic potency of GBM chemotherapy via altering parenchymal/cerebral spinal fluid clearance rate.

Patients with glioblastoma (GBM) face a poor prognosis with a median survival of less than two years. Escalating the dose of chemotherapy is often impossible due to patient comorbidities; thus, we focused on modulating brain clearance as a mechanism to enhance drug accumulation. Given the recently identified interconnectivity between brain parenchymal fluid and cerebral spinal fluid (CSF), we reasoned enhancing drug concentration in the CSF also increases drug concentration in the parenchyma where a GBM resides. To improve drug accumulation in the CSF, we impair the motility of ependymal cell cilia. We identified FDA-approved therapeutics that interact with cilia as a "side effect." Therapeutics that inhibit airway cilia also inhibit ependymal cilia. Multiple cilia-inhibiting drugs, when administered in combination with GBM chemotherapy temozolomide (TMZ), significantly improved the overall survival of mice bearing orthotopic GBM. Combining TMZ with lidocaine results in 100% of animals surviving tumor-free to the study endpoint. This treatment results in a ~ 40-fold increase in brain TMZ levels and is well-tolerated. Mice bearing MGMT methylated, human PDX orthotopic GBM also responded with 100% of animals surviving tumor-free to the study endpoint. Finally, even mice bearing TMZ-resistant, orthotopic GBM responded to the combination treatment with 40% of animals surviving tumor-free to the study endpoint, implying this strategy can sensitize TMZ-resistant GBM. These studies offer a new concept for treating malignant brain tumors by improving the accumulation of TMZ in the CNS. In the future, this regimen may also improve the treatment of additional encephalopathies treated by brain-penetrating therapeutics. SIGNIFICANCE: We exploit the interconnectivity of parenchymal and cerebral spinal fluid to enhance the amount of temozolomide that accumulates in the central nervous system to improve the survival of mice bearing brain tumors.

The 46.1 Antibody Mediates Neurotensin Uptake into the CNS and the Effects Depend on the Route of Intravenous Administration.

Central nervous system (CNS) exposure to blood-borne biotherapeutics is limited by the restrictive nature of the brain vasculature. In particular, tightly sealed endothelial cells of the blood-brain barrier (BBB) prevent the uptake of protein and gene medicines. An approach to increase the bioavailability of such therapeutics is harnessing the BBB endothelial cells' own receptor-mediated transcytosis (RMT) mechanisms. Key to this process is a targeting ligand that can engage a BBB-resident RMT receptor. We recently identified an antibody, named 46.1, that accumulates in the mouse brain after intravenous injection. To further characterize the brain targeting and penetrating properties of clone 46.1, we conjugated neurotensin (NT) to an scFv-Fc form of the antibody (46.1-scFv-Fc-LongLinker-NT). While centrally administered NT decreases the core body temperature and locomotor activity, effects attributed to two spatially segregated brain areas, systemically administered NT has limited effects. Hence, NT can be used as a model therapeutic payload to evaluate the brain penetration of BBB-targeting antibodies and their capability to accumulate in discrete brain areas. We demonstrate that intravenously administered 46.1-scFv-Fc-LL-NT can elicit transient hypothermia and reduce drug-induced hyperlocomotion, confirming that 46.1 can deliver drug cargo to the CNS at pharmacologically relevant doses. Interestingly, when two intravenous administration routes in mice, retro-orbital and tail vein, were compared, only retro-orbital administration led to transient hypothermia. We further explored the retro-orbital route and demonstrated that the 46.1-scFv-Fc-LL-NT could enter the brain arterial blood supply directly from the retro-orbital/cavernous sinus. Taken together, the 46.1 antibody is capable of transporting drug cargo into the CNS, and at least of a portion of its CNS accumulation occurs via the cavernous sinus-arterial route.

Identification of Brain ECM Binding Variable Lymphocyte Receptors Using Yeast Surface Display.

Extracellular matrix (ECM) is a rich mixture of proteins and glycans secreted by cells. This includes typical ECM structures such as collagen and heparin as well as glycosylated, secreted proteins such as growth factors and peptidases. Certain components of ECM are ubiquitous among all tissue; however, each biological tissue also displays unique variations that can be identified using biopanning techniques. Here we describe using a variable lymphocyte receptor (VLR) yeast surface display library to identify selective binders to brain ECM by combining ECM biopanning with a rapid ELISA-based screen using clonal VLRs isolated directly from the yeast surface. Finally, potential ECM-binding candidates can be verified by immunostaining murine brain sections with VLRs released from the yeast surface. These methods provide a framework for the identification of tissue-selective ECM-binding VLRs using yeast surface display techniques and could easily be adapted for other binding scaffolds or ECM from other tissues.

Antibody-Targeted Liposomes for Enhanced Targeting of the Blood-Brain Barrier.

The blood-brain barrier (BBB) hinders therapeutic delivery to the central nervous system (CNS), thereby impeding the development of therapies for brain injury and disease. Receptor-mediated transcytosis (RMT) systems are a promising way to shuttle a targeted therapeutic into the brain. Here, we developed and evaluated an RMT antibody-targeted liposomal system. A previously identified antibody, scFv46.1, that binds to the human and murine BBB and can pass through the murine BBB by transcytosis after intravenous injection was used to decorate the surface of liposomes. Using an in vitro BBB model, we demonstrated the cellular uptake of scFv46.1-modified liposomes (46.1-Lipo). Next, the biodistribution and brain uptake capacity of 46.1-targeted liposomes were assessed after intravenous administration. Our results showed that 46.1-Lipo can lead to increased brain accumulation through targeting of the brain vasculature. Initial rate pharmacokinetic experiments and biodistribution analyses indicated that 46.1-Lipo loaded with pralidoxime exhibited a 10-fold increase in brain accumulation compared with a mock-targeted liposomal group, and this increased accumulation was brain-specific. These studies indicate the potential of this 46.1-Lipo system as a synthetic vehicle for the targeted transport of therapeutic molecules into the CNS.

Site-Directed Modification of Yeast-Produced Proteins Using Expressed Protein Ligation.

Expressed protein ligation (EPL), using non-self-cleaving inteins, allows for the site-specific addition of customized chemical moieties to the termini of proteins. In this way, protein activity can be preserved while functionalizing the target protein with a wide range of chemical handles. Here, we describe methods for EPL-based modification of proteins produced by yeast, employing an engineered, non-self-cleaving intein known as 202-08. Methods for EPL modification of both yeast surface displayed and secreted proteins with bioorthogonal chemical groups are described. These methods allow for the site-specific modification of intein-fused proteins produced in yeast.

Identification of variable lymphocyte receptors that can target therapeutics to pathologically exposed brain extracellular matrix.

Diseases that lead to blood-brain barrier (BBB) disruption will pathologically expose normally inaccessible brain extracellular matrix (ECM) to circulating blood components. Therefore, we hypothesized that brain ECM-targeting moieties could specifically target the disrupted BBB and potentially deliver therapies. Variable lymphocyte receptors (VLRs) that preferentially associate with brain ECM were identified from an immune VLR library via yeast surface display biopanning coupled with a moderate throughput ECM screen. Brain ECM binding of VLR clones to murine and human brain tissue sections was confirmed. After systemic administration, P1C10, the lead brain ECM-targeting VLR candidate, specifically accumulated in brains with mannitol-disrupted BBB and at disrupted BBB regions in two different intracranial glioblastoma models. We also demonstrate P1C10's ability to deliver doxorubicin-loaded liposomes, leading to significantly improved survival in glioblastoma-bearing mice. Thus, VLRs can be used to selectively target pathologically exposed brain ECM and deliver drug payloads.

Site-Specific Antibody Functionalization Using Tetrazine-Styrene Cycloaddition.

Biologics, such as antibody-drug conjugates, are becoming mainstream therapeutics. Consequently, methods to functionalize biologics without disrupting their native properties are essential for identifying, characterizing, and translating candidate biologics from the bench to clinical practice. Here, we present a method for site-specific, carboxy-terminal modification of single-chain antibody fragments (scFvs). ScFvs displayed on the surface of yeast were isolated and functionalized by combining intein-mediated expressed protein ligation (EPL) with inverse electron-demand Diels-Alder (IEDDA) cycloaddition using a styrene-tetrazine pair. The high thiol concentration required to trigger EPL can hinder the subsequent chemoselective ligation reactions; therefore, the EPL reaction was used to append styrene to the scFv, limiting tetrazine exposure to damaging thiols. Subsequently, the styrene-functionalized scFv was reacted with tetrazine-conjugated compounds in an IEDDA cycloaddition to generate functionalized scFvs that retain their native binding activity. Rapid functionalization of yeast surface-derived scFv in a site-directed manner could find utility in many downstream laboratory and preclinical applications.

Yeast display biopanning identifies human antibodies targeting glioblastoma stem-like cells.

Glioblastoma stem-like cells (GSC) are hypothesized to evade current therapies and cause tumor recurrence, contributing to poor patient survival. Existing cell surface markers for GSC are developed from embryonic or neural stem cell systems; however, currently available GSC markers are suboptimal in sensitivity and specificity. We hypothesized that the GSC cell surface proteome could be mined with a yeast display antibody library to reveal novel immunophenotypes. We isolated an extensive collection of antibodies that were differentially selective for GSC. A single domain antibody VH-9.7 showed selectivity for five distinct patient-derived GSC lines and visualized orthotopic GBM xenografts in vivo after conjugation with a near-infrared dye. These findings demonstrate a previously unexplored high-throughput strategy for GSC-selective antibody discovery, to aid in GSC isolation, diagnostic imaging, and therapeutic targeting.

Modular Three-component Delivery System Facilitates HLA Class I Antigen Presentation and CD8(+) T-cell Activation Against Tumors.

Cell-mediated immunotherapies have potential as stand-alone and adjuvant therapies for cancer. However, most current protocols suffer from one or more of three major issues: cost, safety, or efficacy. Here we present a nanoparticle delivery system that facilitates presentation of an immunogenic measles antigen specifically in cancer cells. The delivery system does not contain viral particles, toxins, or biologically derived material. Treatment with this system facilitates activation of a secondary immune response against cancer cells, bypassing the need to identify tumor-associated antigens or educate the immune system through a primary immune response. The delivery system consists of a stealth liposome displaying a cancer-specific targeting peptide, named H1299.3, on its exterior surface and encapsulating H250, an immunogenic human leukocyte antigen class 1 restricted peptide. This targeted-nanoparticle facilitates presentation of the H250 peptide in major histocompatibility complex class I molecules. Activation is dependent on the targeting peptide, previous antigen exposure, and utilizes a novel autophagy-mediated mechanism to facilitate presentation. Treatment with this liposome results in a significant reduction of tumor growth using an aggressive LLC1 model in vaccinated C57BL/6 mice. These data provide proof-of-principle for a novel cell-mediated immunotherapy that is scalable, contains no biologically derived material, and is an efficacious cancer therapy.

Introduction of plasmid encoding for rare tRNAs reduces amplification bias in phage display biopanning.

Amplification bias is a major hurdle in phage display protocols because it imparts additional, unintended selection pressure beyond binding to the desired target. One potential source of amplification bias is the inherent lack of codon optimization that occurs within phage display libraries. Here we present a method that reduces amplification bias by addition of a plasmid that encodes six low abundance tRNAs into K91 Escherichia coli. This new strain, termed K91+, is used to amplify phage during the selection process. We demonstrate the importance of rare codon usage in phage production, and our method produced an overall increase in uniformity of phage production in a random library. Both of these variables are improved in E. coli K91+ compared with the parental K91 strain. This simple solution, requiring only a commercially available plasmid and an additional antibiotic, can reduce amplification bias in phage display protocols.

Identification of a novel lysosomal trafficking peptide using phage display biopanning coupled with endocytic selection pressure.

Methods to select ligands that accumulate specifically in cancer cells and traffic through a defined endocytic pathway may facilitate rapid pairing of ligands with linkers suitable for drug conjugate therapies. We performed phage display biopanning on cancer cells that are treated with selective inhibitors of a given mechanism of endocytosis. Using chlorpromazine to inhibit clathrin-mediated endocytosis in H1299 nonsmall cell lung cancer cells, we identified two clones, ATEPRKQYATPRVFWTDAPG (15.1) and a novel peptide LQWRRDDNVHNFGVWARYRL (H1299.3). The peptides segregate by mechanism of endocytosis and subsequent location of subcellular accumulation. The H1299.3 peptide primarily utilizes clathrin-mediated endocytosis and colocalizes with Lamp1, a lysosomal marker. Conversely, the 15.1 peptide is clathrin-independent and localizes to a perinuclear region. Thus, this novel phage display scheme allows for selection of peptides that selectively internalize into cells via a known mechanism of endocytosis. These types of selections may allow for better matching of linker with targeting ligand by selecting ligands that internalize and traffic to known subcellular locations.

Associations between demographic variables and multiple measles-specific innate and cell-mediated immune responses after measles vaccination.

Measles remains a public health concern due to a lack of vaccine use and vaccine failure. A better understanding of the factors that influence variations in immune responses, including innate/inflammatory and adaptive cellular immune responses, following measles-mumps-rubella (MMR) vaccination could increase our knowledge of measles vaccine-induced immunity and potentially lead to better vaccines. Measles-specific innate/inflammatory and adaptive cell-mediated immune (CMI) responses were characterized using enzyme-linked immunosorbent assays to quantify the levels of secreted IL-2, IL-6, IL-10, IFN-α, IFN-γ, IFN-λ1, and TNF-α in PBMC cultures following in vitro stimulation with measles virus (MV) in a cohort of 764 school-aged children. IFN-γ ELISPOT assays were performed to ascertain the number of measles-specific IFN-γ-secreting cells. Cytokine responses were then tested for associations with self-declared demographic data, including gender, race, and ethnicity. Females secreted significantly more TNF-α, IL-6, and IFN-α (p<0.001, p<0.002, p<0.04, respectively) compared to males. Caucasians secreted significantly more IFN-λ1, IL-10, IL-2, TNF-α, IL-6, and IFN-α (p<0.001, p<0.001, p<0.001, p<0.003, p<0.01, and p<0.02, respectively) compared to the other racial groups combined. Additionally, Caucasians had a greater number of IFN-γ-secreting cells compared to other racial groups (p<0.001). Ethnicity was not significantly correlated with variations in measles-specific CMI measures. Our data suggest that innate/inflammatory and CMI cytokine responses to measles vaccine vary significantly by gender and race. These data further advance our understanding regarding inter-individual and subgroup variations in immune responses to measles vaccination.

Detection of vaccinia virus-specific IFNγ and IL-10 secretion from human PBMCs and CD8⁺ T cells by ELISPOT.

High-throughput in vitro assays, which rapidly and succinctly assess the immune status of large cohorts of individuals, are essential tools for conducting population-based studies, including vaccine research. The enzyme-linked immunospot (ELISPOT) assay has emerged as a sensitive, reliable high-throughput tool to measure functional recall immunity by assessing the frequency of antigen-specific cytokine-secreting lymphocytes present in peripheral blood mononuclear cells (PBMCs). For the past 10 years, ELISPOT method has been the dominant platform and a standard for the cell-mediated immune (CMI) assays. ELISPOT assays are used extensively as a measure of CMI response to vaccines, including smallpox (vaccinia), following primary or secondary vaccination. Here, we present detailed methodology for using ELISPOT assays to detect the frequency of cytokine secreting vaccinia-specific lymphocytes including optimized protocols for growing, titrating, and inactivating vaccinia virus; isolating, cryopreserving, and thawing human PBMCs; and finally, detecting vaccinia-specific IL-10 and IFNγ secreting lymphocytes, as well as CD8(+) IFNγ T cells following in vitro stimulation of PBMCs with vaccinia virus. The methods presented below, although optimized for vaccinia virus, emphasize principles that can be generally applied to create ELISPOT assays capable of assessing the immune status as well as antiviral CD8(+) T cell response of individuals following primary or secondary vaccination with other licensed or novel vaccines.

Correlations between vaccinia-specific immune responses within a cohort of armed forces members.

Widespread vaccination with vaccinia virus (VACV) resulted in the eradication of smallpox; however, the licensed VACV-containing vaccines are associated with adverse events (AEs), making them unsuitable for certain high-risk populations. A better understanding of the host immune response following smallpox vaccination could result in vaccines with similar immunogenicity profiles to pre-eradication vaccines with a lower incidence of AEs. To study the immune response to VACV, we recruited 1,076 armed forces members who had been vaccinated with one dose of Dryvax(®). We measured multiple VACV-specific immune responses: neutralizing antibody titer, the level of 12 secreted cytokines in peripheral blood mononuclear cell (PBMC) cultures (IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-12p40, IL-12p70, TNF-α, IFN-γ, IFN-α, IFN-ß, and IL-18), and the number of IFN-γ- and CD8(+) IFN-γ-secreting cells. We analyzed these data to determine correlations between immune response measures. We detected a strong proinflammatory response in concert with a Th-1-like cytokine response pattern at a median time point of 15.3 mo following primary vaccination. We also detected correlations between neutralizing antibody titer and secreted IL-2, as well as secreted IFN-γ (p=0.009 and p=0.0007, respectively). We also detected strong correlations between the proinflammatory cytokines IL-1ß, TNF-α, IL-6, and IL-12p40 (p<0.0001). These results further advance our knowledge of vaccinia-specific cellular immune responses. Notably, vaccine-induced proinflammatory responses were not correlated with neutralizing antibody titers, suggesting that further attenuation to reduce inflammatory immune responses may result in decreased AEs without sacrificing VACV immunogenicity and population seropositivity.

Genetic polymorphisms in host antiviral genes: associations with humoral and cellular immunity to measles vaccine.

Host antiviral genes are important regulators of antiviral immunity and plausible genetic determinants of immune response heterogeneity after vaccination. We genotyped and analyzed 307 common candidate tagSNPs from 12 antiviral genes in a cohort of 745 schoolchildren immunized with two doses of measles-mumps-rubella (MMR) vaccine. Associations between SNPs/haplotypes and measles virus-specific immune outcomes were assessed using linear regression methodologies in Caucasians and African-Americans. Genetic variants within the DDX58/RIG-I gene, including a coding polymorphism (rs3205166/Val800Val), were associated as single-SNPs (p≤0.017; although these SNPs did not remain significant after correction for false discovery rate/FDR) and in haplotype-level analysis, with measles-specific antibody variations in Caucasians (haplotype allele p-value=0.021; haplotype global p-value=0.076). Four DDX58 polymorphisms, in high LD, demonstrated also associations (after correction for FDR) with variations in both measles-specific IFN-γ and IL-2 secretion in Caucasians (p≤0.001, q=0.193). Two intronic OAS1 polymorphisms, including the functional OAS1 SNP rs10774671 (p=0.003), demonstrated evidence of association with a significant allele-dose-related increase in neutralizing antibody levels in African-Americans. Genotype and haplotype-level associations demonstrated the role of ADAR genetic variants, including a non-synonymous SNP (rs2229857/Arg384Lys; p=0.01), in regulating measles virus-specific IFN-γ Elispot responses in Caucasians (haplotype global p-value=0.017). After correction for FDR, 15 single-SNP associations (11 SNPs in Caucasians and 4 SNPs in African-Americans) still remained significant at the q-value<0.20. In conclusion, our findings strongly point to genetic variants/genes, involved in antiviral sensing and antiviral control, as critical determinants, differentially modulating the adaptive immune responses to live attenuated measles vaccine in Caucasians and African-Americans.